IN VITRO MUTAGENESIS IN GRAPE AND ITS VALIDATION USING MOLECULAR MARKERS
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Date
2015
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DIVISION OF FRUITS AND HORTICULTURAL TECHNOLOGY INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
Abstract
The present investigation entitled “In vitro mutagenesis in grape and its validation using molecular markers”
was undertaken during the years 2010-2013. The experiments were carried out at the Division of Fruits and
Horticultural Technology, Indian Agricultural Research Institute, New Delhi. The field-grown 20-year-old
mother plants of the four juice and wine making grape genotypes, viz. Pusa Navrang, H-76-1 (Hur x Cardinal),
Pearl of Csaba and Julesky Muscat were selected for the study. Experiments were conducted on comparative in
vitro multiplication of the genotypes. The second experiment was conducted to standardize the in vitro
mutagenesis and to isolate putative mutants. These mutants were identified based on the morphological and
biochemical parameters based on the morphological parameters, which were then subjected to molecular
analysis molecular analysis, viz. RAPD (Random Amplified Polymorphic DNA) and SSR (Simple sequence
repeat) for the detection of genetic polymorphism among grape mutants and parent genotypes thus identifying
true mutants.
Highest culture in vitro establishment recorded in the genotype Pusa Navrang (45.34%) followed by in
Julesky Muscat (44.00%). Whereas, lowest culture establishment (39.96%) was recorded in the Pearl of Csaba.
Among the different growth regulators tried for highest culture establishment (73.85%), minimum day to
axillary bud sprout (9.31) and rooting (16.46%), maximum roots length (6.28 cm), highest number of roots
(8.03) and shoots per explant was noted with 2.0 mg l-1 BAP + 0.2 mg l-1 NAA treatment, followed by 4.0 mg l-
1 BAP + 0.2 mg l-1 NAA. The lowest (31.30%) culture establishment at 45 day after inoculation was noted in
the treatment comprising 2.0 mg l-1 kinetin. Multiplication rate was recorded maximum in Pusa Navrang (5.6
per sub-culture) followed by Pearl of Csaba (5.1 per sub-culture). Among the growth treatments, the maximum
multiplication rate (8.5 per sub-culture) was found in the treatment 2.0 mg l-1 IBA + 200 mg l-1AC. The
plantlets in the sterilized coco peat + vermiculite + perlite (2:1:1) in the glass jar (T1) were found to be the
effective means of in vitro plantlet hardening which gave the highest survival (85.97%).
In irradiation experiment the highest mean survival was noted in the control (83.78%) over other irradiation
dosages except 5 Gy doses (80.95%), which was at par with the control. The gamma irradiation dose of 10 Gy
gave the survival of 46.66%, which was significantly different higher to other dosages. The 10 Gy irradiation
dose was recorded as a lethal dose 50 (LD50). However, as the irradiation dose was further increased, the
explant survival significantly decreased. The highest shoot bud sprouting was noted in Pusa Navrang (57.59%)
followed by Pearl of Csaba (55.59%), while it was lowest in Julesky Muscat (51.23%). Lower irradiation dose
5 Gy (89.73%) gave good shoot bud sprouting compared to higher doses. Mean shoot abnormalities was
significantly the highest in H-76-1 (50.29%) followed by Julesky Muscat (49.38%), which were at par with
each other. Among the irradiation doses, 5 Gy gave only about 3.61% shoot abnormalities, which further
increased in vitro shoot abnormalities, i.e. LD50 dose (10 Gy) as 50.59%, (15 Gy) 67.750% and (20 Gy)
78.56%. In general, as the irradiation dose increased the mean leaf abnormalities were increased.
Initially 26 RAPD and 11 SSR primer used for screening and out of these four RAPD and six SSR markers
were found polymorphic for diversity analysis of the in vitro developed mutants and parent genotyes. The size
of the RAPD and SSR amplification products ranged from the 250 to 2500 bp and 110 to 290 bp, respectively.
Primers OPA01, OPP04 (RAPD) and VVMD-14 (SSR) were the unique primers. The treatment 15 Gy induced
two solid mutants followed by the LD50 dose (10 Gy); while in lower irradiation dose; mutant was similar to
mother plants. In Pusa Navrang, two primers OPP02 (RAPD) and VVMD-21 (SSR) differentiated uniquely. In
Pearl of Casaba, OPA01 (RAPD) and VMC8G9 (SSR) were the unique primers. Nine gamma rays induced
mutants were identified in H-76-1 genotype. Whereas, three solid mutants were identified out of eight putative
mutants in Julesky Muscat. Results suggest that RAPD-OPA1 and SSR-VVMD-14 were the most informative
primers for genetic analysis for the grape genotypes and their putative mutants.
It can be concluded that MS medium supplemented with BAP (2 mg l-1
) + NAA (0.2 mg l-1
) gave good culture
initiation, while MS medium with IBA (2 mg l-1
) + AC (200 mg l-1
) was effective in rapid multiplication
through repetitive micro-cuttings. Hardening of rooted plantlets in glass jar with polypropylene (PP) cap was a
better strategy. The LD50 of gamma rays was observed at 10 Gy irrespective of genotypes. Putataive mutants
generated showed sufficient genetic variability. Results suggest that RAPD-OPA1 and SSR-VVMD-14 were
the most informative primers for genetic variation analysis for the four genotypes and their mutants.
Description
t-9312
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