Agric. Sci. Digest, 23 (1) : 29 - 31, 2003
EXUDATION AND BROWNING IN TISSUE CULTUREOF POMEGRANATE
Ashutosh A. Murkute, Shanti Patil and MayakumariTissue Culture Laboratory, Department of Agricultural Botany,
College of Agriculture, Nagpur- 440 001, India
ABSTRACTThe 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. Severaltreatments were tried including use of adsorbents and antioxidants with different explants viz., leafsegment, 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 ofexplants thrice, at an interval of 24 hrs. controlled browning in all explants used, except cotyledon. Thecotyledon explant was deprived of any browning secretion. .
Tissue culture techniques arebecoming increasingly popular nowadays asalternative means of vegetative propagationand conventional plant breeding. Themicropropagation provides a hew way toproduce a large quantity of. elite clones frommicro plant parts and somaclonal variation isas excellent method to get desired charactersin plants by screening. Exploiting thesefacilities, a huge progress has been made indeveloping the protocols for number of plantspecies including fruits, vegetable andornamental plants. But these techniques receivea set back by certain physiological processeswhich hinders the success of new technique,particularly in perennial fruit crops.
All the tissues have phenoliccompounds including growing cells. Oxidationof phenolic compounds released from the cutends of explants by polyphenoloxidases,peroxidases cause lethal. browning of explantand culture medium (Bhat and Chandel, 1991).The problems associafed with oxidation ofpolyphenols are almost coupled in crops likepomegranate, mango, apple etc. In some
. species the establishment of explants frequentlyrequires special procedures to escape or avoidproblems associated with oxidation ofpolyphenols. In this study, therefore an attemptwas made to control browning of cultures to
get successful establishment of explants ofpomegranate, Pl.!nicagranatumcv. Ganesh bytrying different methods.
The study was carried out usingdifferent explants viz., nodal segment, shoottip, leaf segment and cotyl~don. The explantsother than cotyledon were taken from 7-8 yearmatured juvenile trees maintained in orchards.The cotyledon explant was taken by raising theseedlings in green house. The MS (Murashigeand Skoog, 1962) basal medium was used inthe study fortifying it with different agents andother ~reatments as mentioned in Table 1 andthe results are presented in the same.
The browning of cultures started within24 hrs. of inoculation in all explants cotyledon(Plate 1 and 2). The browning secretions werefirst noticed at the cut surfaces of explants andthe leaching gradually spread into surroundingmedia. The growth of explants was completelystopped and the cultures died within three daysof inoculation. The cotyledon explant did notshow any leaching from cut ends and gotestablished well. The response of browning orblackening results from damaging of cellsfollowed by mixing of cellular contents such asenzymes and their substrates that are normallyheld apart. They might have a role in protectinginjured 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 Leafsegment segment
Cotyledon
A) MS+ Activated charcoal1) 1 g L-\2) 2 g L-t3) 3 9 L-t
B) MS + Ascorbic acid1) 50 mg Ll2) 100 mg L-l31150 mg L-t
C) Subculture of explants1) 1~ day after inoculation2) 1~ and 2nd day after inoculation3) 1". 2"" ~nd 30d d~y after inoculation
++++++
++
++++++
++
++++
+
++++++
++
++++++
++
++++
+
++++++
++
++++++
++
++++
++++++
High browningModerilte browningNo browningBrowning 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 opinionto control browning by subculturing. -Theexception of cotyledon explant for recordingno leaching may be due to the reason that newyoung cells in cotyledon are devoid of suchchemicals.
In conclusion, it is suggested either touse cotyledon as explant or subculturing ofother explants (leaf, shoot tip and nodalsegment explant) for better establishment ofcl.lltures by preventing browning caused by theoxidation of phenolics secreted.
Vol. 23, No.1, 2003
(Compton and Preece, 1986). The resultsobserved indicated that addition of externalagents Le., antioxidants (ascorbic' acid) andadsorbents (activated charcoal) cdl.lld not proveefficient in controlling browning of cultures indifferent concentrations and recorded high tomoderate browning. When explants weresubcultured at regular intervals, it gaveencouraging results. The subcl.llturing of explantconsecutively thrice at an interval of 24 hrs.controlled browning completely. N'aik et ai.(1999) were also ableto overcome this problemby transferring the expl.ants to a fresh mediumdevoid of any external agent. Broome and
REFERENCESBhat, 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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