STUDIES ON SOME BIOCHEMICAL AND MOLECULAR ASPECTS OF CYTOPLASMIC MALE STERILITY (CMS) IN PEARL MILLET
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Date
2002
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MPKV, UNIVERSITY LIBRARY RAHURI
Abstract
The present investigation is an attempt to understand the
phenomenon of CMS in pearl millet, Pennisetum glaucum L. by evaluating the
isolated
respiratory activities in vegetative tissues and [mitochondria in various lines and
detecting the polymorphism and heterogeneity in mtDNA of CMS-fertility-restorer
system of RHRBH 8609. The two CMS systems viz., CMS system I, consisting of
RHRB 1A, RHRB IB, RHRB 138R, RHRBH 8609, and CMS system II, consisting of
RHRB 5A, RHRB 5B, RHRB 458R, RHRBH 8924, were employed in the present
investigation for the isolation of mitochondria from their etiolated seedlings. The
total respiration was measured in the vegetative tissues and isolated mitochondria
and in anthers at different stages of development. The cyanide-sensitive and
cyanide-insensitive (alternative oxidase) respirations were measured in vegetative
tissues and isolated mitochondria by O2 uptake studies. Further, the assays of
cytochrome c oxidase and succinate dehydrogenase were performed only in
isolated mitochondria. The mtDNAs of the presently employed CMS system were
amplified and characterized by RAPD analysis using a PCR machine with a total of
44 decamer primers supplied by Operon Technologies Inc., USA.
The cyanide-sensitive and cyanide-insensitive respirations in
vegetative tissues were low in both the CMS lines than their respective maintainer,
restorer and hybrid lines. The O2 uptake at premeiotic, meiotic and postmeiotic
stages of anther development increased from premeiotic to postmeiotic stage in all
the lines except the CMS lines. The O2 consumption decreased from meiotic to
postmeiotic stage in the anthers of CMS lines which suggested that the tapetum
degeneration at postmeiosis probably decreased the metabolic activity and thereby
the respiratory activity. The mitochondria isolated by the laboratory standardized
method from all the lines were 92-94% intact. The content of mitochondrial proteins
was comparatively higher in the CMS than their respective maintainer, restorer and
hybrid lines of both the CMS systems indicating that some additional
polypeptide(s) are synthesized in the CMS lines. In the mitochondria isolated from
both the CMS systems, the cyanide-sensitive and cyanide-insensitive respiration
was low in the CMS than their respective maintainer, restorer and hybrid lines. The
lowered total and cyanide-sensitive respiration in the vegetative tissues and
isolated mitochondria in CMS lines indicates that there is a dysfunction in any one
of the respiratory chain complexes possibly associated with the CMS phenotype.
The lower levels of cytochrome c oxidase (complex IV) and succinate
dehydrogenase (complex II) activities were observed in both the CMS lines than
their respective maintainer, restorer and hybrid lines. The lower cytochrome c
oxidase activity in mitochondria of CMS lines indicates the mutation in the gene(s)
encoding this enzyme with altered function. The lower levels of nuclearly encoded
enzymes, viz., alternative oxidase and succinate dehydrogenase, in the CMS lines
the.
may possibly be a coincidental event occurring with|CMS phenotype.
The mtDNA isolated by the laboratory standardized method was
sufficiently pure with good yield for RAPD analysis. The RAPD analysis of the
mtDNAsfrom the CMS-fertility-restorer system of RHRBH 8609 with a total of 44
random primers using PCR amplification indicated that 26 primers synthesized 852
fragments of which only 90 appeared to be polymormphic with an average of 10%
polymorphism indicating the more conserved sequences in mtDNA. Amongst the
twenty six primers employed in the present investigation, the random primer
OPI20 detected maximum polymorphism (26%). The ten primers generated
distinguishing differences in a male sterile and its isonuclear maintainer line,
whereas fifteen primers generated differences in a male sterile and restorer line.
The four primers synthesized unique bands in the mitochondria of a CMS line.
In conclusion, the lowered respiration in CMS lines indicates the
mutation in the gene encoding cytochrome c oxidase that may be one of the
probable biochemical causes of a CMS phenotype. The RAPD analysis of mtDNA
with PCR amplification suggested that the CMS phenotype may lie in one of the
unique fragments amplified by the four operon random primers.
(Page No. 1-130)
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