http://www.sidthomas.net/pdf/paperpdfs/108.pdf

Bringing medicinal plants into cultivation: opportunities and challenges for biotechnology

In vitro manipulation and propagation of medicinal plants

A protocol is described for rapid and large-scale propagation of the woody aromatic and medicinal shrub Vitex negundo by in vitro culture of nodal segments from mature plants. Of the three different cytokinins – N6-benzyladenine (BA), kinetin, and thidiazuron – evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.0 mg/l was most effective in inducing bud break. Although callus-free multiple-shoot formation was a function of cytokinin activity alone, faster bud break coupled with an enhanced frequency of shoot development (92%) and internode elongation were dependent on the synergistic influence of gibberellic acid (GA3) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l). The frequency of shoot proliferation was markedly influenced by the explanting season. By repeated subculturing of nodal segments harvested from the in vitro-formed axenic shoots on MS containing 1.0 mg/l BA and 0.4 mg/l GA3, prolific shoot cultures free from proximal callusing and showing a high-frequency multiplication rate were established. The percentage shoot multiplication (98–100%) as well as the number of shoots per node (six to eight) were highest during the first three culture passages, after which there was a gradual decline in shoot development. Rooting was best induced (94%) in shoots excised from proliferated shoot cultures on half-strength MS medium augmented with an optimal combination of indole-3-acetic acid and indole-3-butyric acid each at 1.0 mg/l. Vermi-compost was the most suitable planting substrate for hardening inside a plant growth chamber and its use ensured high-frequency survival (93%) of regenerated plants prior to outdoor transfer. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics as well as vegetative and floral morphology.

Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high-frequency axillary shoot proliferation.

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA3) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil).

Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb

In vitro clonal multiplication of safed musli (Chlorophytum borivilianum Sant. et. Fernand.), a rare Indian medicinal herb, has been achieved on Murashige and Skoog's (MS) medium supplemented with 22.2 μM benzyladenine using young shoot bases as explants. Shoots multiplied at a rate of four-fold every 3 weeks. All shoots rooted when transferred to MS medium with 3/4-strength inorganic and organic constituents and 9.8 μM indolebutyric acid and 67% of the micropropagated plants were successfully established in pots. Such plants produced normal fasciculated storage roots as in wild plants.

Micropropagation of safed musli (Chlorophytum borivilianum), a rare Indian medicinal herb

An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl−1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds.

Micropropagation of Sterculia urens Roxb. - an endangered tree species.

Chlorophytum borivilianum Sant. et Fernand. (‘Safed Musli’), an endangered Indian medicinal herb, is valued for its tuberous roots reputed to have aphrodisiac properties. Farmers in India cultivate this medicinal herb on a commercial scale because of its high economic value. To cater to the growing demand for planting material, a highly reproducible field-tested and cost-effective micropropagation scheme has been developed. Best shoot multiplication was achieved on agargelled Murashige and Skoog medium containing 22.2 μM 6-benzylaminopurine (BA) and 3% sucrose. Phytagel™ at 0.2% showed slightly better response than BDH agar in terms of shoot multiplication, but the use of BDH agar was preferred due to its low cost. With the optimized conditions, more than 15 000 plantlets could be produced in 20 wk. Plantlets subjected to hardening under agro-shadenet conditions during the monsoon months of high humidity showed better survival rate and growth compared to plantlets hardened in vitro and subsequently transferred to the greenhouse for acclimatization. Rate of plantlet survival was 87 and 90% under open field and agro-shadenet conditions, respectively. Plantlets grown ex vitro under agro-shadenet and field conditions produced tuberous roots which could be grown in the next season as a secondary propagule. We concluded that in vitro production of ‘Safed Musli’ was cost-effective compared to conventional propagation and holds great potential for commercial production.

Scaling-up production and field performance of micropropagated medicinal herb ‘Safed Musli’ (Chlorophytum borivilianum)

A method for somatic embryogenesis and plant/et regeneration in ‘Safed Musli’ (Chlorophytum borivilianum) is described. Callus was induced from immature zygotic embryos inoculated on MS medium containing 1.0 mgl−1 2,4-D. Initially, callus was slow growing, soft, watery and slimy. After six weeks of growth and subsequent subculture on MS medium containing 0.5 mgl−1 2,4-D, yellow, compact, hard, nodular, shiny somatic embryos were developed. Embryogenic cultures were maintained by repeated subculturing every four weeks on MS medium containing 0.25 mgl−1 2,4-D. Concentration of 2,4-D was further reduced to 0.1 mg−1 for maturation of these embryos. Plantlets could be recovered from 20 per cent of these somatic embryos when inoculated on auxin free MS medium and the cultures were transferred to light. Precocious germination of somatic embryos and profuse rooting was frequently observed.

Somatic embryogenesis and plantlet regeneration in ‘Safed Musli’ (Chlorophytum borivilianum)

In vitro clonal multiplication of ‘sated Musli’ (Chlorophytum borivilianum) a rare Indian madcinal herb has been achieved on MS medum supplemented with 22.2 μM BA using young shoots. All shoots produced roots on 314 strength MS medium containing 9.8 μM IBA. Sixty seven per cent of p1antlets could be established in pots and soil. Multiplication was also achieved through somatic embryogenesis. Callus raised from zygotic embroys produced somatic embroys on MS medium containing 4.53 μM 2, 4–0. Somatic embroys were maintained on low concentration (1.13 μM) of 2, 4–0. A few somatic embroys (20 per cent) produced plants when inoculated on a medium without any growth regulator. Precocious germination of somatic embroys and profuse rhizogenesis was frequently observed.

Ashish Dave

Papers

Micropropagation of safed musli (Chlorophytum borivilianum), a rare Indian medicinal herb

Micropropagation of Sterculia urens Roxb. - an endangered tree species.

Scaling-up production and field performance of micropropagated medicinal herb ‘Safed Musli’ (Chlorophytum borivilianum)

Somatic embryogenesis and plantlet regeneration in ‘Safed Musli’ (Chlorophytum borivilianum)

In vitro conservation strategies for a rare medicinal herb-safedmusli (Chlorophytum borivilianum sant. et fernand.)