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

Micropropagation of Sapindus mukorossi Gaertn

N S Philomina* & J V S Rao

Department of Botany, S. V. University, Tirupati 517 502, India

Received 13 April 1999; revised 16 December 1999

Bud break and multiple shoots were induced in apical and axillary 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 successfu lly acclimatized and transferred to soil.

Forest trees in general have proved to be difficult to mass propagate by tissue culture. Some success however has been achieved in a few woody tree species. Importance of plant tissue culture for mass propagation of forest trees I ike Eucalyptus 1, Sandal wood2 and Rose wood3 has already been demonstrated. Micropropagation by the method of organogenesis and by multiple shoot production of axillary meristems of seedling explants have been reported in Leucaena4

, Albizia5 and Acacia6. Shoot tip

cultures were established from germinated seedlings in Redsanders7 and Teak8

• So far there are very few reports of Sapindaceae like Sapindus trifoliatus9

established by tissue culture. Sapindus mukorossi Gaertn . or Sapindus detergens

Roxb, soapnut is a perennial tree belonging to the family Sapindaceae, indigenous to northern India. Oil from the seed kernel of soapnut is of interest to the soap industry. The oil is quite useful industrially because of its most valuable phytochemicals like saponins or triglycerides10

• The exhausted cake is used as a filler and fertilizer and the shells for making lignin based adhesives or boards 11

• Vegetative propagation of soapnut did not yield satisfactory results and propagation through seed is unreliable because the per cent survival of the seedlings proved to be meagre due to heavy incidence of mortality at seedling stage in the natural . habi-at12

•

Micropropagation of soapnut tree is at a stage of infancy in forest tree species which has great importance in the soap industry and social forestry programmes. In this communication for the first time an in vitro micropropagation method for Sapindus

*Present address: Dr. N.S. Philomina, Oo. Smt. Y. Elizabethamma, H.S.G. II P.A. Head Post Office, Cuddapah 516 001, India

mukorossi tree using apical and axillary meristem explants has been presented.

Materials and Methods Seeds of soapnut were obtained from Biotechnology

Research Centre for Tree Improvement (BIOTRIM), Andhra Pradesh Forest Department Nursery, Tirupati and soaked in cone. H2S0 4 for 90 min and washed thoroughly with running tap water. The seeds were surface sterilized with 0.1 % HgCI2 for 15 min and rinsed several times with sterile distilled water. Agar water medium (0.8%) without growth regulators was used for seed germination. Seeds inoculated on this medium were incubated at 24° ± 2° C in the dark or light at a photon flux density of 15 11Em·2 S of white fluorescent tubes for thirty days after which seedlings were used for apical and axillary meristems dissection . The apical and axillary meristems (2-4 mm) were collected from one month old aseptic seedlings and cultured on Murashige and Skoog13

(MS) basal medium containing 2% sucrose and lower concentrations of BAP or KIN ranging from 0.4, 0.8, 1.7 and 2.6 1-l.M, higher concentrations ranging from 4.4,8.8 and 13.2 1-l.M, combination of BAP and KIN 0.4 or 0.4 1-l.M, 13.2 and 13.2 11M and combination of BAP or KIN with auxin naphthalene acetic acid (NAA) 0.4 and 0.4 11M to 13.2 and 13.2 11M were used for inducing muliple shoots. For muliplication and elongation of established shoots different combinations of GA, (0.5 and 2.8 11M) alone, . . combination of BAP and GA3 (0.4 and 0.5 1-l.M, 0.8 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, 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 at 25° ± 2° C in the light (16hr photoperiod) . Rooted plantlets were acclimatized gradually in a green house. Results are mean of three culture cycles with 20 replicates per experiment.

Results and Discussion Experiments on explant type, shoot tips and

axillary meristems for multiple shoot formation were tested. In all the BAP concentrations tested, 0.4 and 0.8f.tM concentrations were more effective for

inducing 6 to 8 multiple shoots within a month from axillary meristems. However on the same medium shoot tips were proliferated and 4 to 6 shoots were formed in 4-5 weeks. Addition of hi gher concentration of BAP (8 .8 and 13 .2 f.!M) to MS medium induced more callus formation in both the explants within a week from the cut surface, and along the surface from the apical to basal part of the explants. Initiation of callus was faster in all the higher concentrations of BAP. In order to test the 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.

[Values 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 2.6 + 4.4 + 8.8 +

13.2 + BAP 0.4+0.4 1.0 ± 0.0 +KIN

13.2+1 3.2 1.0 ± 0.0 BAP 0.4+0.4 + +NAA

13 .2+13.2 +

Culture response scored 30 days after inoculation +=callusing on the ex plants

from apical and axillary meristems were separated and recultured on to the same shoot multiplication media (MS with 0.4 or 0 .8 f1M BAP) and shoot multiplication was determined after second and third subcultures. Highest number of shoots (8-1 0) were recorded from a single explant within three weeks (Fig. I). No increase in shoot multiplication was observed by prolonging the culture period beyond sixth subculture.

Shoots obtained by this method were divided into 5-8mm nodal explants with single axillary bud for further proliferation to increase the number of shoots. These buds proliferated into 5 to 8 multiple shoots in 4 weeks on MS medium with BAP 0.4 and 0 .8f.l.M individually. Experiments conducted with BAP in combination of kinetin and auxin showed single shoots with callus formation. Of the two cytokinins BAP was most effective for inducing bud break and shoot proliferation in apical and axillary meristem (Table 1) . Similar results were reported in Madhuca I if. [' 14 at1 ow .

Within 8 weeks of culture, the regenerated shoots elongated upto 2-3 em in height. Prolonged culture on the same medium did not increase the shoot length . For shoot elongation the shoots were separated and grown on MS medium with GA3 (0.5 , 2.8J.1M)in combination with BAP (0.4, 0 .8 f.l.M) treatments. The shoots were elongated upto 5 to 7 em in 4 weeks in all the BAP and GA3 treatments. Lower concentrations of BAP (0.4 or 0.8 JlM) were favorable for bud proliferation and application of GA 3 to these shoots

1.0 ±0.0 2.0±0.119 3.1 ±0.119 1.0 ± 0.0 + 1.9±0.115

+ + 2.0±0.115 + + + + + +

1.0 ± 0.0 2.1 ±0.122 2.7±0.169

1.0 ± 0.0 2.3 ±0.123 2.9 ± 0.217 + + +

+ + +

Table 2-Effect of auxins on in vitro rooting of regenerated shoots of S. mukorossi after 30 days of inculbation

Auxins

Control IBA

NAA

IBA + NAA

Concentrations ( J..LM)

0.0 0.4 2.4 3.4 4.9 0.5 2.6 5.3 7.9

0.4 (each) 2.4 (each)

* 20 replicates/treatment repeated thrice +=callusing at the basal end

Mean percentage of rooting (± SE) *

0.0 10.5 ± 0.275 18.9 ±0.260 68.0 ± 0.478 59.0 ± 0.366

+ + + + + +

increased their length. For elongated multiple shoot formation with a combination of BAP (0.4JlM) and GA3 (2.8 JlM) was optimum. In the present study combination of GA3 with a cytokinin was effective in inducing shoot elongation . Different concentrations of GA3 alone failed to increase the shoot elongation however when GA3 was applied in combination with BAP effectiveness of gibberellic acid was improved in causing shoot elongation . Application of GA3 to in vitro regenerated shoots increased their length in Azadirachta indica15

•

The regenerated shoots were transferred to MS medium with IDA and NAA of different concentrations for rooting. Among these concentrations the 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 2.4~ induced callus at the base of the shoots with poor rooting after 30 days of incubation (Table 2). A combination of IBA with NAA inhibited roots formation and showed only callus at the basal cut ends.

Regenerated plantlets were transferred to plastic containers fill ed with vermiculite. During first week the potted plantlets were covered with polythene bags to provide high humidity. Transplantation success was 60% (Fig. 3). Plantlets were subsequently transferred to larger pots and gradually acclimatized to outdoor conditions. In the present study multiplication by multiple shoot methods from shoot tip or ax ill ary meristems was developed for successful in vitro propagation of Sapindus mukorossi an economically important tree.

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