Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of <Emphasis Type="Italic">Fritillaria imperialis</Emphasis> L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with higher level of cytokinin (1 mg l⁻¹ BAP). In contrast, auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with lower level of cytokinin (0.1 mg l⁻¹ BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.     

Responses of antioxidant defense system of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don. to paclobutrazol treatment under salinity

The effect of paclobutrazol, a plant growth regulator, on antioxidant defense system was investigated in Catharanthus roseus (L.) G. Don. plants subjected to NaCl stress. The growth parameters were significantly reduced under 80 mM NaCl treatment; however, this growth inhibition was less in paclobutrazol-treated (15 mg l⁻¹ plant⁻¹) plants. The non-enzymatic antioxidants ascorbic acid and reduced glutathione were affected under NaCl stress and they increased significantly under paclobutrazol treatment when compared to NaCl treated as well as control plants (P ≤ 0.05). The activity of antioxidant enzyme ascorbate peroxidase showed a significant enhancement under salinity stress. The catalase activity decreased in roots of NaCl-treated plants, but recovered with paclobutrazol treatment. The results suggested that paclobutrazol have significant role in contributing salt stress tolerance of C. roseus by improving the components of antioxidant defense system.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Micropropagation and induction of autotetraploid plants of <Emphasis Type="Italic">Chrysanthemum cinerariifolium</Emphasis> (Trev.) Vis

Rapid propagation technology was established and optimized in vitro for Chrysanthemum cinerariifolium (Trev.) Vis., an important botanical insecticide plant with a huge international market. A large number of buds could be induced directly from epicotyl and hypocotyl explants on Murashige T; Skoog F. J. Plant. Physiol. 15: 473–479; (1962) medium [Murashige and Skoog (MS) medium] supplemented with 0.3 mg l⁻¹ benzyladenine (BA) and 0.3 mg l⁻¹ α-naphthaleneacetic acid (NAA). Root induction and development could be observed within 15 d after inoculation on 1/2 MS medium supplemented with 0.2 mg l⁻¹ indole-3-acetic acid (IAA) and 0.1 mg l⁻¹ rooting powder (ABT). Furthermore, a polyploid breeding study in vitro was reported to obtain superior breeding lines with high yield and good quality. Autotetraploid lines of C. cinerariifolium were obtained by colchicine treatments and identified by root-tip chromosome determination and stoma observation. The chromosome number of the autotetraploid plantlet was 2N = 4x = 36. Obtained autotetraploid lines will be of important genetic and breeding value and be used for further selection and plant breeding.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

Biokinetic parameters and behavior of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during anaerobic growth

The biokinetics of glucose metabolism were evaluated in Aeromonas hydrophila during growth in an anaerobic biosystem. After approx 34 h growth, A. hydrophila metabolized 5,000 mg glucose l⁻¹ into the end-products ethanol, acetate, succinate and formate. The maximum growth rate, μ _m, half saturation coefficients, K _s, microbial yield coefficient, Y, cell mass decay rate coefficient, k _d, and substrate inhibition coefficient, K _si were 0.25 ± 0.03 h⁻¹, 118 ± 31 mg glucose l⁻¹, 0.12 μg DNA mg glucose⁻¹, 0.01 h⁻¹, and 3,108 ± 1,152 mg glucose l⁻¹, respectively. These data were used to predict the performance of a continuous growth system with an influent glucose concentration of 5,000 mg l⁻¹. Results of the analysis suggest that A. hydrophila will metabolize glucose at greater than 95% efficiency when hydraulic retention times (HRTs) exceed 7 h, whereas the culture is at risk of washing out at an HRT of 6.7 h.     

Morphactin influences guggulsterone production in callus cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis>

Guggulsterone, a hypolipidemic natural agent, is produced in resin canals of the plant Commiphora wightii. In this study, the efficacy of different plant growth regulators was evaluated for optimizing its production. Morphactin was found to be effective in enhancing the accumulation of guggulsterones in callus cultures. Maximum callus growth was recorded on medium containing morphactin (0.1 mg l⁻¹) and 2iP (2.5 mg l⁻¹), whereas maximum guggulsterone production occurred when the calluses were cultured on medium containing 0.1 mg l⁻¹ morphactin and 1.0 mg l⁻¹ 2iP. Morphactin and 2iP interacted significantly to enhance the callus growth and guggulsterone production by about 8-folds in one-year-old cultures. However, the effect of morphactin on callus growth and guggulsterone production was not uniform over the levels of 2iP tested. Such an effect of morphactin has never been reported on the production of secondary metabolites.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

In vitro flower induction in roses

Summary Vegetatively propagated plantlets of six rose cultivars were induced to flower in vitro on media containing full-strength Murashige and Skoog (MS) inorganic salts, Gamborg's B5 organic elements with 400 mg l⁻¹ myo-inositol, and different phytohormone combinations of 6-benzyladenine (BA) with α-naphthaleneacetic acid (NAA); thidiazuron (TDZ) with NAA; and zeatin (ZT) with NAA. The most efficient flower bud induction (49.1% and 44.1%) was obtained on media supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.1 mg l⁻¹ (0.54 μM) NAA or 0.5 mg l⁻¹ (2.28 μM) ZT and 0.1 mg l⁻¹ (0.54 μM) NAA for cultivar Orange Parade. Scanning electron microscopy (SEM) showed that in vitro flower bud induction occurred mostly between 15 and 30 d in induction medium through the normal flower development processes. With TDZ and ZT as the best choice for flower induction in all six cultivars tested, different rose cultivars varied in their responses to phytohormone treatments. Our study also revealed that the total time from original culture and subculture time before flower induction were two very important factors for in vitro flower induction. Plantlets 156–561 d from original culture and subcultured for 45 d were the best for flower induction. These authors contributed equally to this work.     

Biodegradation of phenol and 4-chlorophenol by the yeast <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l⁻¹ phenol alone and 350 mg l⁻¹ 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l⁻¹ could not be degraded in the presence of 350 mg l⁻¹ 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l⁻¹ supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l⁻¹ with the presence of 100–160 mg l⁻¹ phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.     

In vitro germination and development of western hemlock dwarf mistletoe

A procedure for in vitro culture of the parasitic flowering plant western hemlock dwarf mistletoe, Arceuthobium tsugense (Rosend.) G.N. Jones subsp. tsugense, is described. A factorial experiment evaluated the effects of media (Harvey's medium (HM) and modified White's medium (WM)), temperatures (15 °C and 20 °C), presence or absence of light, and plant growth regulators (the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the cytokinin 6-benzylaminopurine (BAP) at varying concentrations (0.001 mg l⁻¹ to 1 mg l⁻¹)). Seed explants germinated in less than one week in culture and produced radicles. Optimal conditions for radicle elongation were WM at 20 °C in the presence of light and without plant growth regulators. Some of the radicles split at the tip to yield callus while others swelled to become spherical holdfasts. Holdfasts were also produced at the tips of radicles, and callus arose from split holdfasts. Factors that promoted holdfast production were Harvey's medium, light, and 2,4-D at 1 mg l⁻¹. Callus development from split radicles and split holdfasts was optimal on WM with 0.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP at 20 °C in the dark. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

In vitro micropropagation of Gymnema sylvestre – A multipurpose medicinal plant

The nature of the explant, seedling age, medium type, plant growth regulators, complex extracts (casein hydrolysate, coconut milk, malt extract and yeast extract) and antioxidants (activated charcoal, ascorbic acid, citric acid and polyvinylpyrrolidone) markedly influenced in vitro propagation of Gymnema sylvestre. A maximum number of shoots (57.2) were induced from 30 day old seedling axillary node explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (1 mg l⁻¹), kinetin (0.5 mg l⁻¹), 1-napthalene acetic acid (0.1 mg l⁻¹), malt extract (100 mg l⁻¹) and citric acid (100 mg l⁻¹). High frequency of rooting was obtained in axillary node explant derived shoots (50%) on half strength MS medium supplemented with IBA (3 mg l⁻¹). The plantlets, thus developed, were hardened and successfully established in natural soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.