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Indian Journal of Experimental Biology

Vol. 38, June 2000, pp. 621-624


Micropropagation of Sapindus mukorossi Gaertn
N S Philomi na* & 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 (GA
3
) 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
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
wood
2
and Rose wood
3
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 Leucaena
4
, Albizia
5
and Acacia
6
. Shoot tip
cultures were established from germinated seedlings
in Redsanders
7
and Teak
8
So far there are very few
reports of Sapindaceae like Sapindus trifoliatus
9
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 trigl yceri des
10
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 unreliabl e
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-at
12

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, Indi a
mukorossi tree using apical and axillary meri stem
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. H
2
S0
4
for 90 min and washed
thoroughly with running tap water. The seeds were
surface sterilized with 0.1 % HgCI
2
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 meri stems
di ssection. The apical and axillary meristems (2-4
mm) were collected from one month old aseptic
seedlings and cultured on Murashige and Skoog
13
(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 GA
3
(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
~ MS + 3.4 J.lM IBA + 2% sucrose after 15 days of culture; 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 regul ators 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 6.00.20 8.00.90 2.00.109 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 inocul ati on
+=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) . Simil ar 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 GA
3
(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 GA
3
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 2.00.119 3. 1 0.119
1.0 0.0 + 1.90.115
+ + 2.00.115
+ + +
+ + +
1.0 0.0 2.1 0.122 2.70.169
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 repli cates/treat ment repeated thri ce
+=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
GA
3
(2.8 JlM) was optimum. In the present study
combination of GA
3
with a cytokinin was effective in
inducing shoot elongation. Different concentrations of
GA
3
alone failed to increase the shoot elongation
however when GA
3
was applied in combination with
BAP effectiveness of gibberellic acid was improved
in causing shoot elongation. Application of GA
3
to in
vitro regenerated shoots increased their length in
Azadirachta indica
15

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 pl antlets were covered with polythene bags
to provide high humidity. Transpl antation 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 axill ary
meri stems was developed for successful in vitro
propagation of Sapindus mukorossi an economi cally
important tree.
References
I Gupta P K, Meht a UJ & Mascarenhas A F, A tissue culture
method for clonal propagation of mature trees of Eulayptus
torelliana and Eucalyptus camaldulensis, Plant Cell Rep, 2
( 1983) 296.
2 Rao PS & Bapat VA, Vegetative propagati on of sandalwood
plants through tissue culture, Can J Bot, 56 ( 1978) 1153 ..
3 Lakshmi Sita G & Raghava Swamy BY, Regeneration of
plantlets from leaf di sc cultures of rose wood: control of leaf
abscission and shoot tip necrosis, Plant Sci, 88 ( 1993) 107.
4 Dhawan V & Bhojwani SS, In vitro vegetat ive propagat ion of
Leucaena leucocephala Landewit, Plalll Cell Rep, 4 ( 1985)
3 15.
5 Upadhyaya S & Chandra N, Shoot and Plantl et formation in
organ and callus cultures of Albizia lebbeck Benth, Ann Bot ,
52 ( 1983) 421.
6 Mittal A, Agarwal R & Gupta SC, In vitro development of
Pl ant lets from axillary buds of Acacia auriculariformis, Plant
Cell Tiss Org Cult , 19 (1990) 65.
7 Lakshmi Sita G, Sreenatha KS & Suj ata S, Plantlet production
from shoot tip cultures of red sandal wood (Pterocarpus
santalinus L), Curr Sci, 7 ( 1992) 532.
8 Sunitibala Devi Y, Mukherjee BB & Gupta S, Rapid cloning
of elite Teak (Tectono grandis L) by in vitro mulipl e shoot
production, Indian J Exp Bioi, 32 ( 1994) 668.
9 Desai HV, Bhatt P N & Mehta A R, Pl ant regeneration of
Sapindus trifoliatus L (soapnut) through somati c
embryogenesis, Plant Cell Rep, 3 ( 1986)-190.
10 Dev I & Guha S R D, Glyceride compos iti on of Sapindus
mukorossi (soapnut) oil , Indian J For, 2( 1979) 261.
II Karnik M G, Sharma 0 P & Dev I, Studi es on the chemi cal
composition and possible utiliti es of soapnuts (Sapindus
mukorossi) Indian For, 8 ( 1971) 462.
12 Troup R S & Sapindaceae, The silviculture of Indian forest
trees, Vol. I (Claredon Press, Oxford U.K (1 92 1) 232.
13 Murashi ge T & Skoog F, A revised medi um for rapid growth
and bi oassays wi th tobacco ti ssue cultures , Physiol Plant, 15
(1962) 473.
14 Rout C R & Das P, Mi cropropagat ion of Madhuca longifolia,
Plant Cell Rep, 12 ( 1993) 5 13.
15 Ramesh K & Padhya M A, In vitro propagation of neem
(Azadirachta indica A. Juss), Indian J Exp Bioi, 28 ( 1990)
932.