Indian Journal of Experimental Biology Vol. 38, June 2000, pp. 621-624

Micropropagation of mukorossi Gaertn

N S Philomina* & J V S Rao Department of Botany, S. V. University, Tirupati 517 502, Received 13 April 1999; revised 16 December 1999

Bud break and multiple shoots were induced in apical and ax illary meristems derived from one month old seedlings of S. mukorossi on Murashige and Skoog (MS) medium supplemented with benzylamino purine (BAP) 0.4 J.1M or 0.8 J.1M

alone. A combination of BAP and gibberellic acid (GA3) 0.4 J.1M and 2.8 J.1M produced elongated multiple shoots from both types of explants. Excised shoots were rooted on MS medium respectively with indole-3-butyric acid (IBA) 3.4 J.1M or 2.4 J.!M. The regenerated plantlets were successfully acclimatized and transferred to soil.

Forest trees in general have proved to be difficult to mukorossi tree using apical and axillary meristem mass propagate by tissue culture. Some success explants has been presented. however has been achieved in a few woody tree species. Importance of tissue culture for mass Materials and Methods 1 propagation of forest trees Iike Eucalyptus , Sandal Seeds of soapnut were obtained from Biotechnology wood2 and Rose wood3 has already been Research Centre for Tree Improvement (BIOTRIM), demonstrated. Micropropagation by the method of Andhra Pradesh Forest Department Nursery, Tirupati organogenesis and by multiple shoot production of and soaked in cone. H2S0 4 for 90 min and washed axillary meristems of seedling explants have been thoroughly with running tap water. The seeds were 4 5 6 reported in Leucaena , Albizia and Acacia . Shoot tip surface sterilized with 0.1 % HgCI2 for 15 min and cultures were established from germinated seedlings rinsed several times with sterile distilled water. Agar 7 8 in Redsanders and Teak • So far there are very few water medium (0.8%) without growth regulators was reports of like Sapindus trifoliatus9 used for seed germination. Seeds inoculated on this established by tissue culture. medium were incubated at 24° ± 2° C in the dark or Sapindus mukorossi Gaertn. or Sapindus detergens light at a photon flux density of 15 11Em·2 S of white Roxb, soapnut is a perennial tree belonging to the fluorescent tubes for thirty days after which seedlings family Sapindaceae, indigenous to northern India. Oil were used for apical and axillary meristems from the seed kernel of soapnut is of interest to the dissection. The apical and axillary meristems (2-4 soap industry. The oil is quite useful industrially mm) were collected from one month old aseptic because of its most valuable phytochemicals like seedlings and cultured on Murashige and Skoog 13 10 saponins or triglycerides • The exhausted cake is used (MS) basal medium containing 2% sucrose and lower as a filler and fertilizer and the shells for making concentrations of BAP or KIN ranging from 0.4, 0.8, 11 lignin based adhesives or boards • Vegetative 1.7 and 2.6 1-l.M, higher concentrations ranging from propagation of soapnut did not yield satisfactory 4.4,8.8 and 13 .2 1-l.M, combination of BAP and KIN results and propagation through seed is unreliable 0.4 or 0.4 1-l.M, 13.2 and 13.2 11M and combination of because the per cent survival of the seedlings proved BAP or KIN with auxin naphthalene acetic acid to be meagre du e to heavy incidence of mortality at 12 (NAA) 0.4 and 0.4 11M to 13.2 and 13.2 11M were seedling stage in the natural . habi-at • used for inducing muliple shoots. For muliplication Micropropagation of soapnut tree is at a stage of and elongation of established shoots different infancy in forest tree species which has great combinations of GA, (0.5 and 2.8 11M) alone, importance in the soap industry and social forestry . . combination of BAP and GA (0.4 and 0.5 0.8 programmes. In this communication for the first time 3 1-l.M, an in vitro micropropagation method for Sapindus and 0.5 1-l.M, 0.4 and 2.8 1-l.M, 0.8 and 2.8 11M) were tried. For root induction, 2-3 em long shoots were transferred to MS medium with IBA or NAA (0.4, *Present address: Dr. N.S. Philomina, Oo. Smt. Y. Elizabethamma, H.S.G. II P.A. Head Post Office, Cuddapah 516 001, India 2.4, 3.4 and 4.9 1-l.M) . Media were sterilized at 15 622 INDIAN J EXP BIOL, JUNE 2000

Ib/sq inch for 20 min. Twenty explants were cultured inducing 6 to 8 multiple shoots within a month from at 25° ± 2° C in the light (16hr photoperiod). Rooted axillary meristems. However on the same medium plantlets were acclimatized gradually in a green shoot tips were proliferated and 4 to 6 shoots were house. Results are mean of three culture cycles with formed in 4-5 weeks. Addition of hi gher 20 replicates per experiment. concentration of BAP (8 .8 and 13 .2 f.!M) to MS medium induced more callus formation in both the Results and Discussion explants within a week from the cut surface, and Experiments on explant type, shoot tips and along the surface from the apical to basal part of the axillary meristems for multiple shoot formation were explants. Initiation of callus was faster in all the tested. In all the BAP concentrations tested, 0.4 and higher concentrations of BAP. In order to test the 0.8f.tM concentrations were more effective for passaging on shoot multiplication, the shoots obtained

Fig. I-Formation of multi ple shoots regenerated from axill ary buds of S. mukorossi after 30 day of culture; Fig. 2 - Rooting of shoots o~ MS + 3.4 J.lM IBA + 2% sucrose after 15 days of cultu re; Fig. 3 -In vitro raised S. mukorossi plant 30 days after transplanting to soil. PHILOMINA& RAO: MICROPROPAGATION OF SAPINDUS MUKOROSSI GAERTN. 623

Table I-Effect of growth regulators on in vitro response of apical and axillary buds derived form one month old aseptic seedlings of S. mukorossi. [V alues are means± SE from 20 replicates/treatments] Growth Number of shoots/culture Shoot length (em) regul ators Apical bud Axillary bud Apical bud Axillary bud (JlM) BAPor KIN 0.4 6.0±0.20 8.0±0.90 2.0±0.109 3.0 ± 0.214 0.8 4.0 ± 0.14 6.0 ±0.20 2.0 ±0.118 3.5 ± 0.248 1.7 1.0 ±0.0 1.0 ±0.0 2.0±0.119 3.1 ±0.119 2.6 + 1.0 ± 0.0 + 1.9±0.115 4.4 + + + 2.0±0.115 8.8 + + + + 13.2 + + + + BAP 0.4+0.4 1.0 ± 0.0 1.0 ± 0.0 2.1 ±0.122 2.7±0.169 +KIN 13.2+1 3.2 1.0 ± 0.0 1.0 ± 0.0 2.3 ±0.123 2.9 ± 0.217 BAP 0.4+0.4 + + + + +NAA 13 .2+13.2 + + + + Culture response scored 30 days after inoculati on +=callusing on the ex from apical and axillary meristems were separated Table 2-Effect of auxins on in vitro rooting of regenerated and recultured on to the same shoot multiplication shoots of S. mukorossi after 30 days of inculbation media (MS with 0.4 or 0.8 f1M BAP) and shoot Auxins Concentrations ( J..LM) Mean percentage of multiplication was determined after second and third rooting (± SE) * subcultures. Highest number of shoots (8-1 0) were Control 0.0 0.0 recorded from a single explant within three weeks IBA 0.4 10.5 ± 0.275 (Fig. I). No increase in shoot multiplication was 2.4 18.9 ±0.260 observed by prolonging the culture period beyond 3.4 68.0 ± 0.478 sixth subculture. 4.9 59.0 ± 0.366 NAA 0.5 + Shoots obtained by this method were divided into 2.6 + 5-8mm nodal explants with single axillary bud for 5.3 + further proliferation to increase the number of shoots. 7.9 + These buds proliferated into 5 to 8 multiple shoots in IBA + NAA 0.4 (each) + 2.4 (each) + 4 weeks on MS medium with BAP 0.4 and 0.8f.l.M individually. Experiments conducted with BAP in * 20 replicates/treat ment repeated thrice combination of kinetin and auxin showed single +=callusing at the basal end shoots with callus formation. Of the two cytokinins increased their length. For elongated multiple shoot BAP was most effective for inducing bud break and formation with a combination of BAP (0.4JlM) and shoot proliferation in apical and axillary meristem GA3 (2.8 JlM) was optimum. In the present study (Table 1) . Similar results were reported in Madhuca combination of GA3 with a cytokinin was effective in . [' 14 I at1if ow . inducing shoot elongation. Different concentrations of Within 8 weeks of culture, the regenerated shoots GA3 alone failed to increase the shoot elongation elongated upto 2-3 em in height. Prolonged culture on however when GA3 was applied in combination with the same medium did not increase the shoot length. BAP effectiveness of gibberellic acid was improved

For shoot elongation the shoots were separated and in causing shoot elongation. Application of GA3 to in grown on MS medium with GA3 (0.5, 2.8J.1M)in vitro regenerated shoots increased their length in 15 combination with BAP (0.4, 0.8 f.l.M) treatments. The Azadirachta indica • shoots were elongated upto 5 to 7 em in 4 weeks in all The regenerated shoots were transferred to MS the BAP and GA3 treatments. Lower concentrations of medium with IDA and NAA of different concentrations BAP (0.4 or 0.8 JlM) were favorable for bud for rooting. Among these concentrations the proliferation and application of GA3 to these shoots regenerated shoots were rooted in IDA (3.4 and 4.9JlM) 624 INDIAN 1 EXP BIOL, JUNE 2000 m 15 days of culture (Fig. 2). IBA and NAA (0.4 and Leucaena leucocephala Landewit, Plalll Cell Rep, 4 ( 1985) 3 15. 2.4~ induced callus at the base of the shoots with 5 Upadhyaya S & Chandra N, Shoot and Plantlet formation in poor rooting after 30 days of incubation (Table 2). A organ and callus cultures of Albizia lebbeck Benth, Ann Bot, combination of IBA with NAA inhibited roots 52 ( 1983) 421. formation and showed only callus at the basal cut ends. 6 Mittal A, Agarwal R & Gupta SC, In vitro development of Regenerated plantlets were transferred to plastic Plant lets from axillary buds of Acacia auriculariformis, Plant Cell Tiss Org Cult, 19 (1990) 65. containers filled with vermiculite. During first week 7 Lakshmi Sita G, Sreenatha KS & Sujata S, Plantlet production the potted pl antlets were covered with polythene bags from shoot tip cultures of red sandal wood (Pterocarpus to provide high humidity. Transplantation success was santalinus L), Curr Sci, 7 ( 1992) 532. 60% (Fig. 3). Plantlets were subsequently transferred 8 Sunitibala Devi Y, Mukherjee BB & Gupta S, Rapid cloning to larger pots and gradually acclimatized to outdoor of elite Teak (Tectono grandis L) by in vitro muliple shoot production, Indian J Exp Bioi, 32 (1994) 668. conditions. In the present study multiplication by 9 Desai HV, Bhatt P N & Mehta A R, Plant regeneration of multiple shoot methods from shoot tip or axill ary Sapindus trifoliatus L (soapnut) through somati c meristems was developed for successful in vitro embryogenesis, Plant Cell Rep, 3 ( 1986)-190. propagation of Sapindus mukorossi an economically 10 Dev I & Guha S R D, Glyceride compositi on of Sapindus mukorossi (soapnut) oil , Indian J For, 2( 1979) 261. important tree. II Karnik M G, Sharma 0 P & Dev I, Studies on the chemi cal composition and possible utilities of soapnuts (Sapindus References mukorossi) Indian For, 8 ( 1971) 462. I Gupta P K, Mehta UJ & Mascarenhas A F, A tissue culture 12 Troup R S & Sapindaceae, Th e silviculture of Indian forest method for clonal propagation of mature trees of Eulayptus trees, Vol. I (Claredon Press, Oxford U.K (1 92 1) 232. torelliana and Eucalyptus camaldulensis, Plant Cell Rep, 2 13 Murashi ge T & Skoog F, A revised medium for rapid growth ( 1983) 296. and bi oassays wi th tobacco ti ssue cultures, Physiol Plant, 15 2 Rao PS & Bapat VA, Vegetative propagati on of sandalwood (1962) 473. plants through tissue culture, Can J Bot, 56 ( 1978) 1153 .. 14 Rout C R & Das P, Mi cropropagation of Madhuca longifolia, 3 Lakshmi Sita G & Raghava Swamy BY, Regeneration of Plant Cell Rep, 12 ( 1993) 5 13. plantlets from leaf di sc cultures of rose wood: control of leaf 15 Ramesh K & Padhya M A, In vitro propagation of neem abscission and shoot tip necrosis, Plant Sci, 88 ( 1993) 107. (Azadirachta indica A. Juss), In dian J Exp Bioi, 28 ( 1990) 4 Dhawan V & Bhojwani SS, In vitro vegetative propagat ion of 932.