Genetic stability of micropropagated and in vitro cold-stored strawberries Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/zc77ss28t

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  • Genetic stability of micropropagated and cold-stored Fragaria x anannasa cultivar 'Pocahontas' was evaluated by Random Amplified Polymorphic DNA (RAPD) assay. Plantlets grown for eight months on M & S medium with 5 μM and 15 μM N6- benzyladenine (BA) were analyzed. No evidence of mutations was seen in over 200 loci that were PCR amplified with 29 of the 30 Operon primers used. Pocahontas in vitro plantlets stored for over 4 years in gas-permeable plastic bags at 4°C also showed no evidence of mutations for the same loci. One primer, OPF-18 (TTCCCGGGTT), generated DNA profiles which were polymorphic for all the BA treated and cold-stored plantlets analyzed. The polymorphism seen was not completely reproducible and no pattern matching any treatment was seen. Southern blots in concert with Hpa II and Msp I restriction enzymes were used to assay for methylation pattern changes. No differences from control plants were seen in most of the samples. One sample from 15 μM BA treatment and one cold-stored sample displayed methylation on the external cytosine in the CCGG restriction site which was not seen in controls or in other samples. Field-grown plants of BA and cold-stored treatments showed methylation patterns similar to that of controls. This indicates that changes seen were due to tissue culture stress. Field grown plants from 5 μM and 15 μM BA treatments were not significantly different in morphological characteristics, except for plant height. Significant interactions between treatments and explant source were seen for some characters. Cold-stored plants were significantly different from the 5 μM BA treatment plants for most morphological characters. Plants showed less vegetative and reproductive vigor and two-thirds of the plants showed late or no flowering. A variety of factors influenced the RAPD-PCR reaction including the reaction volume, primer, and MgCl₂. Optimization for each sample and primer was found to be important.
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