STUDIES ON PLANT REGENERATION IN MARIGOLD (TAGETES SPP.) THROUGH IN VITRO CULTURE OF MALE AND FEMALE GAMETOPHYTES
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Date
2017
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Division of Floriculture and Landscaping ICAR-Indian Agricultural Research Institute
Abstract
Marigold (Tagetes spp.) is one of the most popular flower crops of commercial
importance throughout the world. It ranks first among the loose flower production in
India. F1 hybrids of Tagetes species are gaining popularity mainly for loose flower
production, ornamental gardening and increased carotenoid production. In spite of its
economic importance, the genetic potentiality of marigold is intensively not explored
in India and F1 hybrid seeds are being imported from other countries with enhanced
seed cost. Development of F1 hybrids in this crop will drastically reduce the cost of
cultivation, increase the area of cultivation and enhance the productivity which is
possible through utilizing homozygous lines in breeding programme. Marigold is a
cross pollinated crop and traditional way of homozygous line development will take
8-9 generations of continuous selfing and it is not possible in the ornamentally valued
petalous male sterile lines. Hence, production of haploids and doubled haploids (DHs)
either through in vitro androgenesis or gynogenesis are needed to produce the
homozygous lines in a single generation. However, reports are not available on
haploid induction in this genus through any means. Hence, the present study was first
initiative to develop a reliable and an efficient protocol for the development of
haploid and doubled haploid (DHs) lines by in vitro culture of male & female
gametophytes, its in vitro multiplication, maintenance and to perform the genetic
analysis of regenerants and parental genotypes using SSR markers.
The suitable length of florets having uninucleate to early binucleate
microspores in different marigold genotypes was standardized for anther culture. The
availability of suitable length of florets in different capitulum sizes was also
evaluated. Direct embryo induction and di-haploid production were standardized in
the tetraploid French cv. Pusa Arpita by excising the ten days old pre-treated anthers
from 3.5-4.0 mm length florets and cultured on enriched MS (EMS) (MS + organic
and inorganic supplements + 5% coconut water) medium supplemented with 2.0 mg/l
BAP + 0.5 mg/l NAA + 45g/l sucrose and incubated in dark for 20 days. Among the
different genotypes, French lines exhibited higher androgenic capacity over African
genotypes. Gynogenic haploid induction was only observed in petalous male sterile
line ‘Local Orange’. Direct parthenogenic embryo germination obtained from the
freshly excised young, mature ovules cultured on EMS medium supplemented with
2.0 mg/l BAP + 0.5 mg/l NAA + 80 g/l sucrose and incubated in dark for 30 days.
Androgenic and gynogenic regenerants were characterized for their ploidy level by
employing cytological and flow cytometry analysis. Nearly 8 and 6 chloroplasts were
observed in a pair of stomatal guard cells of di-haploid ‘Pusa Arpita’ and haploid
‘Local Orange’ respectively as compared to 16 and 12 in their donor parent. Doubled
haploids were produced in ‘Pusa Arpita’ and ‘Local Orange’ shoots by culturing on
0.005% and 0.01% colchicine supplemented medium for 30 hours exposure,
respectively.
Micropropagation protocol was standardized by using nodal segments in
genotypes viz. Pusa Arpita (PA), Pusa Basanti Gainda (PBG), Seracole Orange (SO)
and Seracole Yellow (SY) and leaf explants in genotypes Pusa Arpita and Pusa
Basanti Gainda. Pre-treatment of nodal segments with Bavistin (0.2%) + Ridomil
(0.2%) + 8-HQC (200 mg/l) for 60 minutes followed by surface sterilization with
0.1% HgCl2 for 4 minutes helped in establishing maximum axenic cultures. Best
establishment of nodal segments were obtained on MS medium supplemented with
2.0 mg/l BAP + 0.05 mg/l NAA + 125 mg/l ascorbic acid. Direct adventitious bud
induction was observed from the leaf explants of Pusa Basanti Gainda and Pusa
Arpita on EMS medium supplemented with 0.5 mg/l BAP + 0.25 mg/l NAA and 2.0
mg/l BAP + 0.5 mg/l NAA respectively. Distal end of mature leaves were having
higher regeneration capacity over proximal ends of immature leaves. MS medium
supplemented with 0.5 mg/l BAP + 0.1 mg/l NAA + 2.5 mg/l AgNO3 was found
optimum for proliferation of quality shoots. Half strength MS medium supplemented
with 0.5 mg/l IBA + 60 g/l sucrose was found best for root induction. Maximum plant
survival was recorded in shoots rooted in liquid culture as compared to solid media.
Hardening of rooted plants was most successful when done in disposable plastic
glasses. Molecular analysis with SSR markers revealed the genetic similarity of leaf
derived regenerants with its mother plant. Genetic diversity analysis with 26 SSRs
was successfully applied to the differentiation of the 12 marigold genotypes used as
donor parents in anther and ovule culture into 3 clusters of African, French and male
sterile lines. The doubled haploids lines developed in this study can further used in
breeding programme as inbred line or as a parent to develop F1 hybrids with higher
economic yield. The established micropropagation protocol paved the way for in vitro
multiplication and maintenance (true-to-type) of breeding lines without any variation.
Description
t9668
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