Dissertations / Theses on the topic 'Plant micropropagation'

The techniques of plant micropropagation have not been successfully applied to all species. This study was carried out with the objectives of developing new techniques for rapidly assessing the relative merits of cultural treatments and identifying fundamental and genotype-specific problems associated with micropropagation. Anatomical characteristics of mlcropropagated Hosta spp. and Paeonia lactiflora were investigated. A root exodermis was present and apoplastic tracer studies indicated it was functionally and anatomically the same as ex vitro root exodermes in the literature. Specialised cells rather than simple wound tissue were present at the plantlet /medium interface An endodermls was present in the shoot base of Hosta plantlets. It is suggested that the basal zone of the shoot is functionally a specialised "root". It is hypothesised that carbohydrate status (or solute potential) of vascular cambla and/or root initial cell Is Important in the induction of adventitious root formation. Growth medium GA, and possibly raised inorganic phosphate, resulted in Increased shoot "health" but inhibited rooting in P. lactiflora cultures, possibly through changes in assimilate partitioning and sucrose uptake. A low mobility esterase isoenzyme was specific to these changes. Water relations are identified as a critical factor in the micropropagation of P. lactiflora. Cold storage of Hosta spp. led to sequential leaf senescence, abscission and changes in isoenzyme patterns. No true dormancy was identified in culture, although it was demonstrated after weaning if a requirement for cold storage was not met. In vitro, "dormancy" was expressed as a reduction in the rate of new leaf production. Removal of this growth inhibition was correlated with the appearance of a highly mobile esterase isoenzyme. The possibility of using this isoenzyme to predict subsequent in vitro growth inhibition and ex vitro dormancy is discussed. The objectives of this study were fulfilled, and the direction of future research is discussed.

The principle hypothesis of this thesis is that hypoxia, in agar-based media, compromises rooting in vitro. From a practical point of view this is important because most plant tissue culture activities require the material to be successfully acclimatised in a nursery environment. Compromised rooting often results in excessive losses at this stage which are costly and inconvenient. In addition, many plants with commercial and/or scientific interest remain unavailable as they are not able to be rooted and acclimatised reliably. The use of agar as a rooting medium has limited the capacity of plant tissue culture to clonally propagate many plants. The thesis begins by demonstrating how poorly some plants respond to agar rooting media. Juvenile Chamelaucium hybrid microcuttings were pulsed with IBA 40 mcg M and then placed for 3 weeks on either M1 (1/2 MS) or aerated in vitro soil-less substrate (IVS) (Chapter 2). IVS had 42-82% rooting at the end of Stage 3 compared with 0-1% in agar. Shoot survival for IVS-rooted microcuttings was significantly greater than M1-rooted shoots. Pulsed shoots placed in IVS showed root primordia after 7 days. In contrast, shoots placed in agar showed no root primordia after 21 days and formed callus but did not root when subsequently placed in IVS for a further 4 weeks. The agar medium almost totally and permanently inhibited the capacity of competent shoots to form root primordia and roots. The effectiveness of different types of aerated and non-aerated media, including IVS, were tested to validate the hypothesis (Chapter 3). Microcuttings from shoot cultures of two Australian plants Grevillea thelemanniana and Verticordia plumosa x Chamelaucium uncinatum were pulsed for 7 days on a high auxin (40 mcg M IBA), agar-solidified medium in the dark. Rooting of the microcuttings was then compared on five experimental substrates: a) standard agar M1 medium (1/2 MS, no hormones, 8 g agar L-1), b) porous-agar medium (1/2 MS, no hormones, 30 g agar L-1, solidified then blended to provide aeration), c) white sand wet with liquid M1, d) white sand with M1 medium containing agar, and e) IVS. A separate experiment involved flushing the IVS soil profile with low or normal oxygen. Low and variable rooting percentages were recorded on the controls on M1 medium. Root induction and average total root length per microcutting at final harvest were significantly higher using the porous media including IVS, blended agar or white sand. The M1 medium and the addition of M1 medium to sand suppressed the percentage rooting and elongation. Flushing the IVS rooting medium with low oxygen also suppressed rooting. The experiments showed that increasing the air-filled porosity of the rooting medium has a positive effect on rooting and this is most likely due to the increased oxygen at the base of the microcutting. The role of ethylene, and the sugar and nutrients in M1 were not investigated. The efficacy of the IVS protocol on a range of Australian herbaceous and woody species was investigated to determine whether the observed benefits were generic or plant specific (Chapter 4). Improved rooting in IVS compared to agar was shown for 28 Australian species and genotypes from the families Liliaceae, Haemodoraceae, Myrtaceae, Thymelaeaceae, Proteaceae, Goodeniaceae and Rutaceae. Twenty-seven of the 28 species rooted in IVS medium at equal or better rates than in M1. In three cases - Actinodium cunninghamii, one of the Pimelea physodes genotypes and one of the Eriostemon australasius genotypes - shoots did not root in M1 but showed good root development in IVS medium. With few exceptions average root length and number in microcuttings rooted in IVS was superior to those in agar medium. To further test the resilience of the hypothesis, it was tested on nodal microcuttings of lentil which are recalcitrant to root in vitro (Chapter 5). The veracity of a published conclusion that inverted lentil microcuttings (with their base in the air) root better because of their altered polarity was also examined. It was found that, as is the case for many species, roots initiated and grew only at the proximal end of the microcutting regardless of its orientation. When the proximal end was in agar (a hypoxic environment) the rooting percentage was low (9-25%) even when the orientation of the microcutting was altered by inverting the culture tube. In contrast, when the proximal end of the microcutting was in an aerobic environment (from the shoot being placed upside down in agar medium or placed normally or upside down in an aerated medium) rooting percentages were higher (62-100%). Given that Stage 2 microcuttings are prepared with the objective to root and acclimatise them to nursery conditions, the duration of this activity becomes important as it can impact on plant quality and costs. The pulsing protocol and the length of time that Stage 3 cultures remain in the culture room during the rooting phase is a component of the unit cost of production of each rooted microcutting. Initially a 7-day IBA pulse was used after which the pulsed microcuttings were transferred to IVS to root. Chapter 6 shows that the pulsing period can be shortened to one day or replaced with a single auxin dip while still achieving high rooting percentages and maintaining plant quality. These materials handling improvements go some way to realising the logistical benefits of ex vitro rooting but without compromising the positive influences of hygiene and a stable environment of the in vitro environment.

The principle hypothesis of this thesis is that hypoxia, in agar-based media, compromises rooting in vitro. From a practical point of view this is important because most plant tissue culture activities require the material to be successfully acclimatised in a nursery environment. Compromised rooting often results in excessive losses at this stage which are costly and inconvenient. In addition, many plants with commercial and/or scientific interest remain unavailable as they are not able to be rooted and acclimatised reliably. The use of agar as a rooting medium has limited the capacity of plant tissue culture to clonally propagate many plants. The thesis begins by demonstrating how poorly some plants respond to agar rooting media. Juvenile Chamelaucium hybrid microcuttings were pulsed with IBA 40 mcg M and then placed for 3 weeks on either M1 (1/2 MS) or aerated in vitro soil-less substrate (IVS) (Chapter 2). IVS had 42-82% rooting at the end of Stage 3 compared with 0-1% in agar. Shoot survival for IVS-rooted microcuttings was significantly greater than M1-rooted shoots. Pulsed shoots placed in IVS showed root primordia after 7 days. In contrast, shoots placed in agar showed no root primordia after 21 days and formed callus but did not root when subsequently placed in IVS for a further 4 weeks. The agar medium almost totally and permanently inhibited the capacity of competent shoots to form root primordia and roots. The effectiveness of different types of aerated and non-aerated media, including IVS, were tested to validate the hypothesis (Chapter 3). Microcuttings from shoot cultures of two Australian plants Grevillea thelemanniana and Verticordia plumosa x Chamelaucium uncinatum were pulsed for 7 days on a high auxin (40 mcg M IBA), agar-solidified medium in the dark. Rooting of the microcuttings was then compared on five experimental substrates: a) standard agar M1 medium (1/2 MS, no hormones, 8 g agar L-1), b) porous-agar medium (1/2 MS, no hormones, 30 g agar L-1, solidified then blended to provide aeration), c) white sand wet with liquid M1, d) white sand with M1 medium containing agar, and e) IVS. A separate experiment involved flushing the IVS soil profile with low or normal oxygen. Low and variable rooting percentages were recorded on the controls on M1 medium. Root induction and average total root length per microcutting at final harvest were significantly higher using the porous media including IVS, blended agar or white sand. The M1 medium and the addition of M1 medium to sand suppressed the percentage rooting and elongation. Flushing the IVS rooting medium with low oxygen also suppressed rooting. The experiments showed that increasing the air-filled porosity of the rooting medium has a positive effect on rooting and this is most likely due to the increased oxygen at the base of the microcutting. The role of ethylene, and the sugar and nutrients in M1 were not investigated. The efficacy of the IVS protocol on a range of Australian herbaceous and woody species was investigated to determine whether the observed benefits were generic or plant specific (Chapter 4). Improved rooting in IVS compared to agar was shown for 28 Australian species and genotypes from the families Liliaceae, Haemodoraceae, Myrtaceae, Thymelaeaceae, Proteaceae, Goodeniaceae and Rutaceae. Twenty-seven of the 28 species rooted in IVS medium at equal or better rates than in M1. In three cases - Actinodium cunninghamii, one of the Pimelea physodes genotypes and one of the Eriostemon australasius genotypes - shoots did not root in M1 but showed good root development in IVS medium. With few exceptions average root length and number in microcuttings rooted in IVS was superior to those in agar medium. To further test the resilience of the hypothesis, it was tested on nodal microcuttings of lentil which are recalcitrant to root in vitro (Chapter 5). The veracity of a published conclusion that inverted lentil microcuttings (with their base in the air) root better because of their altered polarity was also examined. It was found that, as is the case for many species, roots initiated and grew only at the proximal end of the microcutting regardless of its orientation. When the proximal end was in agar (a hypoxic environment) the rooting percentage was low (9-25%) even when the orientation of the microcutting was altered by inverting the culture tube. In contrast, when the proximal end of the microcutting was in an aerobic environment (from the shoot being placed upside down in agar medium or placed normally or upside down in an aerated medium) rooting percentages were higher (62-100%). Given that Stage 2 microcuttings are prepared with the objective to root and acclimatise them to nursery conditions, the duration of this activity becomes important as it can impact on plant quality and costs. The pulsing protocol and the length of time that Stage 3 cultures remain in the culture room during the rooting phase is a component of the unit cost of production of each rooted microcutting. Initially a 7-day IBA pulse was used after which the pulsed microcuttings were transferred to IVS to root. Chapter 6 shows that the pulsing period can be shortened to one day or replaced with a single auxin dip while still achieving high rooting percentages and maintaining plant quality. These materials handling improvements go some way to realising the logistical benefits of ex vitro rooting but without compromising the positive influences of hygiene and a stable environment of the in vitro environment.

The objective of this study was to improve the existing shoot multiplication protocol for Verticordia grandis (McComb, Arthur & Newll, 1986; Newell, Growns & McComb, 2005) and to investigate and establish reliable root induction and acclimatisation protocols to enhance survival of micropropagated plantlets. It was envisaged that these protocols would be successful in micropropagation, growth and survival of different V. grandis clones and possibly applicable to other Verticordia species. The elongation of in vitro Verticordia shoots on multiplication media was improved by reducing the concentration of BAP from 1μM to 0.25 μM, which resulted in a more uniform shoot length of 4.5 – 5 cm; necessary for root induction experiments. The root induction protocol was optimised by determining the appropriate auxin concentration (80μM indole butyric acid; IBA) with an exposure time of 6 days. Acclimatisation and survival was greatly improved by transferring the IBA pulsed shoots to ex vitro conditions consisting of a free draining and aerated substrate (a mixture of peat and perlite 1:3) in crack pots. These were initially placed into a greenhouse (with controlled temperature & light conditions) in order to maintain high humidity. Over time humidity was reduced and after 112 days the plantlets were transferred to larger pots, containing fresh soil (peat/perlite/sand = 1:1:1) and placed in a shade house with a regular watering regime. Long-term survival was monitored and after 252 days survival was over 70%. The declining survival rates after this time has made it evident that field performance and long-term survival needs further investigation. The application of the improved shoot multiplication and root induction protocols on other V. grandis clones produced survival rates of 0 to 62.5% (depending upon clone) over 252 days.

Thesis (MTech (Horticulture)--Cape Peninsula University of Technology, 2017.
A study was conducted to investigate the effects of various media composition and wounding treating on the in vitro propagation of Argyroderma subalbum and A. testiculare explants derived from mature plants, antioxidants and plant growth regulators (PGR) concentrations. One experiment consisted of 3 medium types including Murashige and Skoog (MS) medium strength, vitamin supplement. Fifteen replicates were used for each treatment. The shoots were then sub-cultured to ten replicate regenerated medium consisting of varying levels and combination of indole-3-acetic acid (IAA) and 10 μM 6-Benzyladenine (BA) supplements. In another experiment consisted of varying levels of auxins with MS medium strength, activated charcoal (AC) and vitamin supplements ten replicates were used for each treatment. Results indicated the positive role of cytokinins types’ 6-Benzyladenine (BA), 2-isopentyladenine (2iP) and Kinetin in inducing callus formation from wounded explants. The highest rate of friable callus formation of wounded explants was observed in media containing vitamin supplementation with BA at 10 μM. Callus formation significantly increased with the addition of vitamins at 10 μM on BA, 2iP and kinetin. With regards to the effects of various media composition and wounding explants on in vitro growth and regeneration of A. subalbum and A. testiculare, significant results were achieved with BA, 2iP and kinetin concentrations on explants discoloration and callus formation. The antioxidant treatment, AC did not reduce explants discoloration, but the induction of the callus was developed furthermore, results showed that IAA with BA concentrations without addition of AC there was significantly difference on both species but A. subalbum dominated with browning intensity (Chapter 3). Only sub-culturing of the explants succeeded in preventing explants discoloration and subsequently increased the number of shoots. The interaction between Indole-3-acetic acid (IAA) concentrations combined with BA resulted in the most effective technique in reducing explants discoloration at the media contact point. This study provides an insight into the contributing factor and methods of overcoming the major problem of phenolic oxidation and promoting the in vitro growth and regeneration of A. subalbum and A. testiculare.

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2018.
Drosanthemum micans and Drosanthemum hallii are endangered succulent shrubs of horticultural and medicinal value. They are restricted to the Succulent Karroo, which is one of the world’s biodiversity hotspots. The species risk extinction from illegal over-harvesting for water-wise gardens, erosion by occasional flush floods from ephemeral rivers, competition from alien invasive species, overgrazing and clearing of land for agriculture and human settlement. Although seeds and cuttings may be used in propagating these species, they often require seasonal collection and planting and cuttings struggle to establish, hence the need for in-vitro propagation as an alternative solution. Thus, the main objective of the study was to develop a method for rapid in-vitro shoot and root multiplication and acclimatization of D. micans and D. hallii. To initiate shoot formation, disinfected leaf and stem nodal explants were cultured in Murashige and Skoog (1962) media supplemented with different rates (0, 10, 20 or 30μM) of 2-isopentyladenine, 6-Benzyladenine and kinetin for D. hallii or 2-isopentyladenine, 6-Benzyladenine and Thiadiazuron for D. micans. Shoots from explants were rooted in varying rates (0, 10, 20 or 30μM) of IAA for root initiation. Three media, which were used in previous studies, were tested for acclimatization of rooted explants in i) vermiculite, ii) sand (50%): vermiculite (50%) or iii) sand (75%): perlite (25%). For quantitative evaluation of plant stress, chlorophyll fluorescence index (Fv/Fm) was measured as a proxy for plant stressf stress. It emerged that stem nodal explants of D. hallii tend to produce multiple shoots whilst leaf explants tended to produce callus when cultured in full-strength Murashige and Skoog (1962). Shoot multiplication was optimal in both D. hallii and D. micans at 10 μM of kinetin. Root formation in both D. hallii and D. micans only occurred when shoots were transferred to a full-strength Murashige and Skoog (1962) media without any phytohormones added. The intensity of tissue browning increased at higher levels of cytokinins, suggesting an interaction of plant growth regulators with exudates from explants. Different acclimatization media tested showed no significant differences in the level of stress (Fv/Fm). It is recommended that Murashige and Skoog (1962) media with10 μM kinetin be used for shoot development and multiplication, followed by transfer of the shoots to fresh full-strength Murashige and Skoog (1962) media without hormones for root development. Acclimatization of the rooted explants was possible in one of the following media: i) vermiculite, ii) sand (50%): vermiculite (50%) or iii) sand (75%): perlite (25%) and in a misted greenhouse (ca. 60% RH), with gradual weekly reductions in humidity by 10% over 2 weeks.

A new, uncomplicated system for the forced ventilation of plants and cultures has been investigated in terms of both its efficiency of ventilation and its effects on the growth and physiology of various plant species, including cauliflower, tobacco, Annona (custard apple) and potato. This new system, which has no moving parts or artificial energy requirement, provides a sustained, pressurised stream of sterile, humidified air (RH = 70-94%) driven by humidity-induced diffusion. This process depends upon the maintenance of a gradient of water vapour across a microporous partition for inducing the diffusion of air into the apparatus. Flows up to 5 cm³ min¯¹ can be produced and the atmosphere in a 60 cm³ culture vessel can be renewed every 12 min Compared to the standard conventional diffusive method of ventilation, e. g. by capping the vessel with a polypropylene disc, this new system has proved to be 18X more efficient in removing accumulated ethylene and in keeping CO₂ and O₂ levels in culture vessels close to atmospheric. This forced ventilation system has also been shown to be very effective in the in vitro cultivation of seedlings or cuttings of cauliflower, tobacco, Annona and potato for improving growth and preventing symptoms of vitrification such as leaf epinasty, reduction of leaf area and production of abnormal stomata. In potato cuttings the induction and production of microtubers have been promoted and the growth of abnormal callus prevented. In Annona cuttings flower bud production, leaf and shoot growth and micropropagation have been promoted and leaf and flower bud abscission have been reduced. In cauliflower, tobacco and Annona the leaf chlorophyll contents, rates of photosynthesis and yields were improved by this forced ventilation. These beneficial effects have been variously attributed to the efficient removal of ethylene, the maintenance of near to atmospheric levels of CO₂ and O₂ by day and night and to the reduction of humidity levels in the vessels to below 100% RH. It is hoped that this new ventilation system, which is comparatively inexpensive and requires very little maintenance might have some useful applications in the field of tissue culture and perhaps particularly in developing countries.

The aim of this research was to examine whether the method of micropropagation and tissue source affects the early growth and development of Paulownia in the first six months following transfer from tissue culture and establishment in soil. This tree species was chosen as it is a fast growing, short-rotation timber tree and able to adapt successfully to new environments. It is easily established in vitro and has been micropropagated using a range of different techniques. Three methods of micropropagation were chosen: callus regeneration, somatic embryogenesis and the third method was inducing root suckers in vitro. The third method was developed during this study and has never been documented in other research. Newly established explants and stabilised explants that had been in culture for over 6 months were used to test the efficacy of these methods. Genotype was also another important aspect to examine, as clones of the same species have shown differing response to being micropropagated. Previous studies have not compared different methods of micropropagation and rarely past the initial stages of laboratory experiments to fully determine the influence they have on the explants development ex vitro. Cultures were sourced from five clones (P1, P2, P3, P4, P5) of mature Paulownia elongata x fortunei stock plants. P1 was first established in vitro and had been micropropagated for five years to induce stabilisation. Newly established explants from clones P1, P2, P3, P4 and P5 had been established in culture for three months before being utilised for micropropagation analysis experiments. Examination of these methods in vitro showed that tissue sources from P1 were the easiest to manipulate and propagate in vitro. Callus regeneration was the most successful in its ability to produce explants and in large quantities. Initial callus experiments showed a significant response in shoot regeneration from stabilised cultures. Subsequent experiments showed a greater response from greenhouse material and newly established cultures, while stabilised cultures failed to produce shoots. Root sucker induction was also successful in stabilised and newly established clones of P1, however, it took a significant amount of time to induce root suckers and the quantity of material produced was limited. Somatic embryogenesis was unsuccessful in regenerating new shoots and the complexity of current methods made it difficult to develop a full protocol in this study. Explants produced from callus regeneration and root sucker induction were transferred to the greenhouse, along with controls from stabilised and newly established cultures. All sources readily produced adventitious roots and there was a 100% survival rate upon transfer to the greenhouse. While initial comparisons showed slight variations in growth factors such as height and floral development, these were not statistically significant. Any slight variation became indistinguishable after two months of growth. Most importantly, after six months, plants from all sources readily produced flowers, indicating that the explants retained the mature phenology of the parent material while being maintained in culture. While callus regeneration and root sucker induction were successful in producing new explants in vitro, these methods had no effect on the overall growth and development under greenhouse conditions. All explants exhibited early flowering, which indicates that they maintained the mature characteristics of the parent material. This is not necessarily an undesirable outcome if the intention is to micropropagate mature tissue while still retaining their mature phenology. Ultimately, the method of micropropagation utilised is determined by what growth characteristic is desired and the purpose for which the plants are being propagated.

The work on Spiraea in vitro shoot cultures was done to determine the feasibility of using light quality to modify endogenous phytohormone balances to decrease apical dominance. Such an effect would enable a reduction in the high levels of exogenous cytokinin benzyladenine (BA) applied in culture and thus reduce potential side-effects. The Spiraea in vitro light quality response was characterized by examining the effects of different light wavelengths on growth. A mixture of red/FR induced rates of shoot proliferation with 0.25 mg/1 BA that were as high as rates obtained under white light with 0.5 mg/1 BA. Shoot quality, as determined by the proportion of shoots 1 cm or longer (useful shoots), was highest under red/FR light. The lowest shoot proliferation rate was observed under blue light. When light wavelengths intermediate between blue and red light (green, yellow, and orange) were applied to explants only minor growth modifications occurred. Green light did not inhibit shoot initiation but inhibited shoot elongation at the 0.5 mg/1 BA level. The efficacy of the light source-filter combinations in the first experiment was studied in two further experiments. With the three light sources (tungsten filament, fluorescent, and metal halide) together with a blue filter, results supported the putative blue light inhibitory effect suggested in the first light quality experiment. Under the red filter, the tungsten filament source induced the highest shoot number means at both BA levels used (0.25 and 0.5 mg/1). Two factors may have contributed to the red/FR effect observed in the first experiment; the time under an incubation light regime before transfer to the treatment regime, and the photon fluence rate of each regime. In the subsequent study to examine these factors, shoot initiation was optimized at the lower BA levels of 0.25 and 0.4 mg/1 when cultures under low fluence red/FR were transferred after four weeks to white light of a higher fluence for one more week. Glyphosate, a known promoter of IAA oxidation, was used to investigate the presumed effect of lowered IAA-cytokinin interactions. Two types of responses to glyphosate occurred, each one dependent on the glyphosate concentration. At the lower glyphosate level (0.087 mg/1), cultures under both light regimes with 0.25 mg/1 of BA, showed a strong inhibition of shoot initiation. This inhibitory effect was overcome in cultures with 0.5 mg/1 of BA and an overall stimulatory response occurred as shoot initiation rates were as much as four-fold higher than in the previous experiments. For both BA levels, changes in shoot number were greater under white light than under red/FR. At the higher glyphosate level (0.2 67 mg/1), the shoot initiation rates were greater than glyphosate-free controls for both BA levels under white light although under red/FR the rates were virtually unchanged from controls. The glyphosate effect investigated for Spiraea cultures appears to be influenced by the levels of the cytokinin BA resulting in pleiotropic effects which depend on the specific concentrations of each component.
Land and Food Systems, Faculty of
Graduate

A growth chamber experiment was carried out for ten weeks to reduce post-rooting dormancy in juneberry micropropagation. An RCBD with a split plot arrangement and three replicates were used. Plantlets subjected to 750 mg/L GA, 100 mg/L BA, and 250 mg/L GA + 100 mg/L BA recorded the greatest leaf number. Pre-rooted ?Thiessen? plantlets recorded the greatest biomass (fresh and dry weight) and root volume. In a second study, a cultivar evaluation was conducted in Absaraka, ND, where ten juneberry cultivars and a native biotype planted were evaluated for plant and fruit characteristics. An RCBD with four replicates was used. The high yielding cultivars for total yield were ?Thiessen?, ?Martin?, ?Parkhill?, ?Pembina?, ?Regent? and Native. ?Thiessen?, ?Martin?, and ?Parkhill? maintained a significant higher marketable yield. ?Thiessen?, ?Regent?, ?Martin?, ?Parkhill? and ?Northline? had the largest fruits, while ?Thiessen? and ?Martin? fruit had the greatest mass.

Thesis (Ph. D.)--West Virginia University, 2010.
Title from document title page. Document formatted into pages; contains ix, 160 p. : ill. (some col.), col. maps. Includes abstract. Includes bibliographical references.

Tissue-cultured shoots and plantlets usually have leaves with non-functional, open stomata and little epicuticular and cuticular wax, resulting in excess evapotranspiration after transplantation. Various strategies were evaluated to decrease ex vitro acclimatization difficulties for 'Silvan' blackberry, including transplanting unrooted shoots, increasing the medium agar concentration from 6 to 9 or 12 g/l and diluting the basal medium. Increased medium agar concentrations and medium dilution did not improve survival or growth. Stomatal function resumed sooner in new leaves of plantlets than shoots. High relative humidity ($>$95%) and low light intensity (90 $ mu$mol s$ sp{-1}$ m$ sp{-2}$) negatively affected stomatal closure both on acclimatizing transplants and greenhouse-grown plants. Guard cells developed on leaves in vitro were physiologically active but had apparent anatomical abnormalities that inhibited closure. A rapid clearing and staining method was developed for examination of foliar morphology using intact in vitro blackberry (Rubus sp. 'Silvan') and strawberry (Fragaria x ananassa Duch. 'Totem') plantlets and sections of greenhouse-grown 'Silvan' and 'Totem' leaves. This method involved three steps: (1) removing the chlorophyll by autoclaving in 80% ethanol; (2) dissolution of the protoplasm using 5% NaOH at 80$ sp circ$C; (3) post-alkali treatment with 75% bleach (4.5% NaClO) at room temperature for tissue-cultured plantlets and at 55$ sp circ$C for greenhouse-grown leaves. Aqueous safranin (10 mg/l) was used for staining.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Biotechnology is an important tool that is used to isolate and characterise genes. It is also used to produce clones that are genetically and phenotypically similar. Many Arabidopsis thaliana transcription factors have been isolated and characterised, but many have yet to be fully described. MYB proteins are members of a super-family of multifunctional transcription factors that can also interact with other transcription factors in the control of pathways. To date, more than 126 AtMYBs have been identified, but most have not been fully characterised, particularly in terms of the molecular role(s) they play in plants. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 and MYB32 have been reported to be negative regulators of general phenylpropanoid metabolism. It has been reported that the five transcription factors mentioned above are likely to negatively regulate flavonoid biosynthesis, even though they may have different target genes. Studies on AtMYB13, AtMYB14 and AtMYB15 reported that they are likely regulators of general phenylpropanoid metabolism. The mentioned roles of the eight AtMYB transcription factors means that they can be manipulated in order to see what effect they have on primary and secondary metabolites in plants. The transcription factors ligated into the pUBI510-GRFCA vector were then used to transform sugarcane callus (Chapter 3). Sugarcane produces sucrose which makes up 70% of the sugar produced in the world, making sugarcane a commercially important and profitable plant. The sugarcane callus was transformed via particle bombardment. The transcription factors AtMYB3, AtMYB6, AtMYB7, AtMYB13 and AtMYB32 were successfully incorporated into the genomic DNA of the sugarcane callus. The data obtained for callus over-expressing AtMYB3, AtMYB13 and AtMYB32 on solid media and the callus in liquid media were contradictory (i.e callus on solid media producing more sucrose than the wildtype whereas the same transgenic line will poduce less sucrose that the wildtype in liquid media or vice versa). However, AtMYB13 transgenic lines produced more sucrose than the wildtype. Transgenic lines of AtMYB7 all produced less sucrose as compared to the wildtype both on solid and in liquid media. The transcription factors which resulted in increased production of starch when over-expressed were AtMYB7 and AtMYB13. The data obtained for AtMYB6 transgenic lines was highly inconsistent in lines grown on same media and across the two media. The effects of these transcription factors in the overall metabolism of the sugarcane callus, either on MSC3 solid or liquid media, could not be fully determined from the GC-MS analysis as there was no consistent phenotypic effect between different transgenic lines for any of the MYB transcription factors used. In Chapter 4, a micropropagation strategy was developed and phytochemicals and their biological activities were determined for the medicinal plant Salvia repens. Salvia plants have been found to be medicinally important due to the secondary metabolites, particularly the essential oils that they produce. The plant extracts have been found to have many biological activities such as antibacterial, anti-inflammatory, antioxidant and anticancer activities. Salvia repens was successfully germinated in vitro,with 60% germination being achieved in MS media containing 1x10-5 times diluted smoke water following scarification for 12 min in 75% (v/v) H2SO4. Success rates of 100% were achieved in the hardening off process when the seedlings were moved into the greenhouse. Germination of S. repens ex vitro was 100% in an autoclaved soil mixture of 1:1 (v/v) sand and vermiculite. Importantly the medicinal value of S. repens produced in vitro or ex vitro was not lost as the GC-MS metabolite analysis showed that the plants produced the chemicals that are medicinally important. Metabolite extracts of S. repens were for the first time reported to be active against fungi with MIC values lower than 1 mg/ml over 4-5 d period against four Fusarium spp. tested. Lastly (Chapter 5), transcription factors AtMYB6 and AtMYB13 were used to trasnform Salvia stenophylla via Agrobacterium-mediated transformation, in order to determine whether the over-expression of these transcription factors could up-regulate the production of medicinally and commercially important secondary metabolites in S. stenophylla. Whilst both A. tumefaciens and A. rhizogenes strains were utilised for the transformation procedure, transformation was only achieved using A. rhizogenes and no transformants could be generated from the A. tumefaciens-treated material. Transgenic hairy roots did not produce any of the medicinally important metabolites. The GC-MS analysis of the transgenic root material identified mainly sugars and other primary metabolites.
AFRIKAANSE OPSOMMING: Biotegnologie is 'n belangrike instrument wat gebruik kan word om gene te isoleer en te karakteriseer. Dit word ook gebruik om klone wat geneties en fenotipies identies is te produseer. Baie Arabidopsis thaliana transkripsiefaktore is al geïsoleer en gekarakteriseer, maar baie moet nog volledig beskryf word. MYB proteïene is lede van 'n super-familie van multifunksionele transkripsiefaktore wat ook interaksie het met ander transkripsiefaktore tydens die beheer van metaboliese weë. Tot op hede is meer as 126 AtMYBs geïdentifiseer, maar die meeste is nie volledig gekarakteriseer nie, veral nie ten opsigtigte van die molekulêre rol(le) wat hulle in plante speel nie. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 en MYB32 is gevind om negatiewe reguleerders van algemene fenielpropanoied-metabolisme te wees. Daar is ook berig dat dié vyf transkripsiefaktore moontlik flavenoied-biosintese negatief kan reguleer, selfs al kan hulle verskillende teikengene hê. Studies op AtMYB13, AtMYB14 en AtMYB15 het berig dat hulle waarskynlik reguleerders van algemene fenielpropanoied-metabolisme is. Die genoemde rolle van die agt AtMYB transkripsiefaktore beteken dat hulle gemanipuleer kan word om te bepaal watter effek hulle op primêre en sekondêre metaboliete in plante het. Die transkripsiefaktore, wat in die pUBI510-GRFCA vektor geligeer was, is toe gebruik om suikerriet-kallus te transformeer (Hoofstuk 3). Suikerriet vervaardig sukrose wat tot 70% van die suiker wat in die wêreld geproduseer word opmaak. Dít maak suikerriet 'n kommersieel belangrike en winsgewende plant. Die suikerriet-kallus is getransformeer deur middel van partikel-bombardering. Die transkripsiefaktore AtMYB3, AtMYB6, AtMYB7, AtMYB13 en AtMYB32 was suksesvol in die DNA van die suikerriet-kallus opgeneem. Data wat verkry was vir kallus wat AtMYB3, AtMYB13 en AtMYB32 ooruitgedruk het op soliede media en kallus in vloeibare medium was teenstrydig (m.a.w. kallus op soilede media wat meer sukrose as die wildetipe op soliede media geproduseer het, terwyl dieselfde transgeniese lyn minder sukrose as die wildetipe geproduseer het in vloeibare medium, en anders om). Nietemin, het AtMYB13 transgeniese lyne meer sukrose geproduseer as die wildetipe. Transgeniese lyne van AtMYB7 het almal minder sukrose geproduseer as die wildetipem op beide soliede en vloiebare media. Die transkriopsiefaktore wat gelei het tot 'n styging in stysel produksie wanneer hulle ooruitgedruk was was AtMYB7 en AtMYB13. Data wat verkry is van die AtMYB6 transgeniese lyne was hoogs veranderlik in lyne wat op dieselfde medium gegroie was en oor die twee media. Die effek van hierdie transkripsiefaktore op die algehele metabolisme van die suikerriet-kallus, hetsy op MSC3 soliede of vloeibaremedia, kon egter nie van die GC-MS analise ten volle bepaal word aangesien daar geen konsekwente fenotipiese effek tussen die verskillende transgeniese lyne vir enige van die gebruikte MYB transkripsiefaktore was nie. In Hoofstuk 4 was ‘n mikropropagerings strategie ontwikkel. Fitochemikalieë en hul biologiese aktiwiteite was ook bepaal vir die medisinale plant Salvia repens. Salvia plante is gevind om medisinaal belangrik te wees as gevolg van die sekondêre metaboliete, veral die essensiële olies, wat hulle produseer. Dit is ook bevind dat die plant-ekstrakte baie biologiese aktiwiteite soos anti-bakteriese, anti-inflammatoriese, anti-oksidant en anti-kanker aktiwiteite het. Salvia repens is suksesvol ontkiem in vitro, met 60% ontkieming wat bereik is in MS media met 1x10-5 maal verdunde rook-water na insnyding vir 12 min in 75% (v/v) H2SO4. Suksessyfers van 100% was behaal in die afhardingsproses wanneer die saailinge na die glashuis verskuif was. Ontkieming van S. repens ex vitro was 100% in 'n geoutoklaveerde grondmengsel van 1:1 (v/v) sand en vermikuliet. Gewigtig het die medisinale waarde van S. repens wat in vitro of ex vitro geproduseer was nie verlore gegaan nie. Die GC-MS data metaboliete analise het aangetoon dat die plante die medisinaal belangrike chemikalieë geproduseer het. Metaboliet-ekstrakte van S. repens was vir die eerste keer na berig aktief teen swamme, met MIK waardes laer as 1mg/ml oor ‘n tydperk van 4-5 d, teen vier Fusarium spp wat getoets was. Laastens (Hoofstuk 5), transkripsiefaktore AtMYB6 en AtMYB13 was gebruik om Salvia stenophylla te transformeer deur Agrobacterium-bemiddelde transformasie, om sodoende te bepaal of die ooruitdrukking van hierdie transkripsiefaktore die produksie van medisinale en kommersieël-belangrike sekondêre metaboliete in S. stenophylla kan verhoog. Alhoewel beide A. tumefaciens en A. rhizogenes stamme gebruik was vir die transformasie proses, kon transformasie slegs deur die gebruik van A. rhizogenes bereik word. Geen transformante kon gegenereer word vanuit die A. tumefaciens behandelde materiaal nie. Transgeniese harigewortels het geen van die medisinaal belangrike metaboliete vervaardig nie. Die GC-MS analise van die transgeniese wortel materiaal het hoofsaaklik suikers en ander primêre metaboliete geïdentifiseer.

The responses of the tepary bean (Phaseolus acutifolius) to in vitro tissue culture systems were documented. Tests were conducted to identify the optimal auxin and cytokinin combinations required for optimal callus growth. Regeneration experiments were conducted to: (1) determine the effect of explant source and age on regeneration, (2) effect of callus age on regeneration, (3) the cultivation status of the explant source, and (4) the effects of nutritional additives on somatic embryogenesis. The callus was easily induced and maintained in all hormonal medias except those containing IAA and 6BA. The results for regeneration were most promising from cultures derived from immature cotyledon tissue. Ammonium Chloride, glutamine and Absisic acid appeared to have little affect on embryogenesis, however the addition of kinetin enabled the embryos to develop to the torpedo stage. Callus age and cultivative status of explant source had no effects on plantlet regeneration.

A mist reactor was used to study plant growth and development under various environmental conditions towards the production of healthy plantlets ready for soil transplant in one step from inoculation. In addition, a 3D type of cultivation via surface attachment of explants to vertically hanging strips inside the mist reactor was also investigated to maximize productivity with minimal footprint. Using carrot as the model species, pre-embryogenic cell suspensions were successfully spray-inoculated onto hanging poly-L-lysine (PLL)-coated nylon mesh to which they then attached and remained for several weeks while they developed into rooted plantlets. To study single step micropropagation from shoot explants to fully acclimatized plantlets, Artemisia annua was used as the model species. Nodal cuttings of A. annua were inoculated onto PLL-coated mesh strips by briefly immersing the strips in the suspension of nodal cuttings. Investigation of medium, phytohormones, CO2, ventilation level and humidity ensued resulting in selection of a preferred final process that reduced physiological aberrations like hyperhydricity and was time efficient. The nodal cuttings that attached to the strips were first misted with half strength shooting medium for 7 days to develop new shoots. Then the new shoots were misted with the rooting medium supplemented with NAA for 12 days to develop roots. Rooted plantlets were acclimatized in the same rooting medium for 9 days to acquire fully functional stomata prior to planting into soil. Taken together this study suggested that fully developed plantlets ready for planting into soil could be obtained in a single step in a bioreactor from embryogenic cells or from nodal explants.

The development of a successful protocol for micropropagating seagrass provides a valuable tool for seagrass-restoration programs and a facility to study their biology (especially their physiology). This work reports on some of the culture requirements of some seagrasses that are commonly found in Western Australia: Posidonia coriacea, P. sinuosa, P. australis and Halophila ovalis. The protocol developed for H. ovalis allows very rapid multiplication and sustainable growth of cultures while the protocol developed for Posidonia requires further development. The culture of Posidonia cariacea proved to be problematic however experimental media that provided insights into its culture conditions. The carbohydrate source was the most important medium component as it affected the development of roots and leaves. The presence of sucrose in the culture media enhanced leaf growth (especially glucose) but decreased the proportion of white roots. More fresh weight, roots, leaves and the proportion of white roots were observed in Posidonia when they were grown in glucose-based media than in mannitol-based media. When mannitol was present in the media, the proportion of white roots was high, which could be attributed to its osmotic effects. Similar responses to sucrose, glucose and mannitol were also observed for P. australis and P. sinuosa. Halophila ovalis was able to grow rapidly on most experimental media. Growth was enhanced by the presence of sucrose in the media and was essential for rapid and sustained growth. Other media components altered the growth of this species, in particular levels of nitrogen (most importantly NH4) influenced root growth and morphology. When H. ovalis is grown in media in moderate or high levels of NH4, root length was significantly reduced and root hair was limited. When NH4 was omitted from the medium, roots were significantly longer and root hairs were prolific. Posidonia coriacea and Halophila ovalis have different growth strategies under natural conditions. H. ovalis is an early succession species that grows rapidly and responds to increased nutrients. P. coriacea is slower growing, colonises later and is Jess responsive to environmental changes than H. ovalis. While the growth responses observed for P. coriacea were significant (in some cases), the differences between means were considerably smaller when compared with H. ovalis. This may be due to the different growth strategies of these species or a lack of fundamental requirement in the conditions under which P. coriacea was grown. Much of what is reported in this thesis for Posidonia will need repeating if the reasons for these differences are identified in the future. In summary, in this thesis I have demonstrated that in vitro propagation of these seagrass species is possible, It is necessary for species-specific protocols to be developed which take into consideration the growth strategies employed by each species. This is particularly significant as many researchers attempt to draw comparisons between species and protocols. The protocols developed in this research increase the knowledge of the biology of these seagrasses and can be incorporated into transplantation protocols in the future.

Doutoramento em Biologia
Slash Pine (Pinus elliottii var. elliottii) and the hybrid (Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis) have a great economic value due to their high growth ratio and resin production. Therefore, it is important to achieve a strategy to propagate this species and the hybrid more rapidly maintaining their characteristics. This project aims to preserve/enlarge the Pinus germplasm collection, provided by the company KLÓN, Innovative Technologies from Cloning, by micropropagation and cryopreservation techniques and analyze the putative changes (genetic stability) in micropropagated plants by flow cytometry, to study plant survival rates, growth and photosynthetic performance after acclimatization. In Chapter I, general aspects of the species and hybrid under study are presented, as well as a brief description of the breeding programs in Pinus spp. and the contribution that innovative techniques of in vitro propagation can give, leading to decades of anticipation on the breeding programs results. It was also described some major aspects to the different micropropagation and cryopreservation techniques, always presenting a review of current knowledge on the use of these techniques in the genus Pinus. Finally, the research objectives of this thesis are presented. Chapter II is dedicated to the application of a micropropagation protocol by proliferation of axillary shoots in the specie Pinus elliottii var. elliottii. This Chapter was divided in two sections. In section II.1 a protocol was optimized for large-scale P. elliottii micropropagation, which describes all the steps from disinfection and seed germination to the production of seedlings in vitro, which were used as explants for shoot induction. Various conditions for induction, shoot elongation and rooting were tested, and a protocol enabling the production of micropropagated plantlets 20 to 22 weeks after germination in vitro has been established. In section II.2 was performed the genetic and physiological characterization of P.elliottii plants micropropagated by the methodology developed in the previous section, in comparison with seedlings. The physiological performance of the plants was evaluated by determination of various parameters associated with photosynthesis and carbon metabolism, such as: chlorophyll a fluorescence; relative water content; gas exchange; pigment and carbohydrate contents. In turn, the genetic characterization was performed by analysis by flow cytometry of putative alterations in DNA content, ploidy level and in cell cycle dynamics. The results indicate that the developed micropropagation protocol for P. elliottii did not induce significative changes, both at physiological and genetic level, in the plants. Chapter III focuses on the optimization of a somatic embryogenesis process for the hybrid Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis, from the initiation to the plants regeneration produced from somatic embryos. For initiation of embryogenic cultures of this hybrid, immature megagametophytes obtained from five open-pollinated plus trees were used as explants. To optimize the process, the effect of genotype in both the initiation and maturation, as the influence of different formulations of basal media and growth regulators in the various stages of the process were evaluated. Throughout the process was assessed the genetic stability of embryogenic masses with different time in culture, and at the end of the produced emblings, in comparison with the mother-trees needles. This protocol allows the production of emblings from somatic embryos not having been detected variability in the DNA content and ploidy level. Chapter IV is dedicated to the preservation of germplasm bank produced for the hybrid under study. Cryopreservation of embryogenic masses is beneficial not only for the preservation of germplasm during the breeding programs development, as well as to avoid the loss of the potential of the embryogenic masses. For the optimization of an embryogenic masses cryopreservation protocol using the slow freezing method, different variations were tested in pretreatments and in the duration of slow freezing step. Pretreatments to which the embryogenic tissue was subjected, did not compromise the maturation capacity of cryopreserved masses. On the contrary, cryopreservation had in some genotypes a beneficial effect. The optimized protocol allowed the regeneration of plants from cryopreserved masses and the process did not induce major genetic changes (embryogenic masses cryo and non cryopreserved were analyzed by flow cytometric). Finally, Chapter V presents the final conclusions of this PhD thesis, gathering the results of this thesis on the propagation and preservation methodologies for the species and hybrid in study and discussion this contribution to the state of art in this field. Future challenges for further research in these areas are presented in this Chapter.
Pinus elliottii var. elliottii e o híbrido, Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis, têm um grande valor económico, devido à sua elevada taxa de crescimento e produção de resina. Surge assim a necessidade de desenvolver estratégias de propagação mais eficientes desta espécie e do híbrido, mantendo as suas características. Este estudo tem como objetivos preservar / aumentar o banco de germoplasma de Pinus, fornecido pela empresa KLÓN, Innovative Technologies from Cloning, utilizando técnicas de micropropagação e de criopreservação, analisar possíveis alterações na estabilidade genética das plantas micropropagadas através de citometria de fluxo, estudar a taxa de sobrevivência de plantas, crescimento e desempenho fotossintético após aclimatização. No Capítulo I foram abordados aspetos gerais da espécie e híbrido em estudo, assim como uma breve descrição dos programas de melhoramento em Pinus spp. e da contribuição que técnicas inovadoras de propagação in vitro podem dar levando a décadas de antecipação dos resultados dos respectivos programas de melhoramento. Foram também descritos alguns aspetos mais importantes relativos às diferentes técnicas de micropropagação e criopreservação existentes, apresentando sempre uma revisão do conhecimento atual sobre o uso destas técnicas no género Pinus. Para finalizar este capítulo, os objetivos de investigação desta Tese são apresentados. O Capítulo II é dedicado à aplicação de uma metodologia de micropropagação (por proliferação de rebentos axilares) da espécie Pinus elliottii var. elliottii. Este capítulo foi dividido em duas secções. Na secção II.1 foi otimizado um protocolo para micropropagação em larga escala de P. elliottii desde a desinfecção e germinação de sementes para a produção de plântulas in vitro. Estas por sua vez são utilizadas como explantes para a indução de rebentos. Foram testadas várias condições para a indução, alongamento e enraizamento de rebentos, tendo sido estabelecido um protocolo que permite a produção de plântulas micropropagadas 20 a 22 semanas após germinação in vitro. Na secção II.2 foi realizada a caracterização genética e fisiológica de plantas de P. elliottii micropropagadas pela metodologia desenvolvida na secção anterior, em comparação com plantas provenientes de sementeira. O desempenho fisiológico das plantas foi avaliado pela determinação de diversos parâmetros relacionados com a fotossíntese e o metabolismo de carbono, tais como a fluorescência de clorofila a, o teor relativo em água, as trocas gasosas, o teor de pigmentos e de carbohidratos. Por sua vez a caracterização genética foi realizada pela análise do conteúdo em DNA e nível de ploidia, e ainda as dinâmicas do ciclo célular, com recurso à citometria de fluxo. Os resultados obtidos indicam que o protocolo de micropropagação desenvolvido para P. elliottii não provoca alterações significativas tanto a nível fisiológico como genético nas plantas. O Capítulo III centra-se na otimização de um processo de embriogénese somática para o híbrido Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis, desde a iniciação até à regeneração de plantas produzidas a partir de embriões somáticos. Para a iniciação de culturas embriogénicas deste híbrido foram utilizados como explantes megagametófitos imaturos obtidos a partir de polinização aberta de cinco árvores plus. Para a otimização do processo foi avaliado o efeito do genótipo tanto na iniciação como na maturação, tal como o influência de diferentes formulações de meios basais e reguladores de crescimento nas diversas fases do processo. Ao longo do processo foi avaliada a estabilidade genética das massas embriogénicas com diferentes tempos de cultura, e no final das plântulas produzidas, em comparação com as agulhas das árvores-mãe, concluindo-se que foi desenvolvido um protocolo que permite a produção de plantas provenientes de embriões somáticos não tendo sido detectada variabilidade ao nível de conteúdo em DNA e nível de ploidia. O Capítulo IV é dedicado à preservação do banco de germoplasma produzido para o híbrido em estudo. A criopreservação de massas embriogénicas é benéfica não só para a preservação de germoplasma durante o desenvolvimento de programas de melhoramento, como para evitar a perda do potencial embriogénico das massas. Para a otimização de um protocolo de criopreservação de massas embriogénicas pelo método de congelamento lento, foram testadas diferentes variações nos pré-tratamentos e na duração do passo de congelação lenta. Os pré-tratamentos a que o tecido embriogénico foi submetido não influenciaram negativamente a capacidade de maturação das massas criopreservadas, apresentando-se a criopreservação até em alguns genótipos com um efeito benéfico. O protocolo otimizado permitiu a regeneração de plantas a partir de massas criopreservadas, para as quais se comprovou que o processo não provocou alterações genéticas, através da análise por citometria de fluxo de massas embriogénicas crio e não criopreservadas. Finalmente, no Capítulo V são apresentadas as conclusões finais da Tese de Doutoramento, onde são realçados os avanços realizados como resultado desta tese nas metodologias de propagação e preservação para a espécie e híbrido em estudo. Neste capítulo são também apresentados os desafios futuros para a continuação da investigação nas áreas de propagação e preservação de Pinus.

Chrysanthemum (Dendranthema grandiflora) is one of the most important plants grown for flower production. There is a need to develop an in vitro system for more efficient mass production of chrysanthemum. Previous reports on chrysanthemum production have revealed that most of the in vitro systems attempted for commercial propagation are uneconomical and labour intensive and hence cannot meet the world market demand for chrysanthemum production. Automation would be advantageous in scaling up production and lowering the cost. This could be effectively achieved through the use of somatic embryogenesis in liquid systems. There have been no reports of such a system in chrysanthemum. The first step towards the development of a suitable system is the generation of a protocol for the induction and multiplication of somatic embryos. Previous repo1is on induction of somatic embryogenesis in chrysanthemum failed to produce a significant number of propagules and the procedure was not repeatable. In this study five factors were systematically investigated for their effect on the induction and multiplication of somatic embryos of D. grandiflora with the aim of establishing a protocol for mass propagation which could be used with liquid systems. Through optimisation of each of the factors ( explant, plant growth regulators, cultivar, the sugar type and inorganic macronutrients concentration) the best protocol was determined. Microscopy techniques were used to confirm the origin of somatic embryos from the surface tissue of the leaf and to assess the cytoplasmic changes in embryogenic cells. Further manipulation of some of the factors of the selected induction protocol was carried out to establish a system for secondary embryogenesis, maturation and plantlet conversion. Induction of somatic embryos was significantly higher with leaf explants of D. grandiflora than with internodes or floral stalk explants. The growth regulators used were 0.5 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg L-1 kinetin in Murashige and Skoog (MS) medium supplemented with 500 mg L-1 casein hydrolysate and 150 mg L-1 L-proline. This combination proved the best of the eight growth regulator combinations and concentrations assessed in this study. Experiments comparing different inorganic macronutrient concentrations revealed no significant difference among the four levels tested (¼, ½, ¾ and full strength) in terms of the number of somatic embryos produced. Somatic embryo production was significantly different between the different chrysanthemum cultivars and differences in the rate of embryo development were also noted. Similarly, sugar type had a significant effect on the induction process and of the six types tested, only two responded positively (sucrose and glucose) and with varying numbers of somatic embryos per explant. Light microscopy revealed that the somatic embryos originated from the hypodermis layer of the leaf, and the globular-stage of the somatic embryos had a well differentiated protoderm. Most of the embryos were loosely attached directly to the leaf explant. When transmission electron microscopy was used on the same culture of somatic embryos, cells with dense cytoplasm could clearly be seen and scanning electron microscopy revealed pro-embryos arising directly from chrysanthemum leaf explant. Taken together, these data show that the type of explant, growth regulators, cultivar, and sugar have a strong influence on the induction and multiplication of somatic embryos of chrysanthemum. Secondary embryos can be produced efficiently in large numbers from primary embryos and the technique has the potential for mass production of chrysanthemum plantlets at reduced labour cost.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Salvia africana lutea is one of 26 Sage species indigenous to Southern Africa from a total of 900 worldwide. The genus Salvia belongs to the Lamiaceae family. Labeled a ‘broad spectrum remedy’ S. africana lutea amongst other sage species is medicinally important. Reports are many highlighting its benefits, which include from alleviating coughs and colds to gynaecological complaints. Studies have revealed in vitro antimicrobial, anti-cancer and antioxidant activity. Plant secondary metabolites fundamentally have a strong bearing on the phytochemical activities a plant may possess. Consequently the environment indirectly affects the phytochemical properties as it influences the variation in the plant metabolome via plant-environment interactions. Five S. africana lutea plant populations, within the Western Cape province of South Africa were sampled and chemotypes and bioactivity tested. Four populations were wild growing in protected areas namely; Brackenfell, Koeberg, Silwerstroomstrand and Yzerfontein, while the fifth was a garden growing population from Stellenbosch. Using gas chromatography hyphenated with mass spectrometry (GC-MS), compounds such as monosaccharides, carboxylic acids and fatty acids were detected. Variation of compounds identified with 80% certainty was compared across all populations. Stellenbosch population showed some compounds that were not present in the other four sites. These compounds were namely; propanoic acid, rythronic acid, 2-keto-1-gluconic acid and 1,3-dibromobicyclon, while this population also did not have xylitol that was detected in all the other four populations. To consolidate the GC-MS findings, analysis on the metabolite profiles (utilizing liquid chromatography linked with mass spectrometry (LC-MS) and nuclear magnetic resonance (1H NMR)) was done. Principal component analysis (PCA) was applied to the NMR data. The partial least squaresdiscriminant analysis (PLS-DA) was used to integrate LC-MS and NMR data sets. All statistics were performed with the SIMCA-P+ 12.0 software. By integrating LC-MS and 1H NMR analyses, large chemotype differences leading to samples grouping by site, suggested strong plant-environment interactions as factors influencing metabolite composition. Signals distinguishing the Stellenbosch profile were in the aromatic part of the 1H NMR spectra. Antimicrobial activity was tested against two Fusarium species. Fusarium is a plant pathogenic species that causes large agricultural losses particularly in the maize crop, one of the staple foods in the African continent. Some species also produce mycotoxins in infected crop and lead to a significant increase in the risk factor of cancers when contaminated foods are consumed. Four high-mycotoxin producing strains from two species F. verticillioides (MRC 826 and MRC 8267); F. proliferatum (MRC 7140 and MRC 6809) were utilized in all in vitro antifungal assays in this study. A preliminary assay using dichloromethane: methanol (1:1 v/v) crude plant extracts of the five populations; Stellenbosch, Brackenfell, Koeberg, Silwerstoomstrand and Yzerfontein, from 2009 and 2011. All test samples exhibited good activity as the minimum inhibitory concentrations (MIC) values ranged from 0.031 mg ml-1 to 0.5 mg ml-1, values below the latter are regarded as strong inhibitors. The Stellenbosch extracts were the most active for both 2009 and 2011 collections, with the best activity against F. verticillioides MRC 8267 and MRC 826 at 0.031 mg ml-1. While the least activity, albeit still a strong inhibitor, was observed from the Yzerfontein extracts with an MIC value of 0.5 mg ml-1. Generally comparison between the two years revealed that samples collected in 2011 were more potent than those in 2009, possibly due to prolonged storage that may have resulted in chemical decomposition. As the Stellenbosch population had shown the best activity as well as a relatively different chemical profile, leaves from these plants were then introduced into tissue culture conditions. Leaf explants were placed on solid plant growth regulator (PGR)-free Murashige and Skoog media and that supplemented with hormones in various combinations. (two concentrations of benzyl adenine (BA) utilized individually and in combination with naphthalene acetic acid (NAA) 4.4 and 8.8, while for NAA 0.27, 2.7 and 5.4.) Namely: 4.4 BA, 8.8 BA, 0.27 NAA: 4.4 BA, 2.7 NAA: 4.4 BA, 5.4 NAA: 4.4 BA, 2.7 NAA: 8.8 BA and 5.4 NAA: 8.8 BA. The PGR combinations did not induce shooting nor rooting, only callus on PGR-free MS media. Antifungal activity of the callus extract was in the same range as the whole plant extracts from which the leaf explants were harvested from, showing no ‘loss’ of activity after introduction to tissue culture conditions. Metabolite profiles using LC-MS, however, did reveal qualitative and quantitative differences, though they appear to not have any bearing on the activity. A bioassay-guided fractionation was then conducted on samples collected from Stellenbosch. This led to the identification of carnosol and carnosic acid being involved in the anti-Fusarium activity of S africana-lutea. A combinational study revealed no synergistic activity of the two compounds against four Fusarium test strains, with fractional inhibitory concentration (FIC) values of 1.5 and 3.0. Antifungal activity of carnosol and carnosic acid was observed to be in the same range (strong inhibitor) as was the callus and whole plant extracts. The study showed variation in population chemotypes and identified two compounds that are involved in S. africana-lutea activity against Fusarium species. It also provided a tissue culture system onto which mass production of the two bioactives may be achieved from, in the development of new fungicides.
AFRIKAANSE OPSOMMING: Salvia Africana lutea is een van die 26 Salie spesies wat inheems aan Suid-Afrika is uit ‘n totaal van 900 spesies wêreldwyd. Die genus Salvia hoort tot die Lamiaceae familie. S. africana lutea word geklassifiseer as ‘n “breë spektrum geneesmiddel”, en medisinaal as belangrik geag tussen die ander salie spesies, want volgens verslag word dit gebruik vir die verligting van hoes en verkoues tot selfs ginekologiese ongesteldhede. Definitiewe biologiese aktiwiteit eksperimente het anti-mikrobiese, antikanker en anti-oksidant aktiwiteite aan die lig gebring. Plant sekondêre metaboliete het fundamenteel ‘n baie sterk verband met die fitochemiese aktiwiteite van ‘n plant. Gevolglik affekteer die omgewing indirek die fitochemiese eienskappe, want dit beïnvloed die variasie in die plant metaboloom deur die interaksies van die plant met die omgewing. In vyf streke binne die Wes-Kaap van Suid-Afrika waar S. africana lutea bevolkings voorkom, is steekproewe gedoen en chemotipes en bioaktiwiteit getoets. Vier bevolkings was wild-groeiende bevolkings in beskermde areas, naamlik; Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein, terwyl die vyfde uit ‘n tuingroeiende bevolking in Stellenbosch geneem is. Deur gas chromatografie gekoppel met massa spektrometrie te gebruik, is primêre samestellings soos monosakkariede, karboksielsure en vetsure gevind. Variasies van samestellings wat met 80% sekerheid geïdentifiseer is, is oorkruis met al die bevolkings vergelyk. Die Stellenbosch bevolking het ‘n paar samestellings geopenbaar wat nie aanwesig was in die ander vier terreine nie. Hierdie samestellings was: propanoësuur, erythroniese suur, 2-keto-1-glukoniese suur en 1,3-dibromobicyclon. Verder het hierdie bevolking geen xylitol gehad nie en dit is in al vier die ander bevolkings gevind. Verdere studies was gedoen met die gebruik van vloeibare chromatografie gekoppel met massa spektrometrie (LC-MS) sowel as kern magnetiese resonansie (1H NMR). Chemiese profiele het hoë variasies getoon, en dus deur te fokus op die aromatiese samestelling streke, het die Stellenbosch terrein duidelik merkbare verskille en punte op die PLS-DA aangetoon. Met die koppeling van NMR data met LC-MS data, is daar gevind dat onderskeidende punte van die NMR PLS-DA wat gegroepeer is met retensie tye die skeiding van die Stellenbosch terrein van ander terreine gedryf het. Dit het onweerlegbaar bewys dat daar variasie binne die vyf bevolkings voorkom en dat Stellenbosch die mees noemenswaardige chemotipe variasie het. Dit blyk uit die anti-mikrobiese eksperimente dat aktiwiteit teen Fusarium heel nuwe belangstelling wek. Fusarium is ‘n plant-patogeniese spesie wat groot landbou verliese veroorsaak veral in die mielie gewasse, een van die stapelvoedsels van die Afrika kontinent. Dit produseer ook mikotoksiene in aangetaste gewasse en hierdie kan lei tot die ontstaan van kankers wanneer besmette voedsel op groot skaal verbruik word. Vier hoë-mikotoksien produserende swamlyne van twee spesies, naamlik F. verticillioides (MRC 826 en MRC 8267) en F. proliferatum (MRC 7140 en MRC 6809) is gebruik in alle in vitro anti-swam ondersoeke in hierdie studie. Die eerste analise het dichloromethan: methanol (1:1 v/v) ongesuiwerde plant ekstrakte bevat van die vyf bevolkings: Stellenbosch, Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein, geneem gedurende 2009 en 2011. Al hierdie toets monsters het goeie aktiwiteit getoon waar die minimum beperkende konsentrasie (MIC) waardes van 0.031 mg ml-1 tot 0.5 mg ml-1 gevarieer het. Waardes laer as laasgenoemde word beskou as sterk inhibeerders. Die Stellenbosch ekstrakte was die mees aktief vir albei jare hierbo genoem, met die beste aktiwiteit teen F. verticillioides MRC 8267 en MCR 826 by 0.031 mg ml-1. Die minste aktiwiteit (hoewel nog ‘n sterk inhibeerder) waargeneem was van die Yzerfontein ekstrakte, met ‘n MIC waarde van 0.5 mg ml-1. Oor die algemeen het ‘n vergelyking tussen die twee jare aangetoon dat die monsters wat in 2011 versamel is veel sterker was dan dié van 2009, moontlik te wyte aan ‘n verlengde bewaringstyd wat moontlik ‘n chemiese dekomposisie ten gevolge gehad het. Omdat die Stellenbosch bevolking die beste aktiwiteit getoon het sowel as ‘n relatief afwykende chemiese profiel, is blare van hierdie plante toe bekendgestel aan weefselkultuur kondisies. Blaar eksplante is op soliede hormoonvrye Murashige en Skoog media geplaas en dit is aangevul met sintetiese auksien Naftaleen asynsuur (NAA) en sitokien Bensiel adenien (BA) individueel en in verskillende kombinasies. Geen wortels of uitloopsels is waargeneem in al die hormoon kombinasies nie maar in die hormoonvrye media het daar egter Kallus in twintig persent van die eksplante voorgekom. Kallus is toe as subkultuur van hormoonvrye MS media gekweek en saamgevoeg en dichloromethan: methanol (1:1v/v) ekstrakte is getoets teen die volgende Fusarium swamlyne MRC 826; MRC 8267; MRC 7140 en MRC 6809. MIC waardes het sterk inhiberende eienskappe getoon met die laagste waarde as 0.025 mg ml-1 teen drie swamlyne: MRC 1740, MRC 8267 en MRC 826, en die hoogste was 0.25 mg ml-1 na 48 uur. Die minimum inhiberende konsentrasie waardes het gestyg na 0.5mg ml-1 na 60 uur, wat ‘n fungistatiese aksie getoon het. Maar van 60 tot 92 uu het waardes egter ‘n swamdodende aksie aangetoon met geen verandering van 0.5mg ml-1 nie. In die identifisering van die bioaktiewe komponente, is die ekstraksie van Stellenbosch se bevolking in dichloromethan: methanol (1:1 v/v) uitgevoer, en met gebruik van vyftig gram van die ekstrak is bioanalise-geleide fraksionering gedoen deur gebruik van ‘n VersaFlash®. Die mees aktiewe fraksie is verder gefraksioneer deur die gebruik van ‘n konvensionele silikajel kolom. Aktiewe fraksies is getoets deur LC-MS te gebruik, en twee verbindings, carnosol en carnosic suur, is geïdentifiseer. Voorbereidende TLC is gebruik om identiteit te bevestig, want fraksies was naas die kommersiele standaarde van die twee verbindings getoets. Sinergistiese aktiwiteit van die twee samestellings is ondersoek deur ‘n antiswam ontleding teen die vier swamlyne uit te voer. Hierdie studie het dus die veronderstelde bestaan van verskillende chemotipes tussen die bevolkings waarvan voorbeelde geneem is, bekend gestel. Veral die Stellenbosch se bevolking het die meeste verskil, heel moontlik omrede die verlengde en hoër versteurings deur die nabyheid van mense. Plant– omgewing interaksies speel ‘n belangrike rol in die metaboloom van plante, wat dan indirek hul eienskappe verander, en in hierdie geval die antiswam aktiwiteit. Die tuingroeiende bevolking was die mees aktief, heel moontlik omrede hierdie aspek. Nietemin was geen bioaktiwiteit verloor waar die mees kragtige bevolking met weefsel kultuur kondisies in aanraking gebring is nie. Dus is dit ideaal vir kommersialisering. Een nuwe belangrike bevinding was die carnosol en carnosic suur wat twee welbekende samestellings is wat meesal geassosieer is met Rosmarinus officinalis en gedokumenteer is vir antioksidant aktiwiteit. Hier dui laasgenoemde samestellings ‘n antiswam aktiwiteit aan teen die getoetste Fusarium swamlyne. Met ‘n gevestigde weefsel kultuur sisteem alreeds in plek, voorsien dit ‘n beginpunt vir die bestudering van hoe hierdie bioaktiewe komponente in massa geproduseer kan word in die ontwikkeling van nuwe swamdodende produkte.

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia
Physiology and biochemistry of in vitro plants can be complex and different when compared with conventional and well known plant ex vitro behavior. Metabolic reprogramming events that occur in a number of in vitro propagated plant species give rise to low ex vitro yields as a handicap for commercial application. Temporary Immersion Bioreactors (TIB) are a method to obtain plantlet morphology and physiology much alike that of in vivo grown plants. Three tropical crops were selected for their economic importance, but also because of their different photosynthetic characteristics, to compare C4 (sugarcane) and C3-CAM (pineapple) facultative photosynthetic pathways to the more common C3-type (plantain) photosynthesis. Experiments performed using plantain and sugarcane allowed to integrate the results obtained when monitoring the oxidative stress response in C3 and C4 metabolism. Pineapple plants propagated in TIB and evaluated during acclimatization under C3 and CAM inducing conditions were used to describe the facultative C3-CAM carbon metabolism and the influence of the environmental conditions on the switch from C3 to CAM. Studies based on the modulation of in vitro conditions which reproduce abiotic stress conditions can be used for understanding the influence of upcoming climate changes on the physiology of different species
FCT

Abstract Plant registration and selection work aimed at identifying the best genotypes for northern landscaping has been carried out in Finland since the 1980’s. In the University of Oulu Botanical Gardens, micropropagation methods have been developed for several woody plant taxa registered during the POHKAS (Northern Hardy Plants) project. Micropropagation is an effective method to conserve valuable genetic characteristics and to produce plantlets from woody species with limited mother stock material and in a limited time period. In this study the long-term field phenology and success of 19 micropropagated shrub and tree taxa was followed in plant selection experiments. Experiments were conducted at four northern field sites presenting different climatic conditions. Of the phenological monitoring parameters, the onset of foliation and flowering in the field revealed a strong relation to spring time temperature, being obviously latest to occur in northernmost site. The gradient between southern and northern sites for autumn phenology was not so obvious. However, between the different genotypes, the greatest differences were observed in the timing of autumn colouration and defoliation. Winter hardiness also showed clear differences between genotypes. Of the success parameters, it was most decisive as winter hardy genotypes had a higher occurrence of flowers and ornamental appearance, for example in Rosa majalis ‘Tornedal’. Some of these hardy genotypes with known characteristics were introduced to northern tourism areas to create examples of sustainable landscaping. Further, a list of potential plants for different northern sites was compiled. Special forms with both scientific and ornamental value are occasionally found in wild species. One example of this is the red-leaved form of a pubescent birch, Betula pubescens f. rubra, which was studied in the plant selection experiments, and was used as a model tree to evaluate the role of anthocyanins in northern plants in a case study of northern birches. In the case study, the red-leaved pubescent birch showed some differences in flavonoid responses and growth rate in comparison to Betula pubescens and Betula pubescens ssp. czerepanovii. Phenology of the B. p. f. rubra was corresponding to that of the B. pubescens. For cultivated woody plants the most important selection criteria for the northern areas are the suitability to local climate i.e. timing of phenological events and winter hardiness. Foliar anthocyanins seem to increase adaptation to northern growing conditions with high light intensity and low temperature.

Orientador: Claudia Regina Baptista Haddad
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-26T21:10:07Z (GMT). No. of bitstreams: 1 Chone_RosanaMarySartor_M.pdf: 46520912 bytes, checksum: 140ac9102b951f96df903dee18f16c8a (MD5) Previous issue date: 2015
Resumo: O cultivo do cafeeiro é de grande importância econômica mundial. O consumo crescente de café arábica tem levado os pesquisadores a buscar variedades que produzam bebidas de melhor qualidade e com baixos teores de cafeína. Os programas de melhoramento genético concentram esforços na busca destas características com destaque para cultivares da espécie Coffea arabica, a mais cultivada no mundo. A embriogênese somática é importante para manter características selecionadas em programas de melhoramento genético e produzir os indivíduos selecionados em grande escala, além de contribuir para estudos fisiológicos e moleculares. A embriogênese somática direta apresenta vantagens pela redução de insumos e mão de obra, devido à eliminação da fase calogênica, porém, apresenta baixa eficiência, quando comparada com a via indireta, para a espécie C. arabica. Apesar da importância da embriogênese somática direta, nada se sabe sobre os fatores que controlam a sua ocorrência em genótipos de C. arabica e tampouco há estudos com utilização de brassinosteróides na fase de indução desse tipo de embriogênese in vitro. O brassinosteróide, 24-epibrassinolídeo, foi utilizado isoladamente e em conjunto com uma citocinina, N6-(2-isopentenyl) adenina (2-iP), para avaliar seus efeitos sobre a embriogênese somática direta e verificar possíveis alterações endógenas no metabolismo desses hormônios durante a indução da embriogênese em explantes foliares da cultivar Mundo Novo de Coffea arabica. Os explantes foram cultivados em meio de cultura MS modificado, adicionado de 2-iP a 10 µM, acrescido ou não de 24-epibrassinolídeo. Foram utilizadas as seguintes concentrações do brassinosteróide: 0,01 µM, 0,10 µM e 1 µM, em dois experimentos realizados em diferentes épocas do ano. Análises de microscopia de luz e microscopia eletrônica de varredura foram realizadas para compreensão dos eventos anatômicos e morfológicos da via de embriogênese relacionada às diferentes combinações hormonais praticadas. Avaliações de variáveis qualitativas e quantitativas dos explantes foliares dos dois experimentos foram realizadas in vitro durante 130 e 240 dias. Na tentativa de compreender melhor a participação dos brassinosteróides na via de embriogênese somática, explantes foliares induzidos apenas com a citocinina, 2-iP, foram submetidos a análises de Ressonância Magnética Nuclear (RMN). Os embriões somáticos obtidos tanto no tratamento com uso exclusivo de citocinina, como no tratamento com citocinina associada ao 24-epibrassinolídeo foram cultivados em meio de cultura para germinação e desenvolveram-se em plântulas. A aplicação do brassinosteróide levou à iniciação do processo de embriogênese somática direta, promovendo a formação de estruturas embriogênicas. Sua aplicação conjunta com citocinina aumentou o número de embriões, o número de explantes com embriões, levou à formação de embriões em condição na qual apenas a aplicação de citocinina foi insuficiente e acelerou a via embriogênica. Promoveu o desenvolvimento de embriões mais bem definidos, com características meristemáticas típicas. O aumento de picos na região do espectro de RMN, característico de moléculas esteroidais, coincidente com o período em que ocorre a embriogênese somática direta, pode ser indicativo da participação de brassinosteróides endógenos durante esse processo. Este trabalho abre perspectivas para a utilização de brassinosteróides em embriogênese somática direta em processos produtivos de C. arabica
Abstract: Coffee cultivation is of worldwide economic importance. The increasing consumption of coffee has led to search for varieties that produce better quality beverages with lower caffeine contents. Genetic improvement have concentrated their efforts on the search for these characteristics with emphasis on cultivars of the species Coffea arabica, the most cultivated in the world. Somatic embryogenesis is important to maintain selected characteristics in genetic improvement and produce selected individuals on a large scale, as well as contributing to physiological and molecular studies. Direct somatic embryogenesis shows the advantages of reduced consumables and less manual labor, due to elimination of the callus phase, but is inefficient with the species C. arabica when compared with the indirect pathway. Despite the importance of direct somatic embryogenesis, nothing is known about factors controlling its occurrence in C. arabica genotypes, nor are there any studies on use of brassinosteroids in the in vitro induction of this type of embryogenesis. The brassinosteroid 24-epibrassinolide was used alone and together with cytokinin N6-(2-isopentenyl) adenine (2-iP) to evaluate its effects on direct somatic embryogenesis and verify endogenous alterations in metabolism of these hormones during induction of embryogenesis in foliar explants of cultivar Mundo Novo of species Coffea Arabica. The explants were cultivated in modified MS culture medium with the addition of 10 µM 2-iP, with or without 24-epibrassinolide. The following concentrations of brassinosteroid were used: 0.01 µM, 0.10 µM and 1 µM, in two experiments carried out at different times of the year. Light microscopy and scanning electronic microscopy analyses were carried out in order to understand anatomic and morphologic events of embryogenic pathway as related to different hormone combinations applied. The qualitative and quantitative variables of the foliar explants were evaluated in the two experiments at points in time from 130 and 240 days. In an attempt to better understand the participation of the brassinosteroids in the somatic embryogenesis pathway, foliar explants induced only by the cytokinin 2-iP were submitted to nuclear magnetic resonance (RMN) analyses. The somatic embryos obtained both using the treatment with the exclusive use of cytokinin and in treatment with cytokinin associated with 24-epibrassinolide were cultivated in a culture medium for germination and developed into plant embryos. The application of brassinosteroid led to initiation of direct somatic embryogenesis process, promoting the formation of embryogenic structures. Its application together with cytokinin increased number of embryos, the number of explants with embryos, led to formation of embryos in a condition in which only application of cytokinin was insufficient and accelerated embryogenic pathway. It also promoted development of much better defined embryos with typically meristematic characteristics. The increase in peaks in the region of the RMN spectrum, characteristic of steroidal molecules, coinciding with the period in which direct somatic embryogenesis occurred, could be an indication of participation of endogenous brassinosteroids during the process. This work widens the perspectives for the use of brassinosteroids in direct somatic embryogenesis for productive processes with C. arabica
Mestrado
Biologia Vegetal
Mestra em Biologia Vegetal

Aspects scientifiques de la production in vitro en relation avec l'activité "recherche-développement" d'un laboratoire industriel (LBV France). Mise au point d'une technique pour 30 variétés. Détermination d'un milieu de culture optimal pour chacune des phases de développement. Recherche de marqueurs précoces de la conformité et de l'identité variétale. Utilisation de l'électrophorèse, application à 14 systèmes enzymatiques. Proposition d'essai de modélisation appliqués à la planification et à la gestion de la production. Simulation de la généralisation de la cinétique de production sur 200 variétés réparties dans 15 genres différents.

Monterey pine (Pinus radiata), a native tree to California and two Mexican islands, is important both ecologically and economically. Outside native stands, Monterey pines are grown for landscaping in California and on plantations around the world. Pitch canker, a disease caused by the fungus Gibberella circinata Nirenberg & O’Donnell (Fusarium circinatum Nirenberg and O'Donnell) is threatening the survival of Monterey pines. The disease currently affects Monterey pines in many parts of the world including the native stands. No effective chemical or biological control is available but some Monterey pines show resistance to the disease. The purpose of this project was to develop a working protocol for producing genetic clones of the resistant pines through micropropagation. These genetic clones will be used for outplanting in places outside the native stands for ornamental and plantation purposes. This project analyzes the results of ten trials with varied parameters and bases the final protocol on the parameters used in the trial that induces the growth of new shoots. The final protocol developed in this project describes, step-by-step, the media preparation for the initiation, plant material collection, surface sterilization of plant material, plating in media and initiation of shoots on explants. The protocol calls for collecting shoot tips with hardened buds that have not yet elongated, then washing the shoot tips in sterile water with Tween 20 for 15 minutes. The shoots tips are then surface sterilized in a 50% bleach solution for 20 minutes. The explants are broken into disks (to minimize damage to the cells) by inserting the tip of a scalpel and tilting it slightly. The initiation media shown to induce growth consists of ½ strength LePoivre basal salt mixture, 5mg/L benzylaminopurine, 3% sucrose and 0.8% agar and is adjusted to a pH of 5.7, then autoclaved for 20 minutes. The explants are inserted into solidified media and incubated in a growth chamber programmed for 16 hours of light and 8 hours of dark with temperatures of 27ºC and 22ºC and light irradiance of 80µEm-2s-1. After 1 month the protocol calls for transferring the growing shoots to elongation media with full LP basal salts and transferring every month. When the number of desired shoots has been reached the forthcoming protocol for rooting can be followed.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Bacterial and fungal pathogens have developed numerous defence mechanisms against antimicrobial chemical agents, and resistance to old and new produced drugs are on the rise. Discovery of natural products derived from plants with diverse chemical structures and novel mechanisms of action to treat these notorious pathogens is a priority. Biotechnology (discussed in Chapter 1) has much to offer as a pharmacological tool and in the general study of medicinal plants. The Genus Salvia (Lamiaceae) has gathered much interest as these plants manufacture a diverse range of secondary metabolites including flavonoids, tannins and terpenoids. Of particular interest are the terpenoids which are largely implicated in the efficacy of Salvia plants as traditional medicines contributing to their pharmacological actions (discussed in Chapter 2). Due to the importance of these plants as herbal remedies, in this study, biotechnological techniques such as tissue culture and Agrobacterium-mediated transformation were applied on Salvia runcinata L.f., a South African medicinal plant, in an attempt to enhance the metabolomic profile and its bioactivity. Like so many other sages, S. runcinata has been used in folk medicine to treat a variety of ailments. Application of biotechnology was viewed as an important value adding platform for this species, assisting with its commercialisation for the cosmeceutical and pharmaceutical industries. Therefore the study had three foci: (1) to determine the seed germination behaviour and optimal conditions for micropropagation; (2) to develop a protocol that would be efficient whilst being simple for genetic transformation; and lastly, (3) to conduct phytochemical studies on in vitro generated S. runcinata transgenic hairy root and in vitro organ cultures by comparing these to glasshouse plants as potential therapeutic sources of natural compounds used in the treatment of infections in plants and humans. Data generated is thus summarised in three research chapters and Chapter 3 describes the formulated procedures assisting with in vitro seed germination and micropropagation of S. runcinata. The efficacy of smoke and scarification treatments for germination improvement was initially tested coupled to the evaluation of different hormonal combinations and different explant types which would aid with inducing adventitious shoot formation in vitro. The most effective germination treatment proved to be a 3 min exposure of seeds to 25% (w/v) H2SO4 combined with a concentration of 10-5 M smoke solution, resulting to more than 80% germination. Shoot proliferation was significantly higher using nodal explants with the addition of 4.43 μM BA. The protocol established in this part of the study is viable for large scale commercial production of S. runcinata as it would yield 1296 to 46656 viable plants in 4 to 6 months from one nodal explant. Micropropagation was applied also as a pre-emptive measure to ease pressure on the wild plants as the demand for S. runcinata is anticipated to increase due to its growing economic value as it is one of two South African sages with epi-α-bisabolol that is sought after by the pharmaceutical and cosmeceutical industries. This makes the protocol developed in this part of the study suitable for ex situ conservation of S. runcinata plantlets. Evaluations on the transgene transfer capacities of two different agropine strains (A4T and LBA 9402) of Agrobacterium rhizogenes to induce hairy root cultures of S. runcinata explants on nodal and leaf explants were conducted (reported in Chapter 4). Hairy roots formed 3 to 4 weeks after inoculation of the explants and these agropine strains showed different abilities for genetic transformation with the LBA 9402 strain producing significantly more roots on each explant compared to the A4T strain (P=0.0075). However, none of the LBA 9402 derived clones and only 2 clones generated through A4T transformation survived subculturing. The polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence and transcription (respectively) of rol A, rol B, rol C and ags genes which are mobilised from the transfer-DNA (T-DNA) fragment of the root-inducing (Ri) plasmid of A. rhizogenes to the plant genome during transformation. The two A4T clones, termed here A4T3 and A4T5, were stably transformed, Southern blot analysis using rol A as a probe further validated the integration of one copy of the rol A gene. Transformed hairy roots, untransformed roots from tissue cultured plants, tissue culture-derived plants and glasshouse-grown plants were profiled for secondary metabolites by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) in Chapter 5. In this part of the study, it is clear that the use of tissue culture as a propagation system did not negatively affect the volatile compound profile of S. runcinata and plants had a similar essential oil content to that reported by Kamatou et al. (2008), leading to a conclusion that in vitro plants maintained their biochemical integrity even under an alternative micro-controlled environment. Similarly to others, Ri-transformation was explored as an avenue to alter secondary metabolism creating inter-clonal variation. Transformed clones were distinguishable, displaying more of some primary metabolites including sucrose, galactose, sorbose and fructose than the leaf extracts. With the current GC-MS methods used, this clear distinction was not obvious at the secondary metabolite level. In general, solvent extracts (acetone and methanol:dichloromethane (MetOH: DCM) (1:1 v/v) exhibited good to moderate antibacterial activity with the minimum inhibitory concentration (MIC) values ranging from 0.39 to 0.78 mg ml-1. However, in vitro plant cultures were the most potent against two Gram-negative bacterial strains: Escherichia coli (ATCC 11775) and Klebsiella pneumoniae (ATCC 13883), and two Gram-positive bacterial strains: Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 12600). The hairy root extracts did not show any activity against fungi, Fusarium subglutinans (MRC 0115) and Fusarium proliferatum (MRC 6908). Micropropagation therefore proves to be an interesting avenue for commercial production of S. runcinata, supplying plants with an improved pharmacological activity. Hence the biotechnological approach applied here is a viable strategy for the production of medicinal bioactives from S. runcinata.
AFRIKAANSE OPSOMMING: Bakterieë en fungi patogene het baie verskeie meganismes ontwikkel teen antimikrobiese chemiese agente, en weerstand teen ou en nuwe chemise stowwe is besig om te vergroot. Daarom is dit belangrik om natuurlike plantaardige produkte met diverse chemiese strukture en unieke werkings meganismes te ontdek waarmee hierdie berugte patogene beveg kan word. Biotegnologie (wat in Hoofstuk 1 bespreek word) kan gebruik word as 'n farmakologiese hulpmiddel in die algemene studie van plante. Die Klas (Genus) Salvia (Lamiaceae) het al baie aandag getrek aangesien hierdie plante 'n wye reeks sekondêre metaboliete vervaardig wat flavonoïede, tanniene en terpenoïede insluit. Veral van belang is die terpenoïde wat betrokke is by die doeltreffendheid van die Salvia plante as tradisionele medisyne, aangesien dit bydra tot hulle farmalogiese aksie (wat in Hoofstuk 2 bespreek word). Aangesien hierdie plante sulke belangrike kruie is, word daar in hierdie studie, biotegnologiese tegnieke soos die kweek van weefsel en Agrobacterium-bemiddelde transformasie op Salvia runcinata L.f. toegepas om die metabologiese profiel en die bioaktiwiteit daarvan te verbeter. Soos baie van die salies is S. runcinata tradisioneel dikwels gebruik om allerhande siektetoestande te behandel. Die toepassing van biotegnologie word beskou as 'n belangrike manier om waarde by te voeg sodat hierdie plant kommersieei deur die kosmetiese en farmakeutiese bedrywe gebruik kan word. Daarom is daar op drie dinge gefokus: (1) die ontkiemings gedrag van saad en die optimale toestande vir mikrovoortplanting (2) die ontwikkeling van protokol wat eenvoudig maar doeltreffend is vir genetiese transformasie, en die (3) fito-chemise studies op in vitro genereerde S. runcinata transgeniese harige wortels en in vitro orgaan kwekings deur om hulle te vergelyk met kweekhuis plante as potentiële terapeutiese bronne van natuurlike samestellings vir die behandeling van infeksies in beide plante en mense. Die data wat gegenereer is, is opgesom in drie hoofstukke, en in Hoofstuk 3 word die prosedures wat gebruik word in die in vitro saad ontkieming en die mikro voortplanting van S. runcinata, bespreek. Die doeltreffendheid van rook en skarifikasie behandeling vir die verbetering van ontkieming is eers getoets en gekoppel aan die evaluering van verskillende hormoonkombinasies en verskillende eksplant tipes wat lei tot die formasie van uitloopsels in vitro. Daar is gevind dat die effektiefste behandeling vir ontkieming, 'n 3-minuut blootstelling van saad aan 25% (w/v) H2SO4 gekombineer met 'n konsentrasie 10-5 M rook oplossing is. Dit het gelei tot meer as 80% ontkieming. Daar was baie meer uitloopsels toe nodale eksplante gebruik is met die byvoeging van 4.43 μM BA. Die proktokol wat hier gevestig is, kan op groot skaal gebruik word vir die kommersiële produksie van S. runcinata, want 1296 tot 46656 lewensvatbare plante kan binne 4 ot 6 maande van een nodale eksplant gemaak word. Mikro voortplanting is toegepas as 'n voorkomende maatreel om die druk op die natuur te verminder omdat daar verwag word dat die vraag na S. runcinata sal toeneem na gelang die groeiende ekonomiese waarde daarvan toeneem. Dit is een van twee Suid-Afrikaanse salies met epi-α-bisabolol wat deur die farmakeutiese en die kosmetiese bedrywe gebruik word. Dit beteken dat die protokol wat hier ontwikkel is, geskik is vir die ex situ bewaring van S. runcinata plante. Die transgeen oordrag van twee verskillende agropien tipes (A4T and LBA 9402) van Agrobacterium rhizogenes is geevalueer (en in Hoofstuk 4 beskryf). Harige wortels het 3 tot 4 weke na die inenting van die eksplante gevorm en hierdie agropien tipes het verskillende vermoëns vir genetiese transformasie getoon, met die LBA 9402 tipe wat baie meer wortels op elke eksplant voorgebring het in vergelyking met die A4T tipe (P=0.03116). Geen van die LBA 9402-afgeleide klone en slegs 2 klone wat deur A4T transformasie genereer is, het oorleef. The polimerase ketting reaksie (PCR) en die teenoorgestelde trenskriptasie-polimerase (RT-PCR) ketting reaksie het die teenwoordigheid en transkipsie (onderskeidelik) van rol A, rol B en rol C en ags gene, wat oorgedra word deur die oordrag DNA (T-DNA) fragment van die wortel induserende (Ri) plasmied van A. rhizogenes na die plant genoom tydens transformasie, bevorder. A4T klone, hier A4T3 and A4T5 genoem, is stabiel transformeer. Southern blot ontleding het met die gebruik van rol A, die integrasie van een kopie van die rol A geen, bevestig. In Hoofstuk 5 is transformeerde harige wortels, ongetransformeerde wortels van weefsel gekweekte plante, weefsel gekweekte plante, en kweekhuis plante deur dun-laag chromatografie (TLC) en gas-chromatografie-massa spektrometrie (GC-MS) geprofiel vir sekondêre metaboliete. In hierdie deel van die studie is dit duidelik dat die gebruik van weefsel kwekery as 'n voortplantsisteem nie 'n negatiewe effek gehad het op die vlugtige samestelling profiel van S. runcinata nie en dat plante 'n sootgelyke essentiële olie inhoud het as wat deur Kamatou et al. (2008) bevind is. Dit lei tot die gevolgtrekking dat in vitro plante hulle biochemiese integriteit behou selfs onder alternatiewe mikro-beheerde omgewings. Ri-transformasie is ondersoek as 'n manier om sekondêre metabolisme te verander om interkloon variasie te skep. Getransformeerde klone kon uitgeken word, aangesien dit meer primêre metaboliete soos sukrose, galaktose en fruktose insluit as die blaar ekstrakte. Hierdie verskil was nie met die huidige GC-MS metodes so duidelik sigbaar op die sekondêre metabolitiese vlak nie. Oor die algemeen toon ekstraksie met asetoon en methanol dichlorometaan (MetOH: DCM) (1:1 v/v) goeie tot gemiddelde antibakteriese aktiwiteit met die minimum remmende konsentrasie (MIC) waardes van 0.39 tot 0.78 mg ml-1. Die in vitro plant kulture het egter sterker weerstand gebied teen twee Gram-negatiewe bakteriese tipes: Escherichia coli (ATCC 11775) en Klebsiella pneumoniae (ATCC 13883), en teen twee Gram-positiewe bakteriese tipes: Bacillus subtilis (ATCC 6051) en Staphylococcus aureus (ATCC 12600). Die harige wortel ekstrakte het geen aktiwiteit teen die swamme, Fusarium subglutinans (MRC 0115) en Fusarium proliferatum (MRC 6908) getoon nie. Mikro-voortplanting is dus 'n interessante manier om S. runcinata kommersieel te produseer aangeien die plante verbeterde farmalogiese aktiwiteit toon. Die biotegnologiese benadering wat hier toegepas word, is 'n praktiese strategie vir die produksie van geneesmiddels van S. runcinata.

Thesis ( PhD. (Biotechnology )) --University of Limpopo, 2005
The marula tree (Sclerocarya birrea subsp. caffra), an indigenous, multipurpose, drought tolerant tree of Africa harbors great economic potential. Acceptance of marula-derived products internationally will directly increase the demand for marula resource. Rapid multiplication of marula trees of superior quality forms the basis of sustainable export growth. In vitro propagation and genetic improvement offer the opportunity for accelerated multiplication of selected tree material as well as to dramatically increase production, quality and efficiencies. The objectives of the study were therefore to develop a protocol for in vitro multiplication of marula and to determine the feasibility of Agrobacterium-mediated transformation of the marula tree. Nodal sections with axillary bud (s) were cultured on Murashige and Skoog (MS) medium supplemented with 4.8μM BA and 2.4μM KN and 0.1% polyvinylpyrrolidone (PVP) to obtain on average 2.5 microshoots per responding explant. The proliferated microshoots were elongated on MS medium supplemented with 1.2μM BA and 1.0μM KN. Elongated microshoots were rooted in MS medium at half salts strength supplemented with 10μM IBA and 0.3% activated charcoal (AC). On average 82% of the shoots rooted. Survival of acclimatized plantlets was 90%. RAPD analysis confirmed intraclonal genetic stability between parent plants and their clones within the limits of the technique.Nodal sections cocultivated with Agrobacterium tumefaciens for 3 days on MS multiplication medium supplemented with 100μM acetosyringone resulted on average in transient expression of 52.5% of the explants with 1.6 blue stained zones per explant. Cocultivated explants on MS selection medium containing 300mgl-1 kanamycin resulted in 1.5% chimeric putative transgenic shoots. This is the first report on the micropropagation and genetic transformation of marula, Sclerocarya birrea subsp caffra.
South Africa’s National Research Foundation Institutional Research Development Program (NRF-IRDP)

O crescente aumento no uso da micropropagação de teca como forma de produção de clones com qualidades genotípicas e fenotípicas selecionadas a partir de árvores de elite, determinou a importância desse método, pois origina plantações com maior qualidade e uniformidade, agregando maior valor ao preço da madeira no mercado. O uso de sementes para a obtenção de mudas é uma técnica menos onerosa, porém resulta em plantas com tamanhos desiguais e não há um padrão na qualidade da madeira, essa técnica depende também da época de produção de sementes e, portanto é restrita a um período do ano. A micropropagação permite a clonagem em larga escala das árvores de elite em tempo e espaço reduzidos, podendo ser realizada em qualquer época do ano, além disso, permite a formação de mudas totalmente livres de pragas e patógenos. Faz-se necessário, maiores estudos com meio de cultura para Tectona grandis, pois os materiais relacionados a esse assunto são escassos. Para que a técnica do cultivo in vitro da teca seja incrementada, objetivou-se nesse experimento, otimizar o crescimento dos explantes de três clones diferentes, testando a eficiência de 6 meios de cultura com diferentes formulações nutricionais e constatar qual deles apresenta a melhor resposta para cada clone. O estudo contou com 6 tratamentos (MS, Básico, M1, M2, M3 e M4), durante oito épocas de avaliação (0, 7, 14, 21, 28, 35, 42 e 49 dias), para três clones de Tectona grandis (61, 62 e 68), com três repetições por tratamento/clone, utilizando o delineamento experimental inteiramente casualizado. A avaliação do crescimento foi feita por meio do peso de matéria fresca (PMF), matéria seca (PMS) e a taxa de crescimento relativo (TCR) proporcionado pelos meios. O PMF foi usado para obtenção do PMS e cálculo da TCR. Baseado nos valores do PMS obtidos, para o clone 61, constatou-se a formulação do meio Básico (PMS = 0,38 g), como a mais eficiente. Para o clone 62, o meio mais responsivo foi M4 (PMS = 0,47 g) e no clone 68, destacou-se o meio M3 (PMS = 0,71 g). Quanto a TCR, não foi encontrada diferença estatística significativa para nenhum dos 6 meios de cultura levando-se em conta os três clones.
The increasing use of teak micropropagation as a way of producing clones with genotypic and phenotypic qualities selected from elite trees, established the importance of this method, it leads to higher crop quality and consistency, adding more value to the price of wood business. The use of seeds to obtain seedlings has a less cost, but it results in plants with unequal sizes and wood quality without a pattern. This technique also depends on the time of seed production and therefore is restricted to a year period. Micropropagation allows cloning elite trees in large-scale and reduced time and space. It can be performed at any time of year, in addition, allows the formation of plants totally free of pests and pathogens. It is necessary more studies with culture medium for Tectona grandis, because the materials related to this subject are scarce. To increase the technique of teak in vitro cultivation, this experiment aimed to optimize the growth and development of explants from three different clones, testing the effectiveness of six culture media with different nutritional formulations and find which one offers the best answer to each clone. The study included six treatments (MS, Basic, M1, M2, M3 and M4) for eight periods (0, 7, 14, 21, 28, 35, 42 and 49 days) for three clones of Tectona grandis (61, 62 and 68) with three replicates per treatment/clone, in a randomized experimental design. The growth assessment was performed by the fresh matter weight (FMW), dry matter weight (DMW) and relative growth rate (RGR) provided by the media. The FMW was used to obtain the DMW and to calculate RGR. Based on DMW values obtained for clone 61, was found that the formulation of Basic medium (DMW = 0.38 g) was the most efficient. For clone 62, the most responsive medium was M4 (DMW = 0.47 g) and to clone 68, M3 (DMW = 0.71 g) was the highlighted medium. As the RGR, it was found no statistically significant difference for any of the six culture media taking into account the three clones.

O objetivo deste estudo foi otimizar a multiplicação de brotações de Eucalyptus globulus Labill., por meio da definição do tamanho de explantes iniciais e de ajustes de nutrientes minerais do meio de cultura. Os meios de cultura utilizados foram o JADS (CORREIA et al., 1995), JADS modificado e o MS. Os tamanhos dos explantes foram definidos por suas massas frescas iniciais e classificados em T1 (0,1430g), T2 (0,2685g) e T3 (0,5180g). Inicialmente, os ajustes dos nutrientes minerais foram realizados de forma individual para cada nutriente e concentração utilizados: nitrogênio (NH4NO3), com suplementação de 45,0 (N1) e 65,0 (N2) mmol L-1; fósforo (KH2PO4), com suplementação de 1,0 (P1) e 2,0 (P2) mmol L-1; cálcio (CaCl2.2H2O), com suplementação 7,5 (Ca1) e 10,0 (Ca2) mmol L-1; e Magnésio (MgSO4.7H2O), com suplementação de 1,5 (Mg1) e 4,5 (Mg2) mmol L-1. Com base nos resultados iniciais, novos ajustes para nitrogênio e cálcio foram realizados: nitrogênio (NH4NO3) e cálcio (CaCl2.2H2O), com suplementação de 45,0 e 7,5 mmol L-1 (N1Ca1), respectivamente, e suplementação de 60,0 e 7,5 mmol L-1 (N2Ca1), respectivamente. Todos os experimentos foram conduzidos em delineamento inteiramente aleatorizado (DIA); com 3 tratamentos e 4 repetições (tamanho do explante); 10 tratamentos e 3 repetições (ajuste inicial de nutrientes minerais) e 4 tratamentos e 6 repetições (ajuste final de nutrientes minerais). As características de crescimento, massas fresca e seca, porcentagem de massa seca e taxa de crescimento relativo (%) das brotações foram avaliadas semanalmente, durante os 28 dias de cultivo in vitro. As características de crescimento das brotações foram pouco afetadas pelo tamanho do explante inicial; no entanto, apresentaram deformações morfológicas em altas concentrações de nitrogênio. Todos os tratamentos apresentaram incrementos de massas seca e fresca. As porcentagens de massa seca foram menores nos tratamentos com maiores concentrações de nitrogênio (MS, N2 e N2Ca1). As maiores taxas de crescimento relativo foram observadas aos 7 dias de cultivo, e decresceram ao longo do período estudado. O meio de cultura JADS, apresentou crescimento das brotações considerado ótimo, porém não máximo. O meio de cultura MS apresentou crescimento das brotações fora dos padrões considerados ótimo e máximo. O meio de cultura JADS modificado, contendo 45,0 (N), 3,0 (P), 7,5 (Ca) e 3,0 (Mg), apresentou crescimento máximo das brotações e próximo ao ótimo.
The aim of this work was to optimize Eucalyptus globulus Labill. shoot multiplication in vitro through the definition of the initial explant size and the adjustment of mineral nutrients in the culture media. The culture media utilized were JADS (CORREIA et al., 1995), modified JADS and MS. The explants fresh weight were 0.1430 g (T1); 0.2685 g (T2) and 0.5180 g (T3). Initially, the mineral nutrient and concentrations utilized were: nitrogen (NH4NO3) with 45.0 (N1) and 60.0 (N2) mmol L-1; phosphate (KH2PO4) with 1.0 (P1) and 2.0 (P2) mmol L-1; calcium (CaCl2.2H2O) with 7.5 (Ca1) and 10.0 (Ca2) mmol L-1 and magnesium (MgSO4.7H2O) with 1.5 (Mg1) and 4.5 (Mg2) mmol L-1. Based on the initials results, new adjustments were made for nitrogen and calcium, and the concentrations utilized were (NH4NO3) and (CaCl2.2H2O) with 45.0 and 7.5 mmol L-1 (N1Ca1) and 60.0 and 7.5 mmol L-1 respectively. All the tests were carried out in a completely randomized design; with 3 treatments and 4 replicates (explant size); 10 treatments and 3 replicates (initial adjustment of mineral nutrients) and 4 treatments and 6 replicates (final mineral nutrient adjustment). The shoot growth characteristics, fresh and oven dry weight, oven dry weight percentage and relative growth rate were evaluated weekly for 28 day culture period. The explant initial size had little effect on the shoot growth characteristics. All the treatments with mineral nutrient adjustments showed fresh and oven dry weight increase. The oven dry weight percentage were lower in the treatments with high nitrogen concentrations (MS, N2 and N2Ca1). The highest relative growth rates were observed at the 7th day evaluation and lowered along the culture period. The JADS culture medium showed shoot growths considered optimum but not maximum. The MS culture medium showed shoot growths not considered optimum or maximum. The adjusted culture media with 45.0 (N), 3.0 (P), 7.5 (Ca) and 3.0 (Mg) showed shoot growth considered maximum and almost optimum.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Pertencente à família Meliaceae, a espécie Trichilia catigua A. Juss é conhecida popularmente como catigua, cataguá, argelim-rosa e mangalto-catingam. Sua casca apresenta propriedades adstringente, inseticida, purgativa, tônica, bactericida, antiinflamatória e antidepressiva. Com o objetivo de propagar a espécie T. catigua foram desenvolvidos experimentos testando o enraizamento de estacas e a micropropagação com explantes de matrizes e sementes. Os experimentos de enraizamento de estacas foram realizados na primavera 2004, verão 2004/2005, outono, inverno e primavera 2005 e primavera 2006. Em todos os experimentos, estacas com aproximadamente 15 cm de comprimento foram coletadas de árvores adultas e preparadas da parte apical e mediana dos ramos. A seguir, foram submetidas aos reguladores vegetais IBA (ácido indolbutírico), NAA (ácido naftalenoacético) e IAA (ácido 3-indolacético), variando as dosagens. Para a avaliação dos experimentos determinou-se a percentagem de estacas enraizadas, não enraizadas e mortas e quando enraizadas, seu comprimento e diâmetro. No experimento primavera de 2004 foram testadas as concentrações de 1000 e 2000 mg L-1 dos reguladores vegetais IBA, NAA e IAA. As avaliações aos 90 dias após sua instalação revelaram maiores percentagens de enraizamento e iguais a 33,33, 25,00, 22,91 e 23,43 % para estacas submetidas a IBA 1000, 2000 mg L-1 e NAA 1000 e 2000 mg L-1, respectivamente. No verão 2004/2005, outono, inverno e primavera 2005 os experimentos foram conduzidos com as concentrações dos reguladores IBA, NAA e IAA iguais a 1000, 2000 e 3000 mg L-1 e as avaliações foram realizadas após 120 dias. Não houve enraizamento no outono e inverno. A análise conjunta dos resultados obtidos na primavera e no verão mostrou percentagem de enraizamento superior na primavera. A maior percentagem de enraizamento, igual a 19,17%...
The Trichilia catigua A. Juss from the Meliaceae family is popularly known as catigua, cataguá, argelim-rose and mangalto-catingam. Its bark has astringent, insecticide, purgativa, tonic, bactericide, anti-inflammatory and antidepressant properties. With the aim of propagate T. catigua, experiments of rooting with stem cuttings and of micropropagation with explants of trees and seeds were carried out. In all the rooting experiments the stem cuttings with approximately 15 cm of length were collected from adult trees and prepared from the apical and intermediate parts. The cuttings were immersed in the vegetable regulators IBA (Indole-3- butyric acid), ANA (Naphthalene acetic acid) and IAA (Indole-3 acetic acid). The rooted stem cutting and not rooted stem cutting percentage and, when rooted, the length and diameter of roots, were evaluated. In the experiment spring 2004 the concentrations of 1000 and 2000 mg L-1 of IBA, ANA and IAA were tested, with evaluations 90 days after installation. The highest rooting percentage were 33,33, 25,00, 22,91 and 23,43% for IBA 1000, 2000 mg L-1 and ANA 1000 and 2000 mg L-1, respectively. In the summer of 2004/2005, autumn, winter and spring of 2005 IBA, ANA and AIA, with concentration of 1000, 2000 and 3000 mg L-1, were tested. The evaluation was carried out at 120 days. No rooting was observed in autumn and winter. The analysis of data from summer and spring showed higher rooting percentage in spring. The highest rooting percentage was obtained with IBA 3000 mg L-1 (19,17%). In the spring 2006 IBA (1000, 2000, 3000, 4000 and 5000 mg L-1) and ANA (1000, 2000, 3000 mg L-1) were tested. The highest rooting percentage (41,67%) was obtained with IBA 5000 mg L-1. In the in vitro cultivation, explantes obtained from trees were submitted to asepsis treatments with HgCl2, CaOCl2 and NaOCl and inoculated in Murashige & Skoog culture medium (MS) with 25%... (Complete abstract click electronic access below)

O E. benthamii apresenta grande aptidão de cultivo no Brasil, principalmente em regiões de clima frio e com geadas frequentes, isso se deve a sua origem a oeste de Sydney na Austrália, onde as temperaturas são muito baixas, com ocorrência de geadas. Levando-se em conta o seu ótimo desempenho silvicultural, genótipos selecionados certamente representam uma excelente alternativa para plantios futuros. A clonagem de genótipos superiores é realizada através da propagação vegetativa de árvores adultas, e requer material fisiologicamente juvenil ou rejuvenescido. Técnicas especiais são necessárias para reverter árvores adultas a juvenilidade e resgatar condições favoráveis para promover o enraizamento e crescimento. Dentre as técnicas de propagação utilizadas para o resgate de plantas adultas destacam-se o corte raso (decepa), anelamento, estaquia, miniestaquia, enxertia e micropropagação. No caso específico dos eucaliptos para obtenção de brotações, o método de resgate mais utilizado pelas empresas florestais é o corte raso da árvore ou anelamento, técnicas que proporcionam uma alta taxa de enraizamento de estacas, devido à eficiente reversão a juvenilidade. Porém, quando existe a necessidade de preservação do indivíduo, justifica-se o uso de técnicas não destrutivas, evitando dessa forma a morte da árvore matriz. O Presente trabalho objetivou após a seleção in loco de fenótipos com características silviculturais superiores, resgatar (através de técnicas não destrutivas), conservar e multiplicar matrizes adultas de E. benthamii, avaliando-se qual(is) técnica(s) apresenta(m) melhor(es) resultado(s) para a clonagem das mesmas. Para tanto, utilizou-se brotações dos primeiros galhos da copa, brotações epicórmicas (megaestaca), brotações que surgiram provenientes do anelamento e brotações oriundas da poda dos galhos da copa, as quais foram submetidas às técnicas de estaquia, enxertia e micropropagação para cada tipo de broto. Apesar de ter ocorrido variação da porcentagem entre os clones, a megaestaca mostrou ser a melhor alternativa para o estabelecimento in vitro de plantas adultas de E. benthamii, sendo recomendada sua utilização para o resgate de material genético adulto, estabelecido em condições de campo. Todas as matrizes de E. benthamii foram resgatadas com sucesso, e no intuito de formar um banco de germoplasma, um minijardim clonal foi instalado com as mudas que apresentaram melhor qualidade, que poderá ser utilizado para futuros testes e formação de mudas.
The E. benthamii presents a great aptitude for cultivation in Brazil, especially in cold climates and with frequent frosts, this is due to your west of Sydney origin in Australia, where temperatures are very low, with frosts. Taking into account their optimal silvicultural performance, selected genotypes will certainly represent an excellent alternative for future plantings. The cloning of superior genotypes is accomplished by vegetative propagation of mature trees, and requires physiologically juvenile or rejuvenated material. Special techniques are necessary to reverse the juvenile and adult trees recover favorable conditions to promote rooting and growth. Among the propagation techniques used for the rescue of mature plants stand out clear-cutting (coppicing), annealing, cutting, minicutting, grafting and micropropagation. In the specific case of eucalyptus to obtain propagules, the most rescue method used for the forestry companies are clearcutting or annealing, techniques that provide a high rate of rooting, due to efficient reversion to juvenility. However, when there is a need to protect the individual, justifies the use of non-destructive techniques, thus avoiding the death of the tree array. The present study aimed to spot after the selection of phenotypes with characteristics superior silvicultural, rescue (through non-destructive techniques) to retain and multiply matrices adult the E. benthamii, evaluating which technique that present the best result for the cloning of the same. So we used to shoot the first cup of twigs, shoots, epicormic shoots that emerged from the annealing and shoots arising from the pruning of the branches of the crown, which were submitted to the techniques of cutting, grafting and micropropagation for each type of bud. Considering the percentage variation between the clones, the epicormic shoots showed to be the best alternative for the in vitro establishment to the E. benthamii adult plants, being recomended its utilization to the adult genetic material rescue, All the tested E. benthamii plants were rescued with sucess, and in the aim to stablish germoplasm bank, a clonal mini garden was instaled with the plantlets that present the best quality.

Microbouturage à partir de vitrosemis : multiplication par fragmentation-élongation. Multiplication par hyper-ramification de bourgeons adventifs induits par caulogénèse sur des vitroplants. Variabilité clonale au cours des différentes phases de la multiplication par bourgeons adventifs

O cultivo in vitro de plantas possibilita o controle de fatores ambientais e nutricionais, facilitando o estudo da interação planta-bactéria e a interpretação de eventuais alterações morfofisiológicas nas microplantas, decorrentes dessa interação. Entretanto, a presença de bactérias endofíticas na micropropagação é quase sempre caracterizada como contaminação microbiana prejudicial, sendo prontamente eliminada com o uso de quimioterápicos. Essa abordagem desconsidera os efeitos benéficos que bactérias endofíticas podem trazer ao desenvolvimento vegetal, aniquilando a possibilidade de se explorar esse potencial no ambiente in vitro. Em geral, devido a sua baixa culturabilidade, as comunidades bacterianas endofíticas requerem o uso de técnicas independentes de cultivo para seu estudo. Neste contexto, o presente trabalho teve como objetivo principal comprovar a presença de uma comunidade bacteriana endofítica em microplantas de abacaxizeiro (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel consideradas axênicas e os efeitos dessa comunidade na morfofisiologia vegetal após antibioticoterapia. Para tanto, técnicas de PCR-DGGE, PCR-ARISA, clonagem, sequenciamento, análises estruturais e ultraestruturais foram utilizadas. Os resultados evidenciaram que existe uma comunidade bacteriana endofítica composta por membros de Alfa, Beta, gama -proteobactérias, actinobactérias e cianobactérias, que coloniza as microplantas. Os resultados convergentes de PCR-DGGE e PCR-ARISA mostraram que essa comunidade apresentou alteração na sua estrutura e diversidade de acordo com seu nicho de colonização e com o período de cultivo da planta hospedeira, sem renovação de meio de cultura. A ação da antibioticoterapia influenciou a estrutura da comunidade bacteriana, promovendo impactos diferenciados em cada grupo bacteriano avaliado. A antibioticoterapia não promoveu a total eliminação das bactérias endofíticas. Contudo, os tratamentos ocasionaram alterações na comunidade bacteriana, resultando na redução do crescimento de raízes e outras alterações histológicas no sistema radicular das microplantas. As análises ultraestruturais comprovaram a presença de bactérias endofíticas colonizando intracelularmente o mesofilo foliar e o córtex de raízes, mesmo após a antibioticoterapia. Dessa forma, conclui-se que em microplantas de abacaxizeiro assintomáticas existe uma comunidade bacteriana endofítica intracelular, rompendo o principal paradigma da cultura de tecidos vegetais, no qual microplantas são obrigatoriamente axênicas.
The in vitro culture of plants by the control of environmental and nutritional factors, allows the study of the plant-bacteria interaction and the interpretation of possible morphophysiological changes in the microplants, from such interaction. However, the presence of endophytic bacteria in micropropagation is often characterized as harmful microbial contamination, which is promptly eliminated by the use of chemotherapeutics. This approach does not take into account the beneficial effects that endophytic bacteria can bring to the plant development, and annihilates the possibility of exploiting this potential in the in vitro environment. In general, due to its low culturability, endophytic bacterial communities require the use of culture-independent techniques for their study. In this context, this work aimed to prove the presence of bacterial endophytes in pineapple (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel microplants considered axenics, and the effects of these in plant morphophysiology after antibiotictherapy. For this, PCR-DGGE, ARISA-PCR, cloning, sequencing, structural and ultrastructural analysis were used. The results showed that there is an endophytic bacterial community, composed of members of , , -proteobacteria, actinobacteria and cyanobacteria, which colonizes the microplants. The converging results of PCR-DGGE and ARISA-PCR showed that this community changed in its structure and diversity according to its niche of colonization and the period of cultivation of the host plant, without renewal of culture medium. The action of antibiotics influenced the bacterial community structure, promoting differential impacts on each bacterial group evaluated. Antibiotictherapy did not promote the total elimination of endophytic bacteria. However, the treatments caused changes in bacterial community, resulting in reduced growth of roots and other histological changes in the root system of the microplants. The ultrastructural analysis confirmed the presence of endophytic bacteria colonizing the intracellularly the leaf mesophyll and the cortex of roots, even after antibiotictherapy. Thus, it is concluded that in asymptomatic pineapple microplants there is an intracellular endophytic bacterial community, breaking the main paradigm of plant tissue culture, in which axenic microplants are mandatory.

Orientador: Carmen Silvia Fernandes Boaro
Banca: João Domingos Rodrigues
Banca: Giuseppina Pace P. Lima
Banca: Marcos Roberto Furlan
Banca: Antonio Natal Gonçalves
Resumo: Pertencente à família Meliaceae, a espécie Trichilia catigua A. Juss é conhecida popularmente como catigua, cataguá, argelim-rosa e mangalto-catingam. Sua casca apresenta propriedades adstringente, inseticida, purgativa, tônica, bactericida, antiinflamatória e antidepressiva. Com o objetivo de propagar a espécie T. catigua foram desenvolvidos experimentos testando o enraizamento de estacas e a micropropagação com explantes de matrizes e sementes. Os experimentos de enraizamento de estacas foram realizados na primavera 2004, verão 2004/2005, outono, inverno e primavera 2005 e primavera 2006. Em todos os experimentos, estacas com aproximadamente 15 cm de comprimento foram coletadas de árvores adultas e preparadas da parte apical e mediana dos ramos. A seguir, foram submetidas aos reguladores vegetais IBA (ácido indolbutírico), NAA (ácido naftalenoacético) e IAA (ácido 3-indolacético), variando as dosagens. Para a avaliação dos experimentos determinou-se a percentagem de estacas enraizadas, não enraizadas e mortas e quando enraizadas, seu comprimento e diâmetro. No experimento primavera de 2004 foram testadas as concentrações de 1000 e 2000 mg L-1 dos reguladores vegetais IBA, NAA e IAA. As avaliações aos 90 dias após sua instalação revelaram maiores percentagens de enraizamento e iguais a 33,33, 25,00, 22,91 e 23,43 % para estacas submetidas a IBA 1000, 2000 mg L-1 e NAA 1000 e 2000 mg L-1, respectivamente. No verão 2004/2005, outono, inverno e primavera 2005 os experimentos foram conduzidos com as concentrações dos reguladores IBA, NAA e IAA iguais a 1000, 2000 e 3000 mg L-1 e as avaliações foram realizadas após 120 dias. Não houve enraizamento no outono e inverno. A análise conjunta dos resultados obtidos na primavera e no verão mostrou percentagem de enraizamento superior na primavera. A maior percentagem de enraizamento, igual a 19,17%... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The Trichilia catigua A. Juss from the Meliaceae family is popularly known as catigua, cataguá, argelim-rose and mangalto-catingam. Its bark has astringent, insecticide, purgativa, tonic, bactericide, anti-inflammatory and antidepressant properties. With the aim of propagate T. catigua, experiments of rooting with stem cuttings and of micropropagation with explants of trees and seeds were carried out. In all the rooting experiments the stem cuttings with approximately 15 cm of length were collected from adult trees and prepared from the apical and intermediate parts. The cuttings were immersed in the vegetable regulators IBA (Indole-3- butyric acid), ANA (Naphthalene acetic acid) and IAA (Indole-3 acetic acid). The rooted stem cutting and not rooted stem cutting percentage and, when rooted, the length and diameter of roots, were evaluated. In the experiment spring 2004 the concentrations of 1000 and 2000 mg L-1 of IBA, ANA and IAA were tested, with evaluations 90 days after installation. The highest rooting percentage were 33,33, 25,00, 22,91 and 23,43% for IBA 1000, 2000 mg L-1 and ANA 1000 and 2000 mg L-1, respectively. In the summer of 2004/2005, autumn, winter and spring of 2005 IBA, ANA and AIA, with concentration of 1000, 2000 and 3000 mg L-1, were tested. The evaluation was carried out at 120 days. No rooting was observed in autumn and winter. The analysis of data from summer and spring showed higher rooting percentage in spring. The highest rooting percentage was obtained with IBA 3000 mg L-1 (19,17%). In the spring 2006 IBA (1000, 2000, 3000, 4000 and 5000 mg L-1) and ANA (1000, 2000, 3000 mg L-1) were tested. The highest rooting percentage (41,67%) was obtained with IBA 5000 mg L-1. In the in vitro cultivation, explantes obtained from trees were submitted to asepsis treatments with HgCl2, CaOCl2 and NaOCl and inoculated in Murashige & Skoog culture medium (MS) with 25%... (Complete abstract click electronic access below)
Doutor

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticultural Sciences in the Faculty of Applied Sciences at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY Supervisor: Dr L Kambizi Co-supervisor: Dr NP Makunga Cape Town December 2013
Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a member of the Rutaceae family, and indigenous to the fynbos botanical biome of the Western Cape of South Africa. It is commonly known as buchu. Extracts as well as powdered leaves have traditionally been used for the treatment of various ailments. The increase in the international demand for A. betulina for health as well as food and beverage benefits, have raised concerns over exploitation of wild populations and the lack of horticultural information necessitates this study to evaluate the propagation of this economical important species. The main objective of this study was to establish a simple and highly productive micropropagation protocol for A. betulina through experimenting with nodal explants. Testing of the effect of various treatments (physical scarification, chemical scarification, GA, stratification, smoke and combinations thereof) on the in vitro germination of A. betulina seeds was done to elucidate the factors which control seed germination. The study revealed that the physical scarification and smoke-induced germination had a significant effect on germination percentages. In terms of germination rate, the radical generally started to appear after approximately 10 days in the physical scarification with smoke treatment. Initial decontamination methods with the exposure of various concentrations of NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather than fungal or bacterial contamination. Interestingly, contamination rates of explants were influenced by the stage of maturity of the explant material. This plant material was used to test different strengths of regeneration media, to ensure that the explants receive ample nutrients. Results made exhibited that ½ MS was the best strength for growing A. betulina nodal explants. Compared comparison between in vitro derived explants and ex vitro collected explants showed that the ex vitro derived explants had significant results, but the explants lost vigour soon after the initial exponential growth leading to the explants dying off. Furthermore, ex vitro decontaminated plant material was not economically viable to continue with. Seedlings derived from germinated seeds appeared to be the preferred method of propagation as this spent the least time in culture and produced a stable plant with an established root system, which is essential during the hardening off process after in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was applied to ½ MS media. For both shoot length and multiple shoot production, a combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant results. Interestingly, a higher phytohormone concentration of NAA is necessary to develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it resulted in multiple adventitious roots, but fewer calli was observed in this treatment. Micropropagation becomes valuable as little attention between subcultures is needed; making it less labour intensive compared to conventional nursery propagation systems where weeding watering and spraying of plants are labour intensive. In the traditional world of medicine, more so in Southern Africa, extracts are prepared by adding boiling water to the plant material; however commercial ethanol is used as an extractant. Establishment of the essential oil quality of the in vitro cultures post exposure to various treatments was done. Analysis of essential oils from A. betulina resulted in the identification of twenty one compounds. The results showed qualitative as well as quantitative differences amongst the samples used in the study. The highest relative concentration of limonene was observed in the callus of nodal explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this treatment making it ideal for limonene production. This suggests that liquid culture with the same treatment may produce more calli making it ideal for the production of limonene.

A cultura de tecidos tem sido uma importante ferramenta amplamente utilizada para diversas finalidades, dentre elas a micropropagação tem merecido destaque devido à rápida produção de mudas saudáveis num espaço físico reduzido. Dentro deste contexto, o termo, plantas axênicas, tem sido difundido como um ideal a ser atingido nesta técnica, havendo a busca cada vez maior de métodos que eliminem de forma eficaz toda e qualquer contaminação que possa, eventualmente, crescer no meio de cultura. No entanto, cada vez mais se observa que a concepção de contaminação dentro da cultura de tecidos deve ser revista, assim como a de planta axênica, pois inúmeros trabalhos têm mostrado, que mesmo em microplantas assintomáticas consideradas axênicas, existe uma comunidade endofítica onipresente e que na sua grande maioria desempenha papel fundamental no estabelecimento, desenvolvimento e aclimatização das culturas, sendo, portanto, microrganismos benéficos que devem ser conservados no cultivo in vitro. Dessa forma, o presente trabalho teve como principal objetivo investigar a microbiota endofítica presente em diversas espécies de microplantas assintomáticas consideradas axênicas, independente de seu protocolo de cultivo in vitro, local, método de micropropagação e manipulação. Para tal, foram utilizadas microplantas assintomáticas, em fase de multiplicação por mais de um ano, provenientes de três laboratórios diferentes, submetidas à caracterização por meio de testes histoquímicos, à análises ultraestruturais, a isolamento em diferentes meios de cultura por dois métodos (trituração e fragmentação), à análises moleculares utilizando a extração do DNA genômico total e PCR-DGGE, além da extração e sequenciamento do DNA dos isolados obtidos. Os resultados obtidos pelo isolamento, testes moleculares e análises ultraestruturais, evidenciaram a presença de uma comunidade endofítica, colonizando os tecidos de diferentes espécies de microplantas, provenientes de três laboratórios distintos. Dessa forma, conclui-se que a inexistência de plantas axênicas deve ser considerada, futuramente, como um aspecto positivo dentro da cultura de tecidos, uma vez que, essa comunidade endofítica pode atribuir características de interesse agronômico às diferentes espécies vegetais.
Tissue culture has been an important tool widely used for various purposes, among them the micropropagation has been highlighted due to the rapid production of healthy seedlings in a small space. Within this context, the term axenic plant has been distributed as an ideal to be achieved in this technique, which the search based on numerous methods that effectively eliminate any contamination that may eventually grow into the culture medium. However, it is seen that the conception of contamination in tissue culture should be reviewed as well as axenic plants, because several studies have shown that even in asymptomatic microplants considered axenic, there is an ubiquitous and that the endophytic community mostly plays a fundamental role in the establishment, development and acclimatization of cultures, therefore, beneficial microorganisms that should be preserved in vitro. Thus, this study aimed to investigate the endophytic microbiota present in several species of micro asymptomatic considered \"axenic\" regardless of their protocol for in vitro culture, location, method of micropropagation and manipulation. For this purpose, were used asymptomatic microplants in multiplication stage by more than one year, from three different laboratories, and were submitted to characterization by histochemistry tests, ultrastructural analysis, isolation in different culture medium by two methods (grinding and fragmentation), molecular analyses using the extraction of total genomic DNA and PCR-DGGE, in addition to extracting and sequencing the DNA of the isolates obtained. The results obtained by the isolation, molecular and ultrastructural analysis, showed the presence of an endophytic community colonizing the tissues of different species of microplants, from three different laboratories. Thus, it was concluded that the inexistence of axenic plants should be considered in future as a positive aspect in the tissue culture, since this endophytic community can give agronomical traits to different crop species.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A mangabeira (Hancornia speciosa Gomes) se destaca entre as árvores frutíferas nativas do cerrado como uma das mais promissoras para programas de exploração sustentável. Apesar do potencial econômico, a falta de informações sobre esta cultura vem restringindo seu cultivo comercial. A propagação sexuada desta espécie é dificultada, pois suas sementes têm curta longevidade e a redução do teor de água prejudica sua viabilidade e vigor. A técnica de micropropagação possibilita a multiplicação em massa de mudas com características geneticamente superiores, uniformes, em espaço físico reduzido e curto período de tempo. Entretanto, ela elimina os microrganismos associados ao tecido vegetal, incluindo mutualistas como os fungos micorrízicos arbusculares (FMA), além dos promotores de crescimento vegetal, que podem melhorar o desempenho da planta sob condições de estresse, além de aumentar o rendimento. A inoculação destes microrganismos durante a micropropagação tem sido recomendada para reduzir o tempo de formação das mudas e de aclimatização, aumentar a tolerância a estresses bióticos e abióticos, resistência a patógenos e porcentagem de sobrevivência das mudas após o transplante. Objetivou-se com este trabalho a aclimatização de plantas micropropagadas de mangabeira com diferentes substratos e câmara úmida, inoculadas com fungos promotores do crescimento vegetal (FPCV) in vitro e ex vitro, associados com o FMA Glomus clarum, em casa de vegetação, para maximizar a produção de mudas a serem utilizadas em programas de formação de pomares e reflorestamento. Os experimentos foram conduzidos na UNESP - Universidade Estadual Paulista, Campus de Ilha Solteira e Instituto Federal Goiano – Campus Rio Verde (IF Goiano – Campus Rio Verde). O material vegetal utilizado na propagação in vitro foi retirado de frutos de diferentes plantas de mangabeira coletados na Fazenda de Ensino, Pesquisa e Extensão da UNESP Campus Ilha Solteira e na Fazenda Gameleira, município de Montes Claros de Goiás - GO. Todos os experimentos foram realizados e conduzidos por 120 dias. A associação do saco plástico como câmara úmida com o substrato Bioplant®, seguido da pulverização de FPCV e inoculação do G. clarum é o método mais adequado para aclimatização de plantas micropropagadas de mangabeira, em casa de vegetação.
Mangabeira (Hancornia speciosa Gomes) stands out among the native fruit trees of the Cerrado as one of the most promising for sustainable exploitation programs. Despite the economic potential, lack of information about this culture is limiting its commercial cultivation. The sexual propagation of this species is difficult as its seeds have a short longevity, and reduced water content impairs their viability and vigor. The micropropagation technique enables the mass multiplication of plants with genetically superior features, uniform, in a reduced physical space and a short length of time. However, it eliminates the microorganisms associated with plant tissue, including mutualistcs, as mycorrhizal fungi (AMF), in addition to plant growth promoters, which can improve plant performance under stress conditions, and increase the yield. Inoculation of these microorganisms during micropropagation has been recommended to reduce the time of seedlings formation and acclimatization, and to increase tolerance to biotic and abiotic stresses, resistance to pathogens and percentage of seedling survival after transplantation. This study aimed the acclimatization of micropropagated plants with different substrates and moister chamber, inoculated with plant growth-promoting fungi (PGPF) vitro and ex vitro, associated with AMF Glomus clarum, in a greenhouse, to maximize seedlings production to be used in the mangabeira and reforestation programs. Experiments were conducted at UNESP- Universidade Estadual Paulista, Ilha Solteira Campus, and Federal Institute Goiano - Rio Verde Campus (IF Goiano - Campus Rio Verde). The plant material used in the in vitro propagation was removed from fruits of different mangabeira plants collected in Teaching, Research and Extension Farm of the UNESP Ilha Solteira Campus and Gameleira Farm, Montes Claros de Goiás-GO. All experiments were performed and conducted for 120 days. The association of plastic bag as a moist chamber with Bioplant® substrate, followed by spray PGPF and inoculation of G. clarum is the most appropriate method for acclimatization of micropropagated plants mangabeira in the greenhouse. The association of plastic bag as moist chamber with Bioplant® substrate, followed by spray PGPF and inoculation of G. clarum is the most appropriate method for acclimatization of micropropagated of mangabeira plants in the greenhouse.
FAPESP: 2012/14489-9

Orientador: Ana Maria Rodrigues Cassiolato
Resumo: A mangabeira (Hancornia speciosa Gomes) se destaca entre as árvores frutíferas nativas do cerrado como uma das mais promissoras para programas de exploração sustentável. Apesar do potencial econômico, a falta de informações sobre esta cultura vem restringindo seu cultivo comercial. A propagação sexuada desta espécie é dificultada, pois suas sementes têm curta longevidade e a redução do teor de água prejudica sua viabilidade e vigor. A técnica de micropropagação possibilita a multiplicação em massa de mudas com características geneticamente superiores, uniformes, em espaço físico reduzido e curto período de tempo. Entretanto, ela elimina os microrganismos associados ao tecido vegetal, incluindo mutualistas como os fungos micorrízicos arbusculares (FMA), além dos promotores de crescimento vegetal, que podem melhorar o desempenho da planta sob condições de estresse, além de aumentar o rendimento. A inoculação destes microrganismos durante a micropropagação tem sido recomendada para reduzir o tempo de formação das mudas e de aclimatização, aumentar a tolerância a estresses bióticos e abióticos, resistência a patógenos e porcentagem de sobrevivência das mudas após o transplante. Objetivou-se com este trabalho a aclimatização de plantas micropropagadas de mangabeira com diferentes substratos e câmara úmida, inoculadas com fungos promotores do crescimento vegetal (FPCV) in vitro e ex vitro, associados com o FMA Glomus clarum, em casa de vegetação, para maximizar a produção de mudas ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Mangabeira (Hancornia speciosa Gomes) stands out among the native fruit trees of the Cerrado as one of the most promising for sustainable exploitation programs. Despite the economic potential, lack of information about this culture is limiting its commercial cultivation. The sexual propagation of this species is difficult as its seeds have a short longevity, and reduced water content impairs their viability and vigor. The micropropagation technique enables the mass multiplication of plants with genetically superior features, uniform, in a reduced physical space and a short length of time. However, it eliminates the microorganisms associated with plant tissue, including mutualistcs, as mycorrhizal fungi (AMF), in addition to plant growth promoters, which can improve plant performance under stress conditions, and increase the yield. Inoculation of these microorganisms during micropropagation has been recommended to reduce the time of seedlings formation and acclimatization, and to increase tolerance to biotic and abiotic stresses, resistance to pathogens and percentage of seedling survival after transplantation. This study aimed the acclimatization of micropropagated plants with different substrates and moister chamber, inoculated with plant growth-promoting fungi (PGPF) vitro and ex vitro, associated with AMF Glomus clarum, in a greenhouse, to maximize seedlings production to be used in the mangabeira and reforestation programs. Experiments were conducted at UNESP- Universidad... (Complete abstract click electronic access below)
Doutor

Au cours du travail présenté dans ce mémoire, nous sommes parvenus à obtenir, de façon systématique, la micropropagation in vitro et l'acclimatation ex vitro non seulement des poiriers juvéniles issus de pépins de "passe crassane", mais aussi du cognassier de Provence et de deux cultivars de poirier, "williams" et "passe crassane". Les conditions de culture concernant les différentes phases de la micropropagation et l'acclimatation ont été étudiées. Plusieurs problèmes d'ordre physiologique (dormance des bourgeons in vitro, anomalie des pousses formées, nécrose des apex) ont pu être surmontés ou limités.

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This work focuses the familiar cajuculture at Serra of Mel (RN) that presents a geographic and climatic structure favourable to the development of the cajuculture; the interaction between the ambient factors and agriculture in the region provides a propitious environment to the culture of the cashew; the objective of present work was associate ambient, economic, social and cultural factors of this municipy with the possibility of a sustainable agriculture associated with the vegetal biotechnology
Este trabalho enfoca a cajucultura familiar no munic?pio de Serra do Mel (RN), o qual apresenta uma estrutura geogr?fica e clim?tica favor?vel ? cajucultura; a intera??o entre os fatores ambientais e a agricultura na regi?o proporciona um ambiente prop?cio ao cultivo do cajueiro; buscou-se com o presente trabalho associar os fatores ambientais, econ?micos, sociais e culturais do munic?pio com a possibilidade de desenvolvimento de uma agricultura sustent?vel integrada ? biotecnologia vegetal

O experimento foi conduzido com o objetivo de avaliar a influência da interação entre o nitrato de amônio e nitrato de potássio sobre as respostas morfogênicas de embriões somáticos de pupunheiras in vitro a fim de otimizar o protocolo de micropropagação da espécie. As concentrações utilizadas foram 0, 825, 1650, 2475 e 3300 mg L-1 de nitrato e amônio e 0, 950 , 1900, 2850 e 3800 mg L-1 de nitrato de potássio, combinadas entre si, perfazendo um total de 25 tratamentos. Os tratamentos foram preparados a partir da solução completa de Murashige e Skoog, devidamente modificado para as proporções desses íons no suprimento de nitrogênio. Utilizaram-se duzentos e cinqüenta embriões somáticos com características morfológicas homogêneas, isentos de raízes e folhas, obtidos a partir de microplantas mantidas em sala de crescimento com temperatura e luminosidade controladas. Foram aferidos dados do comprimento da parte aérea e da raiz, o número de raízes, brotações e folhas, a porcentagem de ramificação da raiz, a porcentagem de raízes finas, medianas e grossas, em quatro períodos de cultura, a cada 60 dias, totalizando 240 dias de cultivo. Ao final desse período, avaliou-se também o teor de proteínas totais solúveis, o teor de clorofila pelo índice SPAD e os teores de macro e micronutrientes nas microplantas. Utilizou-se delineamento estatístico inteiramente casualizado e os dados foram submetidos ao teste de Bartlett a 5% e análise de variância (ANOVA) a 1% e 5% de probabilidade de erro ou foi elaborada uma matriz de correlação de Pearson a 1% e 5% de probabilidade de erro, conforme o caso. Os resultados permitiram inferir que aos 240 dias de cultivo as microplantas passam a investir mais em formação de parte aérea e aumentam a porcentagem de raízes finas, e funcionais. Os tratamentos que melhor favoreceram a formação de proteínas foram aqueles com concentração de 2475 mg L-1 de NH4NO3 ou com 2850 mg L-1 de KNO3. Os maiores índices SPAD ocorreram nos tratamentos com até 1650 mg L-1 de NH4NO3 combinado com até 1900 mg L-1 de KNO3. Diferentes combinações dos sais de NH4NO3 e KNO3 podem favorecer a absorção de cada nutriente. Conclui-se que os resultados obtidos no trabalho podem contribuir com a melhoria do protocolo de micropropagação de B. gasipaes, na medida em que permitem estabelecer o melhor tratamento para maximização de uma resposta específica desejada para cada momento do processo de cultivo dos embriões somáticos de pupunheiras, como enraizamento, crescimento da parte aérea, formação de proteínas e formação de brotações, facilitando dessa forma a otimização da produção de microplantas com características desejáveis e, conseqüentemente, sua aclimatização e desenvolvimento ex vitro.
This study aimed to evaluate the influence of the interaction between ammonium nitrate and potassium nitrate on the morphogenetic responses of peach palm somatic embryos in vitro cultivated, to optimize the micropropagation protocol for this species. The concentrations used were 0, 825, 1650, 2475 and 3300 mg L-1 of ammonium nitrate and 0, 950 , 1900, 2850 and 3800 mg L-1 of potassium nitrate, in all possible associations, totalizing 25 treatments. The treatments were prepared with the complete solution of Murashige and Skoog, with modified proportions of these ions in relation to the nitrogen supply. Two hundred and fifty somatic embryos with homogeneous morphological characteristics, without roots and leaves, obtained from microplants from a controlled temperature and luminosity room, were used in the experiment. Every 60 days, during 240 days, the length of shoot and root, the number of roots, propagules and leaves and the root architecture were measured. At the end of 240 days of cultivation, it was also analyzed the total soluble proteins, the foliar chlorophyll by a chlorophyll meter equipment (SPAD-502) and the macronutrients and micronutrients concentrations in the microplants. The experiment was conducted in a randomized design. The data were subjected to Bartlett´s test at 5% and variance analysis (ANOVA) at 1% and 5% of probability of error, or it was made a correlation matrix at 1% and 5% of probability of error, according to each analysis. The results showed that at 240 days of cultivation the microplants spent more energy building the shoot part and raised the percentage of thin, functional root. The best treatments for proteins formation were those with 2475 mg L-1 of NH4NO3 or with 2850 mg L-1 of KNO3. The highest SPAD index occurred in the treatments with at most 1650 mg L-1 of NH4NO3 associated with at most 1900 mg L-1 of KNO3. Different associations of NH4NO3 e KNO3 may favor the absorption of each nutrient. We conclude that the results obtained may contribute to the optimization of the B. gasipaes micropropagation protocol, as it is possible to establish the best treatment for maximization of a specific answer for each moment of the somatic embryos cultivations process, such as rooting, shoot growth, propagules and protein formation, and thus increase the optimization of microplants production with wanted characteristics and hence its acclimatization and ex vitro development.