The role of activated charcoal in plant tissue culture.

Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.

Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny

Gene technology for grain legumes: can it contribute to the food challenge in developing countries?

Due to the commercial importance of some conifer species, advances in conifer biotechnology often appear to overshadow equally significant advances in the biotechnology of angiosperm forest trees. However, progress with some hardwood forest trees has been just as promising as that made with conifers, and in some areas, has surpassed conifer biotechnology, particularly in the past few years. Until relatively recently, progress with in vitro propagation and gene transfer in hardwood forest trees was confined primarily to the genus Populus. Similarly, compared to other hardwood species, the greatest progress has been made both in the areas of genomics and modification of wood quality traits in this genus. However, the advances in in vitro propagation, in general, and somatic embryogenesis, in particular, have brought mass clonal propagation of other top commercial hardwood trees, in particular Eucalyptus, closer to reality and gene transfer systems have been reported for a number of them. While by far the most extensive application of genomic tools, including genomic sequencing, expressed sequence tags, transcript profiling and molecular markers, has also been made with Populus, these tools are now being applied to wider range of hardwood species. Just as with conifers, potential biotechnology applications for hardwood forest species include development of trees with faster growth, altered wood quality, and insect and disease resistance. In addition, some hardwood species are being manipulated for such non-traditional uses as phytoremediation. Given these advances and the worldwide importance of the products derived from them, it is likely that in vitro propagated and transgenic hardwood forest trees will have just as great an impact on commercial forestry and our environment as the top coniferous species.

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Hardwood tree biotechnology

An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.

In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using Thidiazuron

Shoot tip explants of cucumber (Cucumis sativus L. cv. Poinsett 76) were cultured in vitro on Murashige-Skoog medium with L-glutamine, ammonium nitrate, adenine sulphate, asparagine, ammonium succinate, potassium nitrate and sodium nitrate as the nitrogen sources along with optimal concentration of 0.044 mM benzyladenine to study their effects on in vitro morphogenesis. The explants grown with 0.068 mM L-glutamine displayed the highest culture response (74.6 %) and greatest shoot number per explant (13.6) at the end of two subcultures. The explants cultured with other nitrogen sources resulted in low culture frequency and low number of shoots per explant accompanied by basal callusing and necrosis.

Glutamine : A suitable nitrogen source for enhanced shoot multiplication in Cucumis sativus L.

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months.

In vitro organogenesis and plant formation in Acacia sinuata

Embryogenic callus was induced from primary leaves of Vigna unguiculata (L.) Walp. in MS medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D). Greenish-white, friable embryogenic calluses were used to establish suspension cultures. A shaking speed of 90 rpm and 0.4 ml packed cell volume per 25 ml medium were found to be optimal for maintaining suspension cultures. Globular, heart-shaped and torpedo-shaped embryos were developed in suspension culture containing 4.52 μM 2,4-D. Maturation of cotyledonary-stage somatic embryos was achieved on 0.05 μM 2,4-D, 5 μM abscisic acid and 3% mannitol. Twenty-two percent of the embryos were converted into plants and survived; survival in the field was 8–10%.

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA3). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Enhanced numbers of multiple shoots were induced from shoot tip explants of cucumber. The effects of amino acids (leucine, isoleucine, methionine, threonine, and tryptophan) and polyamines (spermidine, spermine, and putrescine) along with benzyladenine (BA) on multiple shoot induction were investigated. A Murashige and Skoog (MS) medium containing a combination of BA (4.44 μM), leucine (88 μM), and spermidine (68 μM) induced the maximum number of shoots (36.6 shoots per explant) compared to BA (4.44 μM) alone or BA (4.44 μM) with leucine (88 μM). The regenerated shoots were elongated on the same medium. Elongated shoots were transferred to the MS medium fortified with BA (4.44 μM), leucine (88 μM), and putrescine (62 μM) for root induction. Rooted plants were hardened and successfully established in soil with a 90% survival rate.

V. Ramesh Anbazhagan

Papers

Glutamine : A suitable nitrogen source for enhanced shoot multiplication in Cucumis sativus L.

In vitro organogenesis and plant formation in Acacia sinuata

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Leucine and spermidine enhance shoot differentiation in cucumber ( Cucumis sativus L.)