Indian Journal of Biotechnology

Vol 9, July 2010, pp 336-337

Short Communications

Somatic embryogenesis from leaf explants

of soapnut (Sapindus mukorossi Gaertn.)

N S Philomina*

Department of Botany, Yogi Vemana University,

Kadapa 516 003, India

Received 5 October 2009; revised 27 November 2009; accepted

25 January 2010

Plant regeneration through somatic embryogenesis has been

developed in an economically important forest tree, Sapindus

mukorossi Gaetin. Calli obtained by culturing young leaf explants

on MS medium containing growth regulators, 2,4-D (6.7 µM)

and Kn (9.0 µM), when subjected to reduced levels of 2,4-D

(2.2µM)+Kn (4.6µM), produced numerous somatic embryos.

Somatic embryos developed into complete plantlets on MS

medium devoid of growth regulators. The regenerated plantlets

were successfully established in the soil with 90% survival

frequency after a few days of acclimatization.

Keywords: Soapnut, somatic embryogenesis, callus, regeneration,

Sapindus mukorossi

Soapnut (Sapindus mukorossi Gaertn.) is an

economically important forest tree and has been

brought under silvicultural practices in southern

parts of India1. Since the pulp of the fruits is

used as a substitute for soap, it has great

importance in the soap industry as well as in Social

forestry programmes2. Conventional propagation

rate through stem cuttings is very slow and

per cent survival of plant progeny raised from

seeds also proved to be meagre due to heavy

incidence of mortality at seedling stage in the

natural habitat. Though in vitro propagation of

soapnut via meristematic cultures and seed cultures

has been reported earlier, there are no reports

on its regeneration through somatic embryogenesis3-5

.

Induction of somatic embryogenesis is an

important route for the regeneration of plants

on a large-scale from cell cultures. Therefore,

the present study was conducted for rapid

multiplication of soapnut through somatic

embryogenesis from leaf explants.

Healthy leaves were taken from 60-d-old

aseptically grown seedlings. The leaves were cut into

small segments (1.5-2.0 cm) to serve as explants.

These were then aseptically cultured on Murashige

and Skoog (MS)6 medium supplemented with various

concentrations of 2,4-dichlorophenoxyacetic acid

(2,4-D; 2.2, 4.5, 6.7 & 9.0 µM) + kinetin (Kn; 1.0, 4.6,

9.0 & 13.9 µM)+agar (0.8% w/v) for callus induction.

To induce somatic embryos, calli produced on the

most suitable concentration of 2,4-D were used.

Only green friable calli were selected for culturing

on MS medium supplemented with a combination

of 2,4-D (2.2 µM)+Kn (4.6 µM) to study the

embryogenic potentialities. For germination of

somatic embryos, MS medium without growth

regulators was used. The cultures were incubated at

24±2ºC and an illumination of 3000 lux was given

with 16/8 h (light/dark) periods. The experiments on

somatic embryogenesis and shoot regeneration were

replicated thrice.

The explants cultured on MS+2,4-D (6.7 µM)+Kn

(9.0 µM) + agar (0.8% w/v) exhibited callus initiation

at the cut ends of the explants after 2-3 d of

incubation. Creamy white callus developed all over

the explant within 2 wks. The callus induction

medium induced excellent callusing (82.5%)

(Fig. la).

The primary calli obtained from the cultured

leaf explants, when sub-cultured on MS medium

supplemented with 2,4-D (2.2 µM) in combination

with Kn (4:6 µM) produced green embryogenic

callus after 7th d of incubation (Fig. 1b). The

embryogenic callus was further differentiated into

somatic embryos within a wk. An average of

40-50 somatic embryos were obtained from 50 mg

of embryogenic callus. Somatic embryogenesis

has already been reported in some woody plants

viz., bamhoo (Dendrocalanus strictus)7, teak

(Tectona grandis)8, amla (Emblica officinalis)

9 and

sandal wood (Santalum album)10

.

The somatic embryos turned bipolar (Fig. 1c), and

showed various stages of embryo development such

as heart shaped (Fig. 1d) and cotyledonary stages

(Fig. 1e). Although a large member of somatic

embryos in the cotyledonary stage were produced,

many reverted to callusing (Fig. 1f). It has been

reported that auxins caused an undesirable callusing

in Wrightia tinctoria cultures11

.

Somatic embryos were transferred to hormone-free

medium (MS+ agar 0.8% w/v) for shoot regeneration.

SHORT COMMUNICATIONS

337

After 5 d of culture the somatic embryos germinated

into 2-3 cm long shoots with nodes, healthy leaves

and tap root system and finally 90% of them

developed into plantlets (Fig. 1g). The regenerated

plantlets were hardened using vermiculite, sand and

soil (1:1:1) without any adjuvants. The regenerated

plants were established in soil with 90% survival

frequency (Fig. 1h). The protocol established in the

present study serves as an alternative means for the

multiplication of soapnut on large-scale in a relatively

shorter time.

References 1 Troup R S, The silviculture of Indian forest trees, Vol I

(Claredon Press, Oxford, UK) 1921, 232.

2 Dev I & Guha S R D, Glyceride composition of Sapindus

mukorossi (soapnut) oil, Indian J For, 2 (1979) 261-263.

3 Philomina N S & Rao J V S, Micropropagation of Sapindus

mukorossi Gaertn., Indian J Exp Biol, 38 (2000) 621-624.

4 Philomina N S & Rao J V S, Multiple shoot production from

seed cultures of soapnut (Sapindus mukorossi Gaertn.),

Phytomorphology, 49 (1999) 419-423.

5 Wiliams E G & Maheswaran G, Somatic embryogenesis:

Factors influencing coordinated behaviour of cells as an

embryogenic group, Ann Bot, 57 (1986) 443-462.

6 Murashige T & Skoog F, A revised medium for rapid growth

and bioassays with tobacco tissue cultures, Physiol Plant, 15

(1962) 473-497.

7 Rao U, Rao I V R & Narang V, Somatic embryogenesis and

regeneration of plants in bamboo, Dendrocalamus strictus,

Plant Cell Rep, 4 (1985) 121-124.

8 Kushalkar R & Sharon M, Direct and indirect somatic

embryogenesis in teak (Tectona grandis L.), Curr Sci, 71

(1996) 712-714.

9 Zhang S, Yao Y, Ren H, Wang K & Zhang S Y, A

preliminary study on rapid propagation in vitro of emblica,

For Res Beijing, 15 (2002) 116-119.

10 Bapat V A & Rao P S, Somatic embryogenesis and plantlet

formation in tissue cultures of sandalwood (Santalum album

L.), Ann Bot, 44 (1979) 629.

11 Purohit S D & Kukda G, Micropropagation of an adult tree,

Wrightia tinctoria, Indian J Biotechnol, 3 (2004) 216-220.

Fig. l—Somatic emhryogenesis in S mukorossi: a, Initiation

of callus from leaf explants on MS medium containing 2,4-D

(6.7 µM) +Kn (9.0 µM); b, Formation of embryogenic callus on

MS medium containing 2,4-D (2.2 µM) + Kn (4.6 µM); c, Bipolar

somatic embryo loosely attached to the embryogenic callus;

d, Heart shaped somatic embryo; e, Cotyledonary stage of somatic

embryo; f, Cotyledonary stage of somatic embryo showing

callusing; g, Complete plant formation from somatic embryo on MS

basal medium; & h, Establishment of regenerated plants in the soil.