Forests are the best source of renewable energy and also provide raw materials for several important industries. Among leguminous trees are of special interest from an ecological point of view, since many of them can readily fix atmospheric nitrogen. Therefore, it is essential to improve our fabaceous forest trees using tissue culture techniques and present plant biotechnological approaches. The application of tissue culture techniques offers novel and quick means of improving trees over the conventional time-consuming breeding methods. In addition to that, height of trees and long life put further hindrance. The genetically superior trees with fast growth rate can be rapidly multiplied through micropropagation. Mascarenhas and Muralidharan (1989) have summarized the work on tissue culture studies of forest trees in India. The first report of tissue culture using shoot tip cultures of a leguminous tree was published by Skolmen and Maps (1976) in Acacia koa. In their studies, shoots were regenerated from shoot tip derived callus though at a very low frequency. From literature survey, it has been noticed that leguminous trees are very recalcitrant for in vitro differentiation. However, there are some positive reports where regeneration has been achieved using juvenile explants excised from in vitro grown young seedlings, for example, Albizia lebbeck (Gharyal and Maheshwari, 1983), Dalbergia sissoo (Mukhopadhyaya and Ram, 1981), Acacia nilotica (Mathur and Chandra, 1983), Leucaena leucocephala (Datta and Datta, 1985; Dhawan and Bhojwani, 1985), Dalbergia latifolia (LakshmiSita et al., 1986), and Sesbania bispinosa (Kapoor and Gupta, 1986). There are very few reports on clonal propagation using explants, excised from field grown mature plants. In these studies, either axillary buds from nodes or apical buds have responded well, for example, in Prosopis cineraria (Goyal and Arya, 1984), Albizia procera (Ror and Datta, 1985), L. leucocephala (Datta and Datta, 1985), and D. latifolia (LakshmiSita et al., 1986).

Seeds of Leucaena were obtained from Pratap Nursery, Dehradun, and were sown in the Botanical Garden of the Department, Delhi University. Young shoots 2–3 cm were excised from 5-year-old trees of Leucaena growing in botanical garden. Shoot pieces were washed with tap water several times and finally surfaced sterilized with chlorine water in the culture room inside a laminar flow cabinet for 10–20 min. Subsequently, they were washed thrice with sterilized distilled water (2–3 min each) to remove all traces of chlorine. Later, just 2–3 mm long shoot tips containing 3–4 leaf primordia were excised from sterilized twigs. All steps from surface sterilization to inoculation were carried out under sterile conditions in a laminar flow placed in the culture room. MS (Murashige and Skoog, 1962), NB (Nitsch, 1969), and B₅ (Gamborg et al., 1968) basal as well as supplemented with various phytohormones (BA, IAA, and NAA) were used. All media contained 3% sucrose (BDH) and 0.8% agar (Difco-Bacto). The pH of all media was adjusted to 5.8 before autoclaving. Approximately 20 ml of the medium was poured into each culture tube and finally autoclaved using standard parameters. All cultures were maintained in the culture room at 25°C temperature and 50–60% relative humidity under continuous cool white light tubes (approx. 500 lux; Philips, India). Observations were taken on various aspects such as initial number of explant inoculated and total number survived, total number of explants callusing and rooting, size, amount, color and texture of calli on relative basis, the number of shoots and roots per explant, and initiation of multiple shoots and their number per explant. To prevent browning of the explant, polyvinylpyrrolidone (PVP) was used at 250–1000 mg/l.

In vitro regeneration leading to plantlet formation is difficult in tree species (Bonga, 1977), plantlets have been obtained in L. leucocephala from shoot tip explants excised from field grown plants during present investigations. These results are of interest since leguminous trees are strongly recalcitrant to in vitro morphogenesis. Among cytokinins, BA is most effective for shoot differentiation in Leucaena. At 3 mg/l, BA produced multiple shoots in the presence of 0.05 mg/l NAA. The regeneration using explants from in vivo growing trees is important for clonal propagation of selected elite species. Therefore, shoot tip, stem and leaf pieces were excised and cultured on different media. Out of various explants, shoot tips were found more responsive. In previous reports, micropropagation of Leucaena from mature trees has been achieved but it was through axillary bud cultures (Datta and Datta, 1985; Dhawan and Bhojwani, 1985). Similar to our results with Leucaena, shoots have been obtained from shoot tips of mature trees of A. procera on MS + BA medium ( Ror and Datta, 1985). Rooting of regenerated shoots in woody species is believed to be difficult (Nemeth, 1986). In D. latifolia, Sankara Rao (1986) developed a long three-step culture procedure to induce rooting in regenerated shoots. However, in Leucaena, excised shoots could be rooted after transferring them to IAA supplemented medium. Like the present observations, excised shoots of D. latifolia (LakshmiSita et al., 1986) and A. lebbeck (Gharyal and Maheshwari, 1983) were also rooted in the presence of IAA. Thus, it is possible to micropropagate Leucaena mature trees. Regeneration of plantlets from old trees is of great importance since the phenotype of selected plants and their desirability is known (LakshmiSita et al., 1986). Among leguminous trees, multiple shoots have been obtained from nodal explants of 30-year-old trees of D. sissoo (Datta et al., 1983). One of the major problems in culturing old tree explants like shoot tips was the leaching out of high amount of brown substances believed to be polyphenols from tissues in the medium. PVP at 250 and 500 mg/l was quite effective in checking browning of shoot tip explants excised from old trees. This ultimately led to the development of plantlets in vitro.

A rapid and efficient method of clonal propagation and micropropagation has been established in L. leucocephala using shoot tip explants excised from mature trees. Multiple shoots can be differentiated on MS + BA medium. Differentiated and well-developed shoots could be rooted on subculturing them on IAA containing fresh medium.