Plant regeneration from various expiants of cultivated Piper species.

Morphogenetic potential of root, leaf, node and internode expiants of 3 cultivated Piper species was investigated to develop a reliable plant regeneration protocol. P. longum (pipli) was the most responsive followed by P. betle (betel vine) and P. nigrum (black pepper). In P. longum the highest number of shoot buds was produced on root expiants followed by node, internode and leaf expiants. In P. betle and P. nigrum adventitious shoot buds differentiated only from internodal and nodal ring regions, respectively. Histological examination in P. longum showed that adventitious shoot buds originate directly from the cortical cells of the root and the internode without an intervening callus phase. Benzyladenine was superior to kinetin for shoot induction and its optimum concentrations for P. longum, P. betle and P. nigrum were 1-2, 10 and 10 µM, respectively. Shoot elongation and rooting were achieved in B5 medium containing 0.5 µM benzyladenine and 1 µM indoleacetic acid, respectively. Regenerated plants were established in soil.

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.).

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (-196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented wiith 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.

In vitro propagation of Allium tuberosum Rottl. ex. Spreng. by shoot proliferation.

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg11). Ia vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.

Low-temperature storage of Rauvolfia serpentina Benth. ex Kurz.: An endangered, endemic medicinal plant.

On a standard shoot culture medium, nodal cultures of Sarpagandha (Rauvolfia serpentina) could be maintained for nine months at 25° C by replacing cotton plugs with polypropylene caps as enclosures for culture tubes. Low temperature incubation of in vitro cultures appeared highly promising because cultures exhibited normal health even after 15 months of storage at 15° C; while 10°C and 5°C were found deleterious to growth of the cultures of R. serpentina.

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.).

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg/l). In vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.

In vitro propagation of Allium tuberosum Rottl. ex. Spreng. by shoot proliferation.

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (-196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented with 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.

Plant regeneration from callus cultures of Piper longum L. by organogenesis.

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l(-1)α- naphthaleneacetic acid and 0.2 mg.l(-1) N(6)-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l(-1) indole-3-acetic acid with 1.5 mg.l(-1) benzyladenine, and 0.1 mg.l(-1) indole-3-acetic acid with 0.2 mg.l(-1) benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l(-1) indole3-acetic acid. The rooted plants were successfully established in soil.

In vitro propagation of Coleus forskohlii Briq., a threatened medicinal plant.

In vitro clonai multip1ication of Coleus forskahlii Briq a threatened plant, has been achieved on MS medium supplemented with Kn (2.0 mg/l) and IAA (1.0 mg/l) using nodal segments as explants, Shoots multiplied at a rate of 12 - fold every six weeks. Rooting was achieved upon transfer of shoots onto MS medium containing IAA (1.0 mg/l). The micropropagated plants were successfully established under field conditions. Forskolin content in tubers of plants obtained by micropropagation was found to be 0.1%, the same as that found in wild plants. This micropropagation procedure should be useful for conservation as well as production of this important plant.

A novel technique to overcome browning in tissue culture.

Experiments conducted using Dioscorea alata L. revealed that an exudate from the cut end of the explants was responsible for browning of the culture medium. Browning did not affect growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Sealing the cut ends with paraffin wax was found to control browning by preventing exudation. This simple technique permitted establishment of cultures in a small volume of medium in about 90 percent of the cases, while in unsealed cultures lethal browning was recorded in 80 percent of the cases. The advantages of this technique over other methods of controlling browning are discussed.

In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc.).

Rhizome buds, excised from threeCurcuma spp., and ginger, inoculated aseptically on MS medium with varying levels of BAP and kinetin, produced multiple shoots. For shoot multiplication, a concentration of 3.0 mg/l BAP was found to be optimum for all the species.In vitro plants were successfully established in the field and were morphologically uniform. A simple method to extend the subculture interval was used and its relevance to germplasm conservation is discussed.