Littérature scientifique sur le sujet « Hairi root cultures »

Agastache rugosa (popularly known as Korean mint) belongs to the Lamiaceae family and comprises 22 species of perennial aromatic medicinal species native to East Asian countries, such as Korea, Taiwan, Japan, and China. A. rugosa contains many phenolic compounds that exhibit pharmacological and physiological activities, including antioxidant, anticancer, antiviral, antifungal, and antibacterial activities. The highest concentrations of rosmarinic acid and its isomers have been reported in the roots of A. rugosa. In this in vitro study, hairy roots of A. rugosa were obtained and the carbohydrates (sorbitol, mannitol, glucose, maltose, galactose, mannose, and sucrose) were evaluated to determine those that were optimal for rosmarinic acid production and hairy root growth. Antioxidant and antibacterial activities of extracts of A. rugosa were also assessed. The best carbon source for A. rugosa hairy root cultures was sucrose, considering biomass productivity (0.460 ± 0.034 mg/30 mL), rosmarinic acid production (7.656 ± 0.407 mg/g dry weight), and total phenolic content (12.714 ± 0.202 mg/g gallic acid equivalent). Antioxidant and antimicrobial activities were displayed by A. rugosa hairy roots cultured in liquid medium supplemented with 100 mM sucrose. Twenty-five bacterial strains, including multidrug-resistant bacteria and one pathogenic yeast strain, were used for antimicrobial screening of A. rugosa hairy roots. The hairy root extracts displayed antibacterial activity against Micrococcus luteus (KCTC 3063) and Bacillus cereus (KCTC 3624). The inhibition of these bacteria was greater using A. rugosa hairy roots with the highest levels of phenolic compounds cultured in the presence of sucrose, compared to hairy roots with the lowest levels of phenolic compounds cultured in the presence of fructose. Considering hairy root biomass, phenolic compound production, and antibacterial activity, sucrose is the best carbon source for A. rugosa hairy root cultures.

A method named “bag culture” was developed for coculturing of Linum persicum (section Syllinum) and L. austriacum (section Linum) hairy roots. For this propose L. austriacum and L. persicum hairy root cultures were established using Agrobacterium rhizogenes in McCown medium. L. persicum hairy roots in bags (1 mm2 mesh) were successfully grown together with L. austriacum hairy roots. The amounts of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (MPTOX) produced by L. persicum hairy root cultures were detected using HPLC. The results indicated that the amounts of both lignans and growth indexes of the two hairy roots decreased, that may be partly due to a competition between the two types of culture in using precursors of biosynthetic metabolites and the amount of culture medium which is available for each hairy root. However, MPTOX (0.17 g/100 g DW) and PTOX (0.02 g/100 g DW) levels of the L. persicum single culture in bag were significantly higher than of the other cultures which may be due to the immobilization effect of the bag.

Bituminaria bituminosa (L.) C.H. Stirt is a perennial species widely distributed in the Mediterranean basin and the Canary Islands. This species is used in folk medicine and currently has considerable pharmaceutical interest for its content in phenylpropanoids, furanocoumarins and pterocarpans. In vitro cultures (shoots and hairy roots) have been performed to obtain plant material useful for the production of these metabolites. Hairy root cultures were successfully established after inoculation of hypocotyls with the LBA 9402 A. rhizogenes strain. The HRPB3 line was selected for further analysis and elicited with chitosan and salicylic acid. All the HRPB3 cultures showed higher polyphenol content and greater DPPH-antioxidant activity than shoots cultured in vitro. The presence of isoflavone daidzein was detected in the hairy root extracts. The cytotoxic effect of HR extracts has been further tested on HeLa cells: the salicylic acid elicited HR exhibited good antiproliferative effects.

Abstract Peganum harmala, Hairy Root Cultures, β-Carboline Alkaloids Hairy root cultures of Peganum harmala were established by genetic transformation with Agrobacterium rhizogenes strain A4. In contrast to suspension cultures the root cultures contained high levels of β-carboline alka­ loids (1 -1.5% of dry mass).

The aim of this study was to elucidate the effect of elicitors and precursors on the production of forskolin from the hairy root cultures of Coleus forskohlii Briq. Hairy root cultures were established from leaf explants by infecting with Agrobacterium rhizogenes strain A4 on MS basal medium. Suspension cultures of hairy root cultures were initiated in MS medium containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L). We investigated the growth of biomass and forskolin production in suspension cultures of hairy roots. The production of forskolin was parallel to the growth of biomass. The maximum production of forskolin was observed after 5 weeks. With the objective to increase the yield of forskolin, abiotic elicitors such as salicylic acid (100 μM and 500 μM), copper sulphate (100 μM and 500 μM), methyl jasmonate (100 μM and 500 μM) and precursors such as α-ketoglutaric acid (0.2 mM and 1.0 mM), L-phenylalanine (0.2 mM and 1.0 mM) were added to hairy root cultures on different days of incubation period and evaluated their effects on production of forskolin. Elicitor, methyl jasmonate (500 μM) and the precursor, L-phenylalanine (1 mM) on day-14 addition significantly enhanced the production of forskolin over the control hairy root cultures C. forskohlii. Given forskolin’s limited commercial supply, this study provides avenues for improving the production of forskolin in the hairy root culture of C. forskohlii.

Background: Recent years have brought the dynamic development in studies of naphthoquinones obtained from plants, in vitro cultures and semi- or total synthesis. This review presents the hairy root cultures approach for producing naphthoquinones and summarizes their most recent anti-cancer investigations. <p> Objective: This review aimed to define biotechnological strategies impacted on naphthoquinones production in hairy root cultures. Up to now the major source of shikonin/alkannin derivatives, rhinacanthins and ramentaceone is isolation from plant material, also derived via biotechnological methods. Moreover, the most recent anti-cancer activity studies on naphthoquinones which could be produced in hairy root cultures were outlined. <p> Methods: For databases survey two selection criteria were used: (i) naphthoquinone could be produced in hairy roots, and (ii) it exhibits anti-cancer properties. <p> Results: Ninety two papers were included in the review, thirty described biotechnological approaches enhancing naphthoquinones production, among them twenty seven were dedicated to hairy root cultures. Forty papers outlined the anti-cancer activity of targeted naphthoquinones including the type of cancer and bioassays description. The synergistic effect of natural naphthoquinones and other anti-cancer therapies was reviewed and toxicity of natural naphthoquinones and plant extracts was discussed. The review highlights tendencies in hairy root investigations and indicates the possible future research directions for improving biotechnological production efficacy. <p> Conclusion: This review demonstrates a great potential of hairy root cultures for naphthoquinones production, which could be furtherly developed for future medical purposes, especially as anti-cancer agents. This area of plant biotechnology will be surely still developed with traditional and new strategies.

Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots

Transformed root cultures of Catalpa ovata were established following shoots infection with four agropine strains of Agrobacterium rhizogenes. Frequency of root formation was dependent on the bacterial strain and the presence of acetosyringone in the incubation medium. It is the first report concerning the possibility of transforming Catalpa ovata by A. rhizogenes. Both transformed and untransformed root cultures of C. ovata were studied for their growth and phenylethanoid glycoside production. As with the roots of intact plants, cis-and trans-verbascoside as well as martynoside were produced in transformed and untransformed root cultures of C. ovata. In hairy roots, total (cis + trans) verbascoside production could be stimulated up to three-fold of that of roots of 6-month-old plants grown in a greenhouse, by using an appropriate root line cultured in liquid 1/2 B5 Gamborg medium containing indole-3-butyric acid (0.1 mg/1) in the dark but not light conditions. Transformed and untransformed root cultures of C. ovata were also found to have 10 times higher martynoside production than roots of intact plants.

Here we present previously unreported glucosinolate production by hairy root cultures of broccoli (B. oleracea var. italica). Growth media greatly influenced the growth and glucosinolate content of hairy root cultures of broccoli. Seven glucosinolates, glucoraphanin, gluconapin, glucoerucin, glucobrassicin, 4- methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin, were identified by analysis of the broccoli hairy root cultures. Both half and full strength B5 and SH media enabled the highest accumulation of glucosinolates. In most cases, the levels of glucosinolates were higher in SH and BS media. Among the 7 glucosinolates, the accumulation of neoglucobrassicin was very high, irrespective of growth medium. The neoglucobrassicin content was 7.4-fold higher in SH medium than 1/2 MS, in which its level was the lowest. The 1/2 B5 medium supported the production of the highest amounts of glucobrassicin and 4- methoxyglucobrassicin, the levels for which were 36.2- and 7.9- fold higher, respectively, than their lowest content in 1/2 MS medium. The 1/2 SH medium enabled the highest accumulation of glucoraphanin and gluconapin in the broccoli hairy root cultures, whose levels were 1.8- and 4.6-fold higher, respectively, than their lowest content in 1/2 MS medium. Our results suggest that hairy root cultures of broccoli could be a valuable alternative approach for the production of glucosinolate compounds.

2013 - 2014
Hyperpigmentation is the process by which an excess of melanin is produced by the skin. Typically, hyperpigmentation occurs as a result of stress, damage or prolonged inflammation of the skin. The most common cause is sun damage, though hyperpigmentation is often a consequence of inflammation following acne, eczema, psoriasis, dermatitis etc. Hyperpigmentation may also occur in the skin due to hormonal changes in the body typically associated with pregnancy or the taking of oral contraception. Beside this medical aspects, the global skin depigmenting product market has been forecast to reach a value of $19.8 billion by 2018, driven by the growing desire for light-coloured skin among both men and women primarily from the Asian, African and Middle East regions. Although products do exist that can actually bleach the skin, these products contain dangerous or toxic ingredients (such as hydroquinone and mercury) and are banned in most countries. Blocking or reducing the accumulation of melanin in the skin can be obtained either by switching off one or more components of the pathway that go from the receptor activation to the enzymatic inhibition of melanin formation catalyzed by the tyrosinase. For this purpose, several antimelanogenic reagents have been developed and discovered nowadays. However, only a few of these inhibitors have been introduced and used due to their problems in cytotoxicity (affecting the cell growth and survival), selectivity, solubility and stability. The present project was aimed at identifying new total plant extracts exerting beneficial effects in skin care, with special emphasis on the development of novel plant-derived actives with hypopigmenting effects. Experimental activities were carried out in collaboration with Arterra Bioscience S.r.l, in the frame of the programme “Dottorato di Ricerca in Azienda”, funded by European Commission and Regione Campania (POR Campania FSE 2007-2013). Arterra is an Italian research-based Biotech company mostly involved in developing new plant-derived extracts to be used as active ingredients with cosmetic application. Hairy root cultures of three different plant species (Cichorium intybus, Brassica rapa subsp. pekinensis and Helianthus annuus) were generated. Hairy roots of Brassica rapa subsp. pekinensis were selected for further studies on the base of a preliminary screening for anti-oxidant activity of a total crude ethanol extract and a sugar/peptides mixture derived from cell walls, coupled to an active growth. Crude ethanol extract and a sugar/peptides mixture derived from cell wall of Brassica rapa subsp pekinensis hairy roots were tested in murine melanoma cells (B16-F1) and human epidermal melanocytes isolated from lightly pigmented adult skin (HEMa-LP), by using a panel of in vitro and in vivo biological assays to assess their role in modulating melanogenesis. Both extracts at different concentrations demonstrated to inhibit the cellular tyrosinase, a key enzyme in melanin production, and to reduce melanin content in murine melanoma cells. In addition, the sugar/peptides mixture of Brassica rapa susp. pekinensis hairy roots significantly inhibited the levels of cyclic adenosine monophosphate (cAMP), an important second messenger within melanogenesis signalling pathway. Furthermore, the same extract significantly decreased the expression of microphtalmia-associated transcription factor (MITF) and its promoter activity of about 30%, analyzed by in vitro reporter (luc+)-assay. Altogether these data indicates that the sugar/peptides mixture isolated from cell wall of Brassica rapa subsp. pekinensis hairy roots might exert its inhibitory effect on melanogenesis through the downregulation of MITF transcription. Furthermore, Brassica rapa subsp. pekinensis ethanol extract was able to enhance the expression levels of important genes encoding for proteins involved into extracellular matrix (ECM) assembly. Finally, a competitive industrial production hairy-root based platform was developed by Brassica rapa subsp. pekinensis hairy root biomass scaling-up and improved extraction procedures. Overall, these results, under pending patent application, will contribute to introduce product and process innovations at Arterra Bioscience s.r.l, for the identification of new and safer plant-derived melanogenesis inhibitors. In general, the developed industrial production platform will be also extended to the screening of actives from other plant species and to the release of novel plant-derived products in different segments of the cosmetic market. [edited by author]
XIII n.s.

In the present research, nicotine alkaloid production by Nicotiana tabacum (tobacco) hairy roots and tropane alkaloid production by Hyoscyamus niger hairy roots were investigated. The first objective of this research was to improve the oxygen mass transfer in hairy root cultures with microbubbles. Oxygen was shown as a critical nutrient for the growth of tobacco and H. niger hairy roots. In a 1-liter fermentor, microbubble dispersion improved the oxygen mass transfer, tobacco hairy root growth, and nicotine production in the medium. In a novel ground-joint column bioreactor, microbubbles enhanced the oxygen mass transfer and the growth of H. niger hairy roots. The second objective of this research was to enhance the release of alkaloids from the hairy roots into the culture medium. In a l-liter fermentor, nicotine concentration in medium was improved by adjusting the medium pH to 6. Unlike the nicotine alkaloid, hyoscyamine concentration in medium was not detectable at medium pH 6, whereas hyoscyamine in medium increased to 42 mg l-1 at medium pH 3. Similar to the hyoscyamine, scopolamine in medium increased from 0.1 to 11 mg l-1 when the medium pH was adjusted from 6 to 3. The release of alkaloids into culture medium provides opportunities to isolate a high-value alkaloid directly from the culture fluid, and reduces the cost of product recovery.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

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.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

In intensive agriculture, harmful soilborne biotic agents, cause severe damage. These include both typical soilborne (clinical) major pathogens which destroy plants (e.g. Fusarium and Phytophthora pathogens), and subclinical ("minor") pathogens (e.g. Olpidium and Pythium). The latter cause growth retardation and yield decline. The objectives of this study were: (1) To study the behavior of clinical (major) and subclinical (minor) pathogens in plant growth substrate, with emphasis on zoosporic fungi, such as Pythium, Olipidium and Polymyxa. (2) To study the interaction between subclinical pathogens and plants, and those aspects of Pythium biology which are relevant to these systems. (3) To adopt a holistic-integrated approach for control that includes both eradicative and protective measures, based on a knowledge of the pathogens' biology. Zoospores were demonstrated as the primary, if not the sole propagule, responsible for pathogen spread in a recirculating hydroponic cultural system, as verified with P. aphanidermatum and Phytophthora capsici. P. aphanidermatum, in contrast to Phytophthora capsici, can also spread by hyphae from plant-to-plant. Synthetic surfactants, when added to the recirculating nutrient solutions provided 100% control of root rot of peppers by these fungi without any detrimental effects on plant growth or yield. A bacterium which produced a biosurfactant was proved as efficacious as synthetic surfactants in the control of zoosporic plant pathogens in the recirculating hydroponic cultural system. The biosurfactant was identified as a rhamnolipid. Olpidium and Polymyxa are widespread and were determined as subclinical pathogens since they cause growth retardation but no plant mortality. Pythium can induce both phenomena and is an occasional subclinical pathogen. Physiological and ultrastructural studies of the interaction between Olpidium and melon plants showed that this pathogen is not destructive but affects root hairs, respiration and plant nutrition. The infected roots constitute an amplified sink competing with the shoots and eventually leading to growth retardation. Space solarization, by solar heating of the greenhouse, is effective in the sanitation of the greenhouse from residual inoculum and should be used as a component in disease management, along with other strategies.