MOLECULAR INVESTIGATIONS ON GROWTH AND DEVELOPMENT OF IN VITRO RAISEDSOMACLONES OF Dianthus caryophyllus L. cv. ‘Master’
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Date
2017
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UHF,NAUNI,SOLAN
Abstract
ABSTRACT
The present investigation aims at “Molecular investigations on growth and development of in vitro raised
somaclones of Dianthus caryophyllus L. cv. ‘Master’”. A standard plant regeneration and in vitro selection protocol was
developed for Dianthus caryophyllus L. cv. ‘Master’. Indirect organogenesis from leaf and nodal explant was carried out.
Callus was induced from leaf explant on solid MS medium supplemented with 2.0 mg/l 2,4-D in combination with 0.5 mg/l
NAA while in case of nodal explant the best treatment for callus induction was found out to be solid MS medium
supplemented with 1.5 mg/l 2,4-D and 1.0 mg/l NAA. Highest percentage of callus was obtained from leaf (94.44%)
explants followed by nodal (79.17%) explants. Shoots were induced from leaf and nodal derived calli on solid MS medium
supplemented with 1.50 mg/l TDZ, 0.25 mg/l Kinetin and 0.25 mg/l NAA with 80.56% and 65.28% shoot bud induction.
Solid MS medium supplemented with 2.00 mg/l Kinetin, 0.25 mg/l NAA and agar concentration of 1.0 per cent was found
best for in vitro shoot multiplication, which resulted in 14.64 average number of microshoots per explant. The regenerated
shoots were rooted in half strength MS basal medium supplemented with 1.5 mg/l IBA, 200 mg/l activated charcoal and
0.6% agar concentration. Gamma rays emitted by unstable nuclei of Cobalt-60 was used for physical mutagenesis of leaf
callus of Dianthus caryophyllus L. cv. ‘Master’. Leaf derived calli were exposed to five different doses of gamma radiations
(10, 20, 30, 40 and 50 Gy) from Cobalt-60 as a source of mutagen. Control showed 100% survival percentage at 4 and 8
weeks. A steady decrease in the survival was observed with increase in the dose of radiation with passage of time. Lethal
dose value (LD50) calculated from percent survival of gamma irradiated calli at different treatments which was found close to
30.5 Gy. For chemical mutagenesis, four different percent (v/v) solutions of EMS were used for two time intervals (15 and
30 minutes) and untreated calli served as control. The highest survival percentage of EMS treated callus was recorded on the
lower concentrations of EMS used for lesser time. The LD50 was approximately 0.16 % (v/v) for 15 minutes EMS treatment
and in case of 30 minutes EMS treatment, LD50 was calculated to be 0.08 % (v/v). There was a decrease in the percent shoot
bud induction, number of microshoots per explant, rooting percentage and decrease in the survival frequency after hardening
with the increase in dose/duration of gamma and EMS treatment in all the treatments than the control. Five variants were
obtained from gamma treated leaf callus induced plants, one mutant from 10 Gy, 2 from 20 Gy, 1 from 30 Gy and 1 from 40
Gy gamma treatment. Similarly, five variants were obtained from EMS treated leaf callus induced plants, obtained with
0.10%, 0.15% and 0.20% EMS treatment for 15 min and 0.10% and 0.15% EMS treatment for 30 min. A total of 20
Randomly Amplified Polymorphic DNA (RAPD) primers, 20 Inter Simple Sequence Repeats (ISSR) primers and 15 Simple
Sequence Repeats (SSR) primers were used for genetic variation studies in selected variants. Only 16 RAPD, 14 ISSR and
10 SSR primers were able to amplify the genomic DNA of mother plant, control and selected variants raised through gamma
and EMS treated leaf callus, respectively. In gamma treated variants, similarity value of 0.57-0.74, 0.50-0.79 and 0.39-0.76
were observed from RAPD, ISSR and SSR studies, respectively which showed high genetic difference. In EMS treated
variants, RAPD studies showed 0.44-0.71 similarity value whereas 0.51-0.80 similarity value was observed in ISSR studies
and SSR studies showed 0.32-0.739 similarity value. Gamma and EMS treated selected variants showed random grouping of
selected variants by all the three markers used for the study which showed variation between the selected variants, mother
plant and control. It is hoped that the in vitro EMS and gamma induced mutations can open up a new approach for the
breeding of carnation cultivar ‘Master’. These results must be regarded as preliminary studies because of the small size
analyzed, the low number of used primers and the low number of generated RAPD, ISSR and SSR bands.
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