Abstract:
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.
