In vitro mass multiplication and variability in stevia (Stevia rebaudiana bertoni)
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Date
2013
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Publisher
CCSHAU
Abstract
The present investigation was undertaken with the objectives like (i) to develop an efficient
and reproducible mass multiplication protocol in stevia (ii) to evaluate the genetic variability among
regenerated plants of Stevia at molecular level (iii) to compare various agronomic traits between tissue
culture raised and conventionally grown plants of stevia. The shoot-tip and nodal segments were used
as explants. Among the different sterilizing agents used, the maximum (100%) survival was recorded
on when nodal explants were treated with HgCl2 (0.1%) for 3 minutes along with bavistin (0.2%) and
streptocyclin (0.2%) for 45 minutes. The nodal and shoot-tip explants were best regenerated on MS
medium+ BAP 2.0mg/l. Among the different media used, MS medium supplemented with 2.0 mg/l
BAP showed the maximum shoot induction (98.0%) in 4.7 days and (74.0%) in 7.1 days using nodal
and shoot tip explants, respectively.MS medium supplemented with BAP 2.0 mg/l was found to be
most effective for establishment of both the explants. The best shoot multiplication medium was MS
medium supplemented with BAP (0.3mg/l) + Kinetin (0.3 mg/l+0.1 mg/l NAA+ 15 mg/l PEG), on
which an average MS basal medium supplemented with 0.3mg/l BAP + 0.3 mg/l Kin + 0.1 mg/l NAA+
15 mg/l PEG was proved to be the best with 40.56 shoots/ explant developed from nodal and shoot-tip
explants after 30 days on culture medium. Maximum rooting (90±7.75%) was observed on the half MS
medium supplemented with 0.5 mg/l NAA with 21.2 roots per shoot in 7.4 days. The survival of in
vitro raised plantlets was reported to be 100% in potting mixture containing sand + soil + FYM (1:1:1)
and 100% in sand + soil + vermi compost (1:1:1) under green house conditions. For genetic fidelity/
variability, each primer generated a unique set of amplification products ranging in size from 100 to
1100 bps. All the bands in all the primers were same and no polymorphism was detected during the
RAPD analysis of in vitro raised plants, which showed that all the plants raised through micropropagation
were true to type or identical to the mother plant. Comparison of various agronomic traits between tissue culture raised and conventionally grown plants of stevia was carried out in the field. Analysis of variance indicated that in vitro raised plants were significantly superior for all agronomic traits over conventionally grown plants of stevia. The survival of tissue culture raised plants was observed to be 90% under field conditions, while 100% survival was reported in the green house.
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Keywords
Planting, Stevia, Biological phenomena, Genetics, Tissue culture, Stevia rebaudiana, Auxins, Vegetative propagation, DNA, Crops