Agric. Sci.

Digest, 23 (1) : 29 - 31, 2003

EXUDATION AND BROWNING IN TISSUE CULTURE
OF POMEGRANATE
Ashutosh A. Murkute, Shanti Patil and Mayakumari
Tissue Culture Laboratory, Department of Agricultural Botany,
College of Agriculture, Nagpur- 440 001, India
ABSTRACT
The browning of cultures is a major obstacle in establishment of expIants in perennial fruit crops,
which subsequently makes the in vitro techniques more cemplicated. Pomegranate Punica granatum L.
has the same problem in establishment of in vitro cultures due to high phenolic contents. Several
treatments were tried including use of adsorbents and antioxidants with different explants viz., leaf
segment, cotyledon, nodal segment and shoot tip. The activated charcoal (adsorbent) and ascorbic acid
(antioxidant) could not alter the extent of browning at various concentrations. The subculturing of
explants thrice, at an interval of 24 hrs. controlled browning in all explants used, except cotyledon. The
cotyledon explant was deprived of any browning secretion. .
Tissue culture techniques are
becoming increasingly popular nowadays as
alternative means of vegetative propagation
and conventional plant breeding. The
micropropagation provides a hew way to
produce a large quantity of. elite clones from
micro plant parts and somaclonal variation is
as excellent method to get desired characters
in plants by screening. Exploiting these
facilities, a huge progress has been made in
developing the protocols for number of plant
species including fruits, vegetable and
ornamental plants. But these techniques receive
a set back by certain physiological processes
which hinders the success of new technique,
particularly in perennial fruit crops.
All the tissues have phenolic
compounds including growing cells. Oxidation
of phenolic compounds released from the cut
ends of explants by polyphenoloxidases,
peroxidases cause lethal. browning of explant
and culture medium(Bhat and Chandel, 1991).
The problems associafed with oxidation of
polyphenols are almost coupled in crops like
pomegranate, mango, apple etc. In some
. species the establishment of explants frequently
requires special procedures to escape or avoid
problems associated with oxidation of
polyphenols. In this study, therefore an attempt
was made to control browning of cultures to
get successful establishment of explants of
pomegranate, Pl.!nicagranatumcv. Ganesh by
trying different methods.
The study was carried out using
different explants viz., nodal segment, shoot
tip, leaf segment and The explants
other than cotyledon were taken from 7-8 year
matured juvenile trees maintained in orchards.
The cotyledon explant was taken by raising the
seedlings in green house. The MS (Murashige
and Skoog, 1962) basal medium was used in
the study fortifying it with different agents and
other as mentioned in Table 1 and
the results are presented in the same.
The browning of cultures started within
24 hrs. of inoculation in all explants cotyledon
(Plate 1 and 2). The browning secretions were
first noticed at the cut surfaces of explants and
the leaching gradually spread into surrounding
media. The growth of explants was completely
stopped and the cultures died within three days
of inoculation. The cotyledon explant did not
show any leaching from cut ends and got
established well. The response of browning or
blackening results from damaging of cells
followed by mixing of cellular contents such as
enzymes and their substrates that are normally
held apart. They might have a role in protecting
injured tissues from infection and decay
30 AGRICULTURAL SCIENCE DIGEST
Table 1. Efiect of di!lerent treatments on browning
Treatments
Shoot tip
Response of explants
Nodal Leaf
segment segment
Cotyledon
A) MS+ Activated charcoal
1) 1 g L-\
2) 2 g L-t
3) 3 9 L-t
B) MS + Ascorbic acid
1) 50 mg Ll
2) 100 mg L-l
31150 mg L-t
C) Subculture of explants
1) 1 day after inoculation
2) 1 and 2
nd
day after inoculation
3) 1". 2"" ~ 3
0d
~ y after inoculation
+++
+++
++
+++
+++
++
++
++
+
+++
+++
++
+++
+++
++
++
++
+
+++
+++
++
+++
+++
++
++
+
+
+++
++
+
High browning
Moderilte browning
No browning
Browning not noticed after inoculation.
Plate 1. Browning of leaf segment explant cultures Plate 2. Browning of nodal stem explant cultures
Zimmerman (1978) are also of same opinion
to control browning by subculturing. -The
exception of cotyledon explant for recording
no leaching may be due to the reason that new
young cells in cotyledon are devoid of such
chemicals.
In conclusion, it is suggested either to
use cotyledon as explant or subculturing of
other explants (leaf, shoot tip and nodal
segment explant) for better establishment of
cl.lltures by preventing browning caused by the
oxidation of phenolics secreted.
Vol. 23, No.1, 2003
(Compton and Preece, 1986). The results
observed indicated that addition of external
agents Le., antioxidants (ascorbic' acid) and
adsorbents (activated charcoal) cdl.lld not prove
efficient in controlling browning of cultures in
different concentrations and recorded high to
moderate browning. When explants were
subcultured at regular intervals, it gave
encouraging results. The subcl.llturing of explant
consecutively thrice at an interval of 24 hrs.
controlled browning completely. N'aik et ai.
(1999) were also ableto overcome this problem
by transferring the expl.ants to a fresh medium
devoid of any external agent. Broome and
REFERENCES
Bhat, S.R. and Chandel,KP.J. (1991). PI. eellReports, 10: 358-361.
Broome, a.c. and Zimmerman, R.H. (1978). Hort. Sci., 23: 151-153.
Compton, M.E. and Preece, J.E. (1986). LA.p.T.e News/ett., 50: 9-18.
Murashige, T.and Skoog, E(1962). Physiol. PI., 15: 473-497.
Nook, S.K. et aJ. (1999). Scientia Hort., 79: 175-183.
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