PhD

Thermosensitive Genic Male Sterile (TGMS) plants which are male sterile
above critical sterility temperature (CST) and male fertile below CST can be
utilized as an efficient system for developing hybrid rice suitable to the state of
Kerala. In this context, a study on ‘Physiological approaches for manipulating
male sterility in thermosensitive genic male sterile system for hybrid rice seed
production’ was conducted at the Department of Plant Physiology, College of
Agriculture, Vellayani during 2015 to 2018 to evaluate the environmental
conditions required for complete male sterility of TGMS plants and to manipulate
the male sterility by using plant growth regulators and also to understand the
molecular mechanism associated with TGMS system.
The male sterile trait of stable TGMS line EC720903 from IRRI,
Philippines, was transferred to red rice variety of Kerala, Jyothi. The CST of
EC720903 was determined as 26.9⁰C and the critical thermosensitive phase was
identified as 15-22 days before flowering. The seeds of BC1F2 plants were used
for the experiment. The seeds were sown in pots in the open field and Rain Out
Shelter (ROS) at monthly interval from June, 2017 to May, 2018 to evaluate the
environmental conditions required for complete male sterility. The newly
developed TGMS line exhibited complete pollen and spikelet sterility throughout
the study period at both the experimental conditions since the average temperature
prevailed during the critical thermosensitive period of TGMS red rice line was
above the CST of 26.9⁰C. Anatomical studies of anther also showed that the
TGMS lines were pollen free which is a preferred character of an ideal TGMS
plant. The TGMS plants inside ROS had higher plant height, number of
productive tillers and they flowered early compared to open field. Hence this low
cost structure which is covered with a UV stabilized transparent sheet can be used
for commercial hybrid seed production using TGMS lines throughout the year.
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To maintain the sterility expression during the critical stages, the potential
of plant growth regulators (PGR), were evaluated at RARS, Ambalavayal. Three
PGRs, ethrel (400 mg L-1, 800 mg L-1 and 1200 mg L-1), salicylic acid (400 mg L-
1, 600 mg L-1 and 800 mg L-1) and maleic hydrazide (MH: 600 mg L-1, 800 mg L-1
and 1000 mg L-1) were applied as foliar spray at two stages viz., panicle initiation
and two weeks after panicle initiation. The PGRs were capable of enhancing
pollen sterility in all the treated TGMS plants. MH induced significantly higher
percentage of pollen sterility (83.49%) compared control plants (19.92%). The
external application of salicylic acid at three different concentrations induced 62-
82% of pollen sterility in rice and ethrel caused more than 75% pollen sterility. It
was also observed that the PGRs applied were not inhibitory to the primary
metabolism of TGMS lines. Hence spraying of MH (1000 mg L-1) two times at
the time of panicle initiation and fifteen days after panicle initiation can be
recommended to maintain male sterility.
The TGMS line was hybridized with two rice varieties as pollen parents,
Aiswarya (red) and Swetha (white) using proximal hybridization to evaluate the
potential of using TGMS line as a female parent in Kerala condition. Seed setting
was significantly high for the cross involving Swetha (40.07%). The F1 progenies
obtained from two crosses along with parents and female parent EC720903 were
sown in pots during April-May to September-October. The F1 progenies attained
early flowering and physiological maturity. The protein content at panicle
initiation stage was also high. Plant height, photosynthetic rate, transpiration rate
and stomatal conductance at panicle initiation stage of F1 progenies of Swetha
were significantly higher. The F1 plants did not show sterility and the seed setting
percentage was in the acceptable range. Complete spikelet sterility was exhibited
by the TGMS line since temperature was higher than CST.
Molecular characterization of TGMS plants maintained at sterility inducing
and fertility inducing conditions was done using samples collected from leaf and
young panicle at ten days after panicle initiation. Protein profiling with SDS-Poly
Acrylamide Gel Electrophoresis showed an enhanced expression of proteins at 25-
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35 kDa, 35-48 kDa and 245 kDa under sterility inducing condition. In the panicle,
increased expression at 17 kDa and presence of protein band at 245 kDa was
observed at fertility and it was between 48-63 kDa under sterility inducing
condition.
Microarray gene expression analysis of TGMS leaf and panicle revealed
that the genes encoding proteins similar to programmed cell death protein 2,
Nuclear ribonuclease Z, hormone degradation and conjugation were up regulated
and genes encoding proteins similar to male sterility protein 2, pollenless3,
pollen-specific protein SF21 and no pollen were down regulated in the TGMS leaf
under sterility inducing condition. The genes involved in hormone degradation
and conjugation and programmed cell death protein 2 and genes encoding ABC
transporter proteins required for exine and sporopollenin synthesis, lipid transfer
protein and wax synthase, sucrose transporter, male sterility protein 2, β-1,3-
glucanase and tapetal programmed cell death were down-regulated in the panicle.
Pathways involved in the IAA, GA, brassinosteroid and jasmonic acid
biosynthesis and carbohydrate synthesis and transport was down-regulated during
sterility inducing condition in the TGMS line leaf and panicle.
The TGMS line can be recommended to the state of Kerala as a female
parent for the development of suitable red rice hybrids because of its stable
sterility. If the temperature of a particular location goes above 27⁰C in a particular
season that period can be used for hybrid seed production. Application of MH
(1000 mg L-1) at the time of panicle initiation and fifteen days after panicle
initiation is more effective in sterility manipulation when there is drop in
temperature. Occurrence of pollen free anthers in the TGMS line during sterility
inducing condition is mainly due to the down-regulation of genes encoding ABC
transporter proteins, lipid transfer protein and strictosidine synthase, glucosemethanol-
choline oxidoreductase, male sterility protein 2, wax synthase and β-
1,3-glucanase.