Kulkarni, p.K.. P.K. Gupta and A.F. Mascarenhas1- Biochemistry
Division, National Chemical Laboratory, Poona 411 008, India.
TISSUE CULTURE STUDIES ON LEUCAENA LBDCQCBEBMA
The main objectives of the present studies were to standardize
tissue culture methods for the rapid multiplication of leucaena
trees and to isolate callus and suspension cultures for evolving
plants resistnat to 'gummosis' diseases or having relatively low
levels of mimosine.
Materials and Methods. Nodal segments were collected from 1 to 2
year old plants. Stem segments (20-30mm) were sterilized with
0.1% HgCl, for 25 min and 10mm nodal segments were inoculated
into different media test tubes (25 x 100mm). Standard tissue
culture methods were used3-'2 for the sterilization of glassware,
plant materials and media.
The main culture media tested were the following. Supplements^
were either added or omitted from Murashige and Skoog's medium
and the media designated as:
MS
MS-1
MS-2
MS-3
Murashige and Skoog's medium3 without kinetin (Kn), indole
acetic acid (IAA) and edamin and with 2% sucrose.
MS supplemented with Kn (0.2 mg/1) + BAP (0.3 mg.l) +
calcium pantothenate (0.1 mg/1) + biotin (0.1 mg/1).
MS (1/2 strength) liquid + IAA + IBA + IPA + NAA (each
added at 2.0 mg/1).
MS (1/2 strength) + activated charcoal (0.25%).
Medium was distributed in 20ml amounts per tube. Semisolid media
(MS-1 and MS-3) contained 0.8% Difco agar. Test tubes containing
liquid medium were provided with a filter paper support. All
cultures were incubated at 25 ± 2°C and were illuminated for 18 h
with light from fluorescent tubes (1000 lux) .
The methods followed for standardization of the medium consisted
of systematically testing the effects of different growth
hormones and other growth regulators to determine their optimum
levels for obtaining a particular response, either for shoot or
root formation.
Results and Piscussion?
Multiple shoot formation: In order to determine the optimal
requirements for shoot formation the effect of cytokinins viz.
kinetin (Kn).and benzyl aminopurine (BAP) were tested at
different concentrations alone or in combination with MS basal
medium. Maximum number of shoots were obtained in medium
containing both Kn and BAP (Fig. 1). This effect was enhanced if
calcium pantothenate and biotin were also supplemented to the
3To whom all correspondence should be addressed.
2NCL Communication No. 3280
above medium (MS-1). Of-naphthalene acetic acid (NAA), 2,4dichlorophenoxy acetic acid (2,4-D) tested individually at
differect concentrations retarded shoot formation and only a
mass of undifferentiated cells (callus) developed from the cut end
of the explant (Fig. 2). On medium MS-1 the dormant buds
elongated and shoots attained a height of 30-40mm within 25-30
days. At this stage the shoots were removed and stem segments
contaning one leaf were excised separately and transferred to
fresh medium, where each segment forms 1-2 shoots within 20-30
days. This sequence of transfer to fresh medium has been
repeatedly carried out without any reduction in the number of
shoots formed at each subculture.
Figure 1
Figure 2
Figure 3
Figure 1:
Figure 2:
Multiple shoots on MS 1 medium
Callus formation on MS-1 medium supplemented with 2,4-
Figure 3:
Rooted plantlet of leucaena on MS-3 medium
D + NAA.
Root formation: The second stage before the formation of
plantlets involves rooting of shoots. To achieve this, elongated
shoots (30-40mm) were excised and treated with MS-2 liquid
containing an auxin mixture for 72 h in dark. Following this
treatment the shoots were transferrred to a semisolid medium
containing 0.25% activated charcoal (MS-3) and incubated in light
25 + 2°C (16 h photoperiod). Within 15-20 days on this medium
rooting resulted in 70% of the shoots (Fig. 3).
Further studies on transfer of these plantlets to field and also
on differentiation of shoots from callus or suspension cultures
to isolate mutants are in progress.
The clonal propagation method developed by us which is suitable
for mature (1-2 years old) trees could immediately find
application in the mass multiplication of naturally occurring
variants characterized by resistance to gummosis or containing
relatively low levels of mimosine.
References:
1Mascarenhas,
A.F., S. Hazra, U. Potdar, D.K. Kulkarni and
P.K. Gupta. 1982. Plant Tissue Cultrue Proc. 5th Inti.
Cong. Plant Tisue and Cell Culture, Tokyo, Japan, Ed.
Fujiwara, A. p. 719.
2
Nadgauda, R.S., A.F. Mascarenhas and V. Jagannathan.
Ind. J. Expt. Biol. 1£, 120.
3Murashige,
T. and F. Skoog.
1962.
1978.
Physiol. Plant, 15:473.