Gossypium raimondii: a source of fertility restorer for Cytoplasmic Male Sterility of Gossypium hirsutum L.

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1 CARYOLOGIA Vol. 55, no. 3: , 2002 Gossypium raimondii: a source of fertility restorer for Cytoplasmic Male Sterility of Gossypium hirsutum L. S.S. MEHETRE*, V.R. PATIL AND A.R. AHER All India Coordinated Cotton Improvement Project, Mahatma Phule Krishi Vidyapeeth, Rahuri (Maharashtra, India. Abstract - Comparative studies of pollen fertility restoration in different triploid hybrids using cytoplasmic male sterile (CMS line of Gossypium hirsutum 76 IH (2n=4x=52;, wild species and perennial cottons were studied. Triploid hybrid (3n=39; D 5 involving G. raimondii (2n=26; D 5 D 5 was vigorous growing and partially fertile. Restoration of pollen fertility observed in this hybrid was attributed to bivalent formation (11.13 II / PMC leading to formation of 40 % fertile pollen. In contrast to the CMS female parent, arrestation of meiosis during early prophase and degeneration of PMCs was not observed in triploid hybrid that indicated existence of fertility restorer factor in G. raimondii. Efforts were made to induce chromosome doubling as well as induce boll setting in this hybrid with the help of different treatments of colchicine and growth hormones, respectively. The fertility restorer factor reported in present report will be an alternative source of restorer gene in G. hirsutum L. cotton added with tolerance to sucking pests, drought and desired fibre properties. INTRODUCTION Release of intra-hirsutum hybrid cotton H-4 in 1971 (PATEL 1971 paved way for the hybrid cotton era that is unique in the global cotton history. Subsequently, several intra G.hirsutum x G. hirsutum and interspecific (G. hirsutum x G. barbadense and Asiatic (G. herbaceum x G. arboreum hybrids became successful in the central and south zones of India (BASU and PARODA One of the main constraints in tetraploid hybrid seed production is its production through manual emasculation and pollination, which increases the cost of hybrid seeds. Genetic Male Sterility (GMS and Cytoplasmic Male Sterility (CMS based hybrids are a few. In certain promising cultivars, the fertility restoration is not complete; therefore, favorable genes enhancing fertility should be accumulated in such a restorers, to attain 100% fertility of the F 1 population. Hence, it was recommended to undertaken intensified * Corresponding author: fax ( , 43208, 43302, 43216; subhashmehetre@rediffmail.com research for identifying additional restorers (ANONYMOUS MATERIALS AND METHODS Cytoplasmic male sterile line 76 IH of G. hirsutum L. and diploid and tetraploid wild Gossypium Spp. were crossed using the hybridization technique of DOAK (1934. The F 1 hybrids along with their parents (Table 1 were raised during the summer of The comparative cyto-morphological studies (MEHETRE and THOMBRE 1981; THOMBRE and MEHETRE 1981 and anther histological studies (MURTHY and WEAVER 1974 were carried out. Pollen viability was tested using differential stain (ALEXANDER Pollen germination test was conducted according to IYENGAR (1939. RESULTS The data (Table 2 revealed that the F 1 hybrid plants were distinct from both the parents in respect of morphological (Figs. a-f and m characters as well as biometrical observation. It was

2 230 MEHETRE, PATIL and AHER Table 1 Details of inter-specific F 1 hybrids between G.hirsutum x wild and perennial cotton obtained and maintained. Sr. No. F 1 hybrids CMS G.hirsutum L 76 IH, 2n=52( as female with male parents 1 G. anomalum 2n=2x=26, (B 1 B 1 2 G. sturtianum 2n=2x=26, (C 1 C 1 3 G. raimondii 2n=2x=26, (D 5 D 5 4 Perennial G. hirsutum, L Exotic 3. 2n=4x=52 ( 5 Perennial G. hirsutum, L Mock 2n=4x=52 ( 6 Perennial G. hirsutum, L Vensil 2n=4x=52 ( 7 Perennial G. barbadense Kidney cotton 2n=4x=52 (A b A b D b D b not possible to study the meiosis in G. hirsutum CMS female parent as the degeneration of PMCs was noticed during pre-meiotic stages. (Fig. g Similar observations (Fig. h were noted in all F 1 hybrid involving G. hirsutum CMS x perennial cotton and wild species (Table 1. However, normal chromosome pairing and separation of bivalent was noticed during first meiotic division. Further, normal separation of chromatids, their dispersal to opposite poles formation of normal sporads (tetrads and normal pollen grain were observed in wild G. raimondii species. This indicated that the meiosis was quite normal hence, it lead to formation of normal fertile, viable pollens(figs. k and l. The hybrid involving G. hirsutum CMS x G. raimondii was partially fertile. In F 1 hybrid on an average I II III were noticed at metaphase-i. Earlier workers BOZA and MADOO (1941 reported I II III IV. Thus the present observation are similar to them. The presence of univalent, unequal separation of chromosomes during anaphase-i further resulted into formation of sporads, containing 3 to 7 sporads. From the pollen formed were of unequal in size (Figs. l and j. Pollen fertility and germination was 40.0 and 28.78%, respectively (Table 3. Earlier workers used male fertile female parent hence they could not come across such type of fertility restoration on CMS background. In contrast, cytoplasmic male sterile line was used as a female parent; hence, fertility restoration of G. raimondii could be detected in the present investigation. In the present study all the other wild species and races (Table 1 failed to restore fertility on CMS background except G. raimondii. Since the triploid hybrid could not set the boll after selfing and back crossing either G. raimondii or G. hirsutum fertile genotype, the inheritance of fertility restoration could not be studied. Efforts were made to induce chromosome doubling as well as induce boll setting with the help of different treatments of colchicines and growth hormones, respectively. The results were encouraging. DISCUSSION Exploitation of heterosis has played a significant role in increasing productivity and production of several crops world over.cms in higher plants is a maternally inherited trait characterized by the inability of the plant to produce viable pollen. Although a number of investigators have demonstrated an association between the CMS phenotype and the mitochondrial genome (WARMKE and LEE 1977; FORD et al. 1980; BOESHORE et al. 1985; WISE et al. 1987, the cause of pollen abortion remains to be determined. In most cases of CMS in higher plants, pollen can be restored by the introduction of a nuclear fertility restorer gene that appears to mask expression of the CMS trait (MACKENZIE and CHASE Male sterility based tetraploid and diploid cottons hybrids are important for sustainable hybrid cotton production. G. harknessii based CMS (MEYER 1973 and Weaver s double recessive GMS lines are used for development of male sterility based hybrids in India (BASU and PARODA However, currently CMS based hybrid seed production has major limitations like sensitivity in their sterility/fertility response to climatic factors, especially temperature (MEYER and MEYER 1965; SARVELLA 1966; MEYER 1969, inadequate bee population for pollen transfer (HASNAM 1990, infertile segregates and poor fertility restoration by R line in spite of consistent sterility and fertility of A and B lines, respectively (ZHAN 1990 and segregation for good and weak fertile plants in F 1 progeny of crosses between restores and CMS and restorer lines as well as inability of fertility enhancing factor to restore fertility in F 1 generation (CHAUDHARY Thus, it appeared that no strong fertility restorer gene having sus-

3 ON GOSSYPIUM RAIMONDII AND RESTORATION OF POLLEN FERTILITY 231 Table 2 Comparison of different morphological characters of parent and hybrid. Sr. No. Character Female Male Hybrid G. hirsutum (CMS G. raimondii Chromosome (2n Plant type Small annual shrubs to large perennial tree, m. long. Perennial shrub 2-3 m, tall densely tomatoes on all young parts including bolls. Perennial shrub m. Stems Erect, flexible bowing becoming naked with age. Hairy under the croup. Fruiting branches Many jointed. 100 joined, leafy Nil Leaves Stipules Bracteoles Flowers Petal spot Absent. Very large, dark red covering almost lower half of petal, small reverse petal spot at base of the outside of the petal. Filaments Twigs and young, densely hairy to glabrous, small to large cordate, 1 2 or less cut into 3-5 lobes. Falcate, not more than 4mm, wide and about 10mm long, caduceus. Triangular or longer than broad, cordate, gashed into usually 4-12mm long acuminate teeth that are more than thrice as long as broad. Small or large, corolla forming a narrow tube (cup or widely expanding. The upper filaments ascending, usually longer than lower once. Anthers Loosely arranged. Arranged regularly but Arranged regularly and not in ranks. loosely. Styles Short. Very long. Straight long. Stigmas United throughout, rarely dividing at the tip never United to the top. United to the top. separated. 14 Capsules Varying size, rounded or tapering to acuminate point, few and inconspicuous oil glands, 3-5 locular, sutures devoid of hairs, 5-11 seeds per loculus, seed possess copious coat of lint hairs or naked with a small tuft at the end. Entire, cordate, acuminate, large, those on the main stem usually 15 cm or more long. Small, attenuate acuminate, caduceus early. Small, broad at the base, but not cordate cut into 15 or more very long almost thread like, tails along the upper margin. Very large, 8 cm or more long, not widely opening, almost tabular pedicel cleavate. Long, so arranged as to produce a spherical androecium, densely pigmented a dark red extending into the anther cases. Tapering, acuminate, 3-5 locular, sutures bearing a line of hairs projecting between the two rows of seeds 4-8 per locales. Seeds free, covered with a dense irregular coat of greenish hairs, up to 10 mm. Long. Distribution Department of catamaran and la liberated, north Peru. 2-5 lobed, shallowly lacinated, lower surface hairy Caduceus, 2-4 mm wide, 8-10mm long. Small, broad at the base densely hairy cut into 15 teeth. Large (7-8 cm, corolla cream colour. Large ( cm, light red. The upper filaments ascending, usually longer than lower once. Sterile, hence did not set any boll. tainable fertility restoration in CMS G. hirsutum is available across the varieties of this species except a report of fertility enhancer factor from G. barbadense (WEAVER and WEAVER Theories put forward on the time and place of the origin of the tetraploid species (ENDRIZZI et al Accordingly, G. barbadense has evolved in South America as a hybrid between G. herbaceum

4 232 MEHETRE, PATIL and AHER Fig. 1 a-c flower size, petal colour and petal spot. a Female parent CMS G. hirsutum L. 2n=4x=52 (AhAhDhDh CMS 76 IH; b F1 hybrid. c, Male parent G. raimondii 2n=2x=26; D5D5. d-g Anthers and stigma. d Non-dehiscent anthers of Female parent CMS G. hirsutum L. 2n=4x=52 (AhAhDhDh CMS 76 IH. e Fertile dehiscent anthers in F1 hybrid. f Normal dehiscent anthers in male parent G. raimondii 2n=2x=26; D5D5. g and h Anther histological section of Non-dehiscent anthers of female parent CMS G. hirsutum L. 2n=4x=52 (AhAhDhDh CMS 76 IH showing degeneration of PMCs before pre meiotic stages. i-j Sterile (green and fertile (red pollen grains in F1 hybrid, i 450x, j 100x. k-m Fertile (red pollen grains in G. raimondii 2n=2x=26; D5D5. k 450x. l 100x. m Leaf shape, petal spot and androeceum shape and colour in G. raimondii.

5 ON GOSSYPIUM RAIMONDII AND RESTORATION OF POLLEN FERTILITY 233 Table 3 Chromosome associations in F1 hybrid and its male. Parentage Meiotic stages Chromosome Association Pollen size Average pollen (% (microns I II III Germination Fertility G. raimondii Metaphase-I F 1 hybrid Metaphase-I and G. raimondii (JOHNSON Seed protein banding pattern (CHERRY et al revealed differences between tetraploid, G. hirsutum, G. barbadense and G. tomentosum. However, their banding patterns were more or less similar to a combination of G. herbaceum (A 1 genome and G. raimondii (D 5 genome than any other A plus D genome combination. It is also further confirmed by comparison of 2C nuclear DNA content of G. herbaceum, G. raimondii and G. barbadense (MEHETRE and NARAYAN Existence of a fertility enhancer in G. barbadense reported earlier (WEAVER and WEAVER 1977 certainly is descendent from G. raimondii that is progenitor of G. barbadense (CHERRY et al. 1970; JOHNSON 1975 and MEHETRE and NARAYAN Present data on chromosome pairing (12.78 I II III indicated close affinity of D chromosomes of G. raimondii with AD chromosomes of G. hirsutum which is confirmatory to ENDRIZZI et al. (1985 and BOZA BARDUCCI and MADOO (1941 who reported average chromosome associations I II III IV. Efforts underway to transfer this gene to G. hirsutum genotypes to build up new diverse restorer line/lines alternative to existing available restorer. Acknowledgments Sincere thanks are due to Hon ble Dr. S.N Puri, Vice-Chancellor, M.P.K.V. Rahuri for his encourgements. Financial assistance for TMC-MMA-1 project from ICAR - New Delhi is gratefully acknowledged. REFERENCES ALEXANDER D.E., 1969 Differential staining of aborted and non-aborted pollen. Stain Tech., 44: ANONYMOUS 1990 Proceedings FAO-ICAR Regional Expert Consultation on Hybrid Cotton, India. Page 1. BASU A.K. and PARODA R.S., 1995 Hybrid Cotton In India A Success Story-APAARI Publication 1995/1. Asia-Pacific Association of Agricultural Research Institutions FAO Regional Office for Asia and the Pacific, Bangkok, pp BOESHORE M.L., HANSON M.R. and IZHAR S., 1985 A variant mitochondrial DNA arrangements specific to Petunia stable sterile somatic hybrids. Plant. Mol. Biol., 4: BOZA BARDUCCI T. and MADOO R.M., 1941 Investigations on the relationship of the Peruvian cotton species G. raimondii. Min. Forn. Direccionde Agric. Y. Ganaderia Bo. 22 Lima Peru. pp. 29. CHAUDHARY M.R., 1990 Status of hybrid cotton in Pakistan. Proceedings of FAO-ICAR Regional Expert Consultation on Hybrid Cotton. pp. 3. India. CHERRY J.P., KATTERMAN F.R.H. and ENDRIZZI J.E., 1970 Comparative studies of seed proteins of species of Gossypium by gel electrophoresis. Evolution, 24: DOAK C.C., 1934 A new technique in hybridizing: Suggested changes in existing methods of emasculating and bagging Cotton flowers. J. Hered., 250: ENDRIZZI J.E., TURECOLTE E.L. and KOHEL R.J., 1985 Genetics, Cytology and Evolution of Gossypium. Adv. Agron., FORD B.G., OILVER R.J.C., LEAVER C.J., GUNN R.E. and KEMBLE R.J., 1980 Classification of normal and male sterile cytoplasms in maize. I. Electrophoresis analysis of variation in mitochondrially synthesized proteins. Genetics, 95: HASNAM, 1990 Present status of cotton research and development in Indonesia. Proceedings FAO-ICAR Regional Expert Consultation on Hybrid Cotton, pp , India. IYENGAR N.K., 1939 Pollen tube studies in Gossypium. J. Genet., 37: JOHNSON B.L., 1975 Gossypium palmeri and a polyphyletic origin of new world cottons. Bull. Torrey Bot. Club., 102: MACKENZIE S.A. and CHASE C.D., 1990 Fertility restoration is associated with loss of a portion of the mitochondrial genome in Cytoplasmic male sterile common bean. The Plant Cell, 2: MEHETRE S.S. and NARAYAN R.K.J., 1997 Evolutionary DNA variation and genome differentiation in Gossypium L. Proc. Indian Natl. Sci. Acad., B63:

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