An improved Agrobacterium rhizogenes-mediated genetic transformation of Withania somnifera (L.) Dunal was developed using the bacterial strain R1000 with leaf segment explants of in vitro raised plantlets. Out of the three strains used (R1000, MTCC 2364 and MTCC 532), the strain R1000 proved to be more efficient than others. Among the different conditions tested, the highest (93.3 %) transformation rate was observed after 3 weeks when the explants were subjected to sonication (15 s) and heat treatment (41 °C for 5 min). Transgenic status of the hairy roots was confirmed by PCR using rol B-specific primers. HPLC analysis showed the ability of hairy roots to synthesize withaferin A and withanolide A, both steroidal lactones of medicinal value. This protocol offers new avenue in A. rhizogenes-mediated hairy root induction and is useful for large-scale production of these bioactive compounds from W. somnifera.

No abstract

No abstract

No abstract

No abstract

An efficient protocol for direct shoot organogenesis has been developed for the medicinal plant Aerva lanata (L.) Juss. ex Schult. Regeneration was achieved from leaf segments of 20 days old in vitro plantlets raised on Murashige and Skoog (MS) medium containing 0.25–2.0 mg L−1 thiadiazuron (TDZ), 3% sucrose, and 0.8% agar. After 21 days of culture incubation, maximum number of shoot organogenesis (23.6 ± 0.16) was obtained on medium containing 1.0 mg L−1 TDZ. The shoots were able to produce in vitro flowers on medium containing 1.0 mg L−1 TDZ in combination with 0.25–0.5 mg L−1   α-naphthaleneacetic acid (NAA). Histological observation showed that the epidermal cells of the leaf explants exhibited continuous cell division led to formation of numerous dome shaped meristematic protrusions and subsequently developed into adventitious shoots. Upon transfer of shootlets to half strength MS medium containing 1.0 mg L−1 indole-3-butyric acid (IBA), around 86% of the regenerated shoots formed roots and plantlets. Rooted plants were hardened and successfully established in the soil at the survival rate of 92%. The regeneration protocol developed in this study provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large scale production of its medicinally active compounds and genetic transformations for further improvement.

Endophytic bacteria were isolated from Withania somnifera (L.) Dunal and their potentials were explored through the in vitro plant regeneration techniques. Initially, indole acetic acid (IAA), phosphate solubilization and siderophore producing isolates were screened out. These isolates were identified using molecular tools like 16s rRNA gene sequences namely Bacillus pumilus AS02, Sphingobacterium thalpophilum AS34, Pseudomonas aeruginosa AS36, Agrobacterium tumefaciens AS38 and Enterobacter aerogenes AS75. Further, the regeneration efficacy of IAA producing endophytic bacteria has been analysed on an endangered plant-Exacum travancoricum Bedd. The leaf and inter-node explants of E. travancoricum with or without bacterial infection were tested on Murashige and Skoog's (MS) medium encompassing 6-benzyladenine (BA; 2.0 mg L − 1 ) and l-tryptophan (0.2%; w/v). Somatic embryo induction was observed on both explants. The leaf explants infected with S. thalpophilum AS34, P. aeruginosa AS36 and E. aerogenes AS75 exhibited highest percentage of somatic embryo induction (93.3%) compared with the inter-node explants (86.6%). The explants without bacterial infection (control) and the presence of exogenous IAA exhibited lower level of response (88.8%, leaf and 75.5%, inter-node). Histological analysis revealed the appearance of globular embryogenic masses on epidermal, sub-epidermal regions of both explants. The development of torpedo, cotyledon stage and protoderm formation without the vascular connection from explants confirmed direct somatic embryogenesis. This was confirmed with scanning electron microscopy. Sub-culturing of cotyledon somatic embryos in MS liquid medium without growth regulators was performed to obtain well developed shoots with roots.

An efficient and simple procedure was systematically developed for inducing direct somatic embryogenesis and plantlet regeneration from leaf sheath explants of Curcuma amada Roxb. A two-step culture system was used to induce somatic embryogenesis. The optimized procedure resulted in direct somatic embryogenesis from 93.3% explants after 3-wk culture. Leaf sheath explants were incubated for 2 wk on medium containing 2.24 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine to initiate direct somatic embryogenesis. Thereafter, these explants were transferred to a medium containing 9.10 μM thidiazuron and 1.33 μM α-naphthaleneacetic acid. Elongated somatic embryos obtained from these cultures germinated readily, and the optimal frequency of plantlet development (86.7%) was achieved when embryos were cultured in darkness on 1/2 strength Murashige and Skoog medium containing 1.44 μM gibberellic acid. Histological and scanning electron microscopic studies showed that the initial cell divisions that led to embryo formation occurred in epidermal and subepidermal cells, followed by the development of globular and elongated structures that appeared to be somatic embryos. The presence of a clear protoderm in the globular structures and procambial strands in the elongated structures confirmed that these structures were true somatic embryos. Plantlets derived from somatic embryos were acclimatized successfully to ex vitro conditions at a survival rate of 87.43% and developed with normal phenotypes.