Plant Cell, Tissue and Organ Culture 31: 257-259, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Micropropagation note

Micropropagat ion of a mature leguminous tree - Bauhinia purpurea

Anjani Kumar Department of Botany, North-Eastern Hill University, Shillong 793 014 India

Received 10 July 1991; accepted in revised form 7 May 1992

Key words: callus, differentiation, plantlets

In vitro techniques have been employed success- fully in the propagation of mature trees. How- ever, these are yet to be extended to many forest trees and plants used for afforestation. Or- ganogenesis in woody legumes has been difficult (Datta & Datta 1985), but limited success has been achieved in recent years (Goyal & Arya 1981; Mukhopadhyay & Mohan Ram 1981; Datta et al. 1982; Gharyal & Maheshwari 1982; Anand & Bir 1984; Datta & Datta 1985; Ravishankar & Jagadish Chandra 1988; Kumar et al. 1991).

Bauhinia purpurea L. is a leguminous forest tree, a popular avenue tree, a useful fuel, a timber yielder and an important medicinal plant (Kumar 1987). The plant, growing in sub- Himalayan tracts of India and Southeast Asia, is seed propagated, but viability of seeds is low (Kumar 1987). In vitro propagation of mature B. purpurea tree by axillary bud culture has been accomplished (Kumar 1987). Adventitious re- generation directly from organs gives a limited number of propagules. This number may be increased many fold by an intervening callus phase, however, at the risk of undesirable vari- ation (Hussey 1983). The present study was aimed at developing a suitable methodology for rapid propagation of mature 'elite' B. purpurea lines.

Stem cuttings (ca 1.0 cm) collected from young branches of 15-18 year old, sexually mature 'elite' Bauhinia purpurea trees were stirred vig- orously with Teepol solution (20% v/v) and then washed thoroughly in running tap water for 1 h followed by several rinses in sterile distilled water. The cuttings were subsequently surface

disinfested with 0.5% (w/v) aqueous mercuric chloride solution for 2min, then rinsed thoroughly (5-7 times) with sterile water. Both the exposed ends of the explants were trimmed to get 0.5 cm segments that were cultured asepti- cally on MS (Murashige & Skoog 1962) medium containing 3% sucrose, 0.8% agar (Qualigens, bacteriological grade) and various concentrations of auxins and cytokinins, either individually or in combinations. The pH of the culture medium was adjusted to 5.8 prior to autoclaving at 121°C and 98kPa for 20min. Callus cultures were initiated by inoculating stem segments in 25 × 150mm culture tube (one segment/tube) con- taining 15 ml of the culture medium. On the 15th day, proliferating stem segments were trans- ferred to 150ml flasks containing 50 ml of the culture medium. Again, the elongation of shoots and their subsequent rooting have been carried out in the culture tubes containing 15 ml of the medium. Non-absorbent cotton plugs, covered with aluminium foil wrap, were used as closure of the culture vessels. The cultures were incu- bated at 24 _+ 2°C with a 12-h photoperiod (cool white fluorescent, 40 ~mol m 2s 1) and 60-70% relative humidity.

For rooting of isolated adventitious shoots, MS medium supplemented with various auxins was used. Each treatment consisted of 20 repli- cates and all experiments were repeated thrice.

Stem segments cultured on MS medium sup- plemented with 10 ~M 2,4-dichlorophenoxy- acetic acid (2,4-D) started proliferating by the 15th day of culture and the entire segment was covered with a mass of friable greenish-white callus within 45 days. Both caulogenesis and

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rhizogenesis were observed in the calluses (8- to 10-week-old) subcultured on MS + a- naphthaleneacetic acid (NAA) and/or kinetin within 30 days of subculture (Table 1). Callus cultures subjected to N A A (0.5-101xM) pro- duced roots after 15 days. The percentage of cultures exhibiting rooting was maximum on cal- lus cultured on M S + 5 p~M NAA (Table 1). Calluses subcultured on MS+kinet in (0.5- 10 p~M) showed organised growth and produced 4-6 green nodular protuberances, i.e. shoot buds, at the surface (Fig. 1A) after 15-20 days of subculture. These eventually developed into shoots when transferred to MS medium devoid of growth regulators (Fig. 1B). The percentage of cultures forming shoots was maximum when cultured on MS + 5 ~M kinetin (Table 1) with 4 to 6 shoot buds per culture. Kinetin concen- trations above 5 IxM suppressed differentiation of shoot buds. The combination of NAA with kinetin resulted in decreased caulogenesis. Such antagonistic auxin-cytokinin effect has also been observed in another leguminous tree Dalbergia sissoo (Kumar et al. 1991). The regenerative

Table 1. Effect of kinetin and NAA on the regeneration frequency of shoots and roots from stem callus of B. pur- purea?

MS + growth regulators Cultures showing (~M) organogenesis ± S.D.

(%)

Kinetin NAA Shoots Roots

0 0 0 0 0.5 0 17±2 0 1.0 0 29±4 0 2.5 0 43±4 0 5.0 0 78±5 0

10.0 0 36±3 0 0 0.5 0 19±2 0 1.0 0 31±3 0 2.5 0 47±4 0 5.0 0 62±4 0 10.0 0 33±3 2.5 0.5 33±4 0 5.0 0.5 50±6 0 5.0 2.5 41±5 0 0.5 2.5 22±2 26±3 0.5 5.0 11±1 39±4

10.0 10.0 0 0

aData scored at the end of 60 days, each treatment consisted of 20 replicates and was repeated three times.

Fig. 1. Micropropagation of B. purpurea. (A) Formation of shoot buds (indicated by arrow) in callus subcultured on MS + 5 IxM kinetin (Bar = 0.5 cm). (B) Subsequent develop- ment of shoot buds into shoot on MS devoid of growth regulators. (C) Root of adventitious microshoots on MS + 5 ~M NAA.

callus has been maintained by subculturing on MS + 10 ixM 2,4-D at 30-day intervals.

Isolated shoots transferred to MS medium sup- plemented with 5 IxM N A A developed roots at the base with little callus within 30 days (Fig.

1C). Plant le ts with 2 - 3 leaves and one or more

deve loped roots were r emoved from cultures and

t r ans fe r red to pots con ta in ing a so i l : s and (1 :1 ) mix ture u n d e r glasshouse condi t ions , with 33 of 60 roo ted shoots surviving. The regeneran ts ob-

t a ined appea r to be no rma l and identical .

Acknowledgements

A u t h o r is thankfu l to Dr. SK Dat ta , Ins t i tu te for

P lan t Sciences, Z/irich (Switzer land) for va luable suggest ions and Dr JM Bonga , CFS, Frederic-

t ion , C a n a d a for critical reading of the manu -

script.

References

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