STUDIES ON SOME BIOCHEMICAL AND MOLECULAR ASPECTS OF CYTOPLASMIC MALE STERILITY (CMS) IN PEARL MILLET
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Date
2002-09-27
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Mahatma Phule Krishi Vidyapeeth, Rahuri-413 722, Dist. Ahmednagar Maharashtra (India)
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 ofRHRB 5A, RHRB 5B, RHRB 458R, RHRBH 8924, were employed in the presentinvestigation for the isolation of mitochondria from their etiolated seedlings. Thetotal respiration was measured in the vegetative tissues and isolated itochondriaand in anthers at different stages of development. The cyanide-sensitive andcyanide-insensitive (alternative oxidase) respirations were measured in vegetative tissues and isolated mitochondria by O2 uptake studies. Further, the assays ofcytochrome c oxidase and succinate dehydrogenase were performed only in isolated mitochondria. The mtDNAs of the presently employed CMS system werexviii amplified and characterized by RAPD analysis using a PCR machine with a total of44 decamer primers supplied by Operon Technologies Inc., USA.The cyanide-sensitive and cyanide-insensitive respirations invegetative tissues were low in both the CMS lines than their respective aintainer,restorer and hybrid lines. The O2 uptake at premeiotic, meiotic and postmeioticstages of anther development increased from premeiotic to postmeiotic stage in allthe 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 standardizedmethod 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 additionalpolypeptide(s) are synthesized in the CMS lines. In the mitochondria isolated fromboth the CMS systems, the cyanide-sensitive and cyanide-insensitive respiration was low in the CMS than their respective maintainer, restorer and hybrid lines. Thelowered total and cyanide-sensitive espiration in the vegetative tissues andisolated mitochondria in CMS lines indicates that there is a dysfunction in any oneof 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 ene(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 ccurring with|CMS phenotype.The mtDNA isolated by the laboratory standardized method was sufficiently pure with good yield for RAPD analysis. The RAPD analysis of thexixmtDNAsfrom the CMS-fertility-restorer system of RHRBH 8609 with a total of 44 random primers using PCR amplification indicated that 26 primers synthesized 852fragments of which only 90 appeared to be polymormphic with an average of 10%polymorphism indicating the more conserved sequences in mtDNA. Amongst thetwenty six primers employed in the present investigation, the random primerOPI20 detected maximum olymorphism (26%). The ten primers generateddistinguishing differences in a male sterile and its isonuclear maintainer line,whereas fifteen primers generated differences in a male sterile and estorer 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 hat may be one of the biochemical causes of a CMS phenotype. The RAPD analysis of mtDNA with PCR mplification suggested that the CMS phenotype may lie in one of the unique fragments amplified by the four operon random primers.
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sowing, intercropping, wheats, tillage equipment, seed drilling, crops, mustard, fertilizers, land resources, chickpeas