Multiple shoot culture and effective material production of Anoectochilus roxburghii / 中草药

Objective: To provide a new material for exploring the products of Anoectochilus roxburghii, and study the effect of explant lengths, BA concentration and light intensities on multiple shoot biomass and effective material accumulation of A. roxburghii; This paper also explored the feasibility of the bioreactor culture on the rapid proliferation of A. roxburghii. Methods: The optimum explant lengths for multiple shoot proliferation were selected, the effect of different BA concentration and light intensities on multiple shoot biomass and effective material accumulation were investigated. Additionally, the comparison was carried out between bioreactor culture and conventional solid culture for multiple shoot biomass and effective material accumulation of A. roxburghii. Results: Explants (5 mm) promoted multiple shoot proliferation, mutiple shoot biomass was significantly higher than explants (1 mm). MS medium supplemented with 2.5 mg/L BA was optimal for multiple shoot biomass accumulation but the maximum productivities of polysaccharide, flavonoid, and total phenolic were found at 2.0 mg/L BA. Multiple shoot biomass and effective material accumulation were more suitable under light condition than under dark condition. The biomass and production of polysaccharide, flavonoid, and total phenolic (678.3, 12.2, and 17.8 mg/L) of multiple shoot at 5 g/L of inoculation density were significantly better than in solid culture. Conclusion: Multiple shoot biomass and productivities of polysaccharide, flavonoid, and phenolic compound are enhanced when 5 mm explants are inoculated in MS medium supplemented with 2.0 mg/L BA and cultured under light condition. In addition, bioreactors are used for large scale propagation of multiple shoot in less time. The accumulation of biomass and bioactive compound during bioreactor culture is higher than in solid culture.

Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L. )

Background The effect of polyamines (PAs) along with cytokinins (TDZ and BAP) and auxin (IBA) was induced by the multiple shoot regeneration from leaf explants of gherkin (Cucumis anguria L.). The polyphenolic content, antioxidant and antibacterial potential were studied from in vitro regenerated and in vivo plants. Results Murashige and Skoog (MS) medium supplemented with 3% sucrose containing a combination of 3.0 µM TDZ, 1.0 µM IBA and 75 µM spermidine induced maximum number of shoots (45 shoots per explant) was achieved. Regenerated shoots elongated in shoot elongation medium containing 1.5 µM GA3 and 50 µM spermine. The well-developed shoots were transferred to root induction medium containing 1.0 µM IBA and 50 µM putrescine. Rooted plants were hardened and successfully established in soil with a 95% survival rate. Twenty-five phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) analysis The individual polyphenolic compounds, total phenolic and flavonoid contents, antioxidant and antibacterial potential were significantly higher with in vitro regenerated plants than in vivo plants. Conclusions Plant growth regulators (PGRs) and PAs had a significant effect on in vitro plant regeneration and also a biochemical accumulation of flavonols, hydroxybenzoic and hydroxycinnamic acid derivatives in C. anguria. Due to these metabolic variations, the antioxidant and antibacterial activities were increased with in vitro regenerated plants than in vivo plants. This is the first report describing the production of phenolic compounds and biological activities from in vitro and in vivo regenerated plants of C. anguria.

Arachis hypogaea L. Direct Organogenesis and SDS - PAGE Analysis in Groundnut.

The present research work was carried out to observe the effect of various concentrations of Benzyl amino purine (BAP) in terms of multiple shoot induction with special emphasis on qualitative and quantitative changes of proteins during multiple shoot formation. From whole embryonated cotyledons of groundnut. The shoot induction medium containing different concentrations of Benzyle amino purine ranged from 5.0 to 25.0mg/l along with lower concentration of Indole acetic acid (IAA).The multiple shoots were observed in all the BAP concentrations at a varying levels. Among the various levels of BAP, 25.0mg/l of BAP plus 0.50mg/l of IAA was found to be the most effective in terms of multiple shoot induction. The growth parameters like plantlet height, fresh and dry weight also highly influenced by the concentration of the BAP. The root induction was achieved in micropropagated shoots by using Indole butyric acid (IBA) at a concentrations of 1.0 to 5.0mg/l. The mean number of roots, root length was highly influenced by IBA. In all the concentrations of IBA the rooting was associated with moderate and heavily associated basal callusing. The quantitative estimation of protein content in regenerated shoots also increased with higher concentrations of BAP. The SDS-PAGE analysis showed that there was a nine visible bands with 82.2, 76.4, 57.5, 27.6, 23.7, 21.4, 18.2, 16.5 and 12.8 kDa were observed in multiple shoots derived from all the concentrations of BAP. Whereas in whole embryonated cotyledonary explants there was a entirely different protein banding

Establishment of Agrobacterium-Mediated Transformation System in Sweet Potato (Ipomoea batatas) by Culture of Leaf Segments for Functional Analysis of ASG-1, an Apomixis- Specific Gene.

Aims: In order to do the functional analysis of apomixis-specific gene (ASG-1), which was isolated from apomictic guineagrass, the sweet potato was used to establish an Agrobacterium-mediated transformation system. Study Design: At first, plant regeneration was achieved from the culture of leaf segments of sweet potato. Based on it, a binary vector pSMA35H2-NG for transformation of ASG-1 was used for establishment of a suitable procedure for plant regeneration of transformants. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2009 and December 2012. Methodology: The leaf segments were used for somatic embryogenesis and plantlets regeneration. For the preliminary transformation, a GUS gene set in pSMA35H2-NG was introduced into the Agrobacterium strain GV3101/PMP9, and the Agrobacterium was used to infect the callus derived from leaf segments of sweet potato “Miyazakibeni” and the callus derived from seeds of rice “Nipponbare”. For the plasmid construction, the GUS was replaced by ASG-1, named as pSMA35H2/ASG1. The resultant plasmid was mobilized into Agrobacterium strain GV3101/PMP9 for transformation. For detection of ASG-1, DNAs of the transgenic plantlets were used for PCR, using the primers designed according to ASG-1 and hygromycin, respectively. Results: 1) When the leaf segments were sterilized with sodium hypochlorite solution of 0.3% and 0.4% for 15 min, 100% of surviving rates was achieved. And the segments cultured on Murashige and Skoog (1962) gave 100% of callus formation rates. 2) When the calli were placed onto Komamine and Nomura (1998) medium for differentiation, somatic embryogenesis was obtained with white color and grain-like tissue, and plantlets with multiple shoot-like tissues were obtained from the somatic embryo. 3) For the preliminary transformation, the calli showed GUS blue spots gradually on the surface. 4) When the pSMA35H2/ASG1 was used to the transformation of the embryogenic calli, the plantlets were developed through multiple shoots. 5) The specific bands of ASG-1 and hygromycin were observed from the PCR products of the plantlets’ DNAs, respectively. Conclusion: Overall the above results, the procedure using the binary vector pSMA35H2/ASG1 containing ASG-1 revealed, as the first case, that Agrobacteriummediated transformation system in sweet potato was established using the culture of leaf segments in this study.

In Vitro Regeneration of Exacum wightianum Arn. (Gentianaceae)- An Endemic Medicinal Plant From The Nilgiris, Western Ghats.

Exacum wightianum Arn. (Gentianaceae) is an endemic medicinal plant from the Nilgiri hills, Western Ghats, Tamil Nadu. Indirect regeneration of E. wightianum was obtained through organogenesis in callus culture. Axial bud explants were found to be best suited for callus induction on MS medium supplemented with BA + NAA (2.0+0.03 mg/L). Multiple shoots originated from callus obtained from the axial buds. were multiplied by subculture on the same medium. Maximum shoot regeneration was obtained on MS medium supplemented with BA with NAA (2.0+0.5 mg/L), and up to 25shoots was observed within 2 weeks.

Cytokinin preconditioning enhances multiple shoot regeneration in Pongamia pinnata (L) Pierre - a potential, non-edible tree seed oil source for biodiesel

An efficient, highly reproducible protocol for multiple shoot induction and plant regeneration of Pongamia pinnata has been successfully developed using cotyledonary node explants. This study also demonstrates that preconditioning of explant stimulates production of multiple shoots from cotyledonary nodes of P. pinnata. The highest direct shoot regeneration (90 percent) with an average of 18.4 +/- 3.1 shoots/explant were obtained when cotyledonary node explants were excised from seedlings germinated on Murashige and Skoog (MS) media supplemented with benzyladenine (BA) 1 mg l-1, and subsequently cultured on MS media with 1 mgl-1 thidiazuron (TDZ). Scanning electron microscope observations of cotyledonary node (CN) explants excised from pre-conditioned and normal seedlings, revealed larger buds with rapid development in BA-preconditioned CN explants. The addition of adenine sulphate significantly increased the average number of shoots per explant. The highest direct shoot regeneration (93 percent) with an average of 32.2 +/- 0.93 shoots/explant was obtained from BA-preconditioned CN when cultured on MS media supplemented with 1 mg l-1 TDZ and 200 mg l-1 adenine sulphate (ADS). Repeated shoot proliferation was observed from BA preconditioned CN explants up to 3 cycles with an average of 15 shoots/explant/cycle when cultured on MS media supplemented with 1 mg l-1 TDZ and 150 mg l-1 L-glutamine, thus producing 45 shoots/CN explant. Shoots were elongated on hormone free MS media and rooted on 1/2 MS media supplemented with 1 mg l-1 of IBA. Rooted shoots were successfully acclimatized and established in soil with 80 percent success. The highly regenerative system developed in this investigation for this important tree could be a useful tool for genetic transformation.