A new source of male sterility in onion (allium cepa l.).
Onion is preferred as a commercial vegetable crop all over the world, since it ranks second in value in the list of cultivated vegetable crops. Area under its cultivation is 3.44 million hectares and its total annual production in the world is 61.6 million tonnes (FAO, 2006).
The most important qualitative genes in the edible alliums are those that cause male sterility. In onion, male sterility was first exploited by Jones and Clarke (1943) using a male sterile specimen of cultivar Italian Red, which they found in breeding plots at Davis, California, in 1925. Fortunately, when this plant was prevented from being cross pollinated, bulbils were produced in the flower head and it could be propagated. Jones and Clarke (1943) published this classical work describing the genetics of male sterility and indicating how it could be used to produce hybrid cultivars. On the basis of this technique, originally developed in onion, male sterility has since been exploited in hybrid breeding of more than 150 crop species (Kale and Munjal, 2005).
The genetic basis of the cytoplasmic male sterility (CMS) in Italian Red is simple: a sterility inducing cytoplasm (S) and a nuclear restorer locus with two alleles, Ms and ms. Male fertility is restored by the dominant allele. The fertility restorer genes were later found extensively in onions collected from various countries (Davis, 1957).
In the 1960s, another CMS cytoplasm, namely T-cytoplasm, was discovered (Berninger, 1965; Schweisguth, 1973). This source of male sterility was found in the French cultivar Jaune Paille de Vertus. The T-cytoplasm is restored by two independently operating restorer systems. The first restorer system comprised of a single locus, A, with two alleles, in which male fertility is restored by the dominant allele. The second restorer system is comprised of two loci, B and C, with complementary gene action. Male fertility in this restorer system is restored only when a dominant allele is present on both B and C loci. Meer van der and Van bennekom (1969) observed thermolability, when analyzing a CMS line, which originated from Polish cultivar Wolska, which is a source of T-cytoplasm. They found 93% male sterile plants at 14 0C and only 10% at 23 0C. This complex fertility restoration has resulted in negligible use of T-cytoplasm for hybrid development. The majority of hybrid onion cultivars produced upto date are using S cytoplasm (Havey, 1995; 2000) as a source of male sterility. This source of male sterile cytoplasm traces back to a single onion plant identified in Davis, California, in 1925 (Jones and Emsweller, 1936). Cytoplasmic male sterility is found to have ascribed to mitochondrial genomes. Many scientists (Courcel et al., 1989; Holford et al., 1991; Havey 1993, 1995; Satoh et al., 1993; Sato, 1998) have identified molecular markers that distinguish normal (N) male-fertile and (S and T) sterile cytoplasms of onions.
In 1970, T-cytoplasm carrying maize hybrids suffered heavy losses due to epidemic of southern corn leaf blight (Pring and Lonsdale, 1989, Levings, 1993). This single incidence has forced plant scientists to think seriously about cytoplasmic uniformity and genetic vulnerability of crop plants (Anonymous,1972).So far, only one type of male sterile cytoplasm (CMS S) has been found useful. Newer types of male sterility needs to be studied which will reduce genetic vulnerability of onions and also useful for commercial production of bulb and seed onions at lesser costs (Gokce et al., 2002).
In our study, we have screened onion population for occurrence of male sterile plant/s, tissue cultured them, studied using molecular markers and by making crosses with other onion varieties.
Materials and Methods
All of the genotypes mentioned in Table 1 were planted in the field during Rabi 2003. After harvesting, the bulbs of these varieties were preserved in cold storage for six months at 40C and 65 % relative humidity. Bulbs were taken out during 1st week of November. Planting of all these bulbs was done in Rabi 2004. All the genotypes bolted and flowered in January 2005. Umbels of these genotypes were checked physically and by acetocarmine treatment for assessing their male sterility.
Genomic DNA was extracted from all the genotypes mentioned in Table 1. A systematic study of cytoplasms of all the genotypes was undertaken using polymerase chain reaction (PCR) and primers as described by Sato (1998) and Engelke and Tatlioglu (2002). The PCR-marker which anchors in the upstream region to the mitochondrial gene cob is referred to as 5' cob-marker (Sato, 1998). These markers are able to distinguish between S and N cytoplasm. T cytoplasm which was not studied by Sato(1998) shows the same amplified product as N cytoplasm. Therefore, Engelke and Tatlioglu (2002) developed a new orfA501-marker that amplifies in S and T cytoplasm of onion, but not in N cytoplasm. The combination of the 5' cob-marker with the orfA501-marker allows to distinguish between these three cytoplasm types in individual plants.
Markers used for classification of cytoplasms are tabulated in Table 2.
PCR reaction mixture used for amplification is shown in Table 3.
Table 3: PCR reaction mixture used for amplification PCR was carried out using Mastercycler gradient from Eppendorf, using amplification conditions (Table 4).
The crosses were made involving suspected male sterile plant as female parent and other varieties (Arka Kirtiman,Arka Pitambar, Udaipur 102, Pusa White Round, Pusa White Flat, Punjab White, Gujarat Local, Phule Safed) as male parent. All these crosses were made in Rabi 2005 and seeds of these crosses were planted in Rabi 2006. Bulbs of these crosses were stored in cold storage until planting in Rabi 2007.
Results and Discussion
All the onion genotypes were screened for presence of male sterility. Results of screening are summarized in Table 1.
All the genotypes have shown male fertility, except V7 and Indam, while JISL5 has shown segregation for male sterility. While screening these genotypes for male sterility, we have got one plant in Agrifound white population which was showing male sterility. We have tissue cultured it and planted 100 plantlets in late kharif 2005. All of these plants flowered subsequently and found out to be male sterile. Subsequently, these plants have been again planted in the next season i.e. late kharif 2006 and confirmed their male sterility.
Results of PCR based analyses of cytoplasm are given in Table 5.
Table 5: PCR based analyses of cytoplasm
Results of Table 5 show that genotypes V7, Arka Kirtiman and Arka Pitambar and Indam possess S cytoplasm, while rest of the genotypes possess N cytoplasm. JISL5 is the only genotype with T cytoplasm. Agrifound White A was showing male sterility in the field, but to our great surprise its cytoplasm was found to be male fertile.
Samples: 1. V7, 2. Agrifound White, 3. Phule Safed, 4.Punjab White, 5. Udaipur 102, 6. Pusa White Flat, 7. Pusa White Round, 8. Gujarat Local, 9 to 15. Agrifound White A plants, 16.Arka Kirtiman, 17. JISL5, M- 100bp ladder.
Genotype Agrifound White A was showing male sterility in the field, while its PCR based molecular character indicated it to have N type cytoplasm. In order to understand the nature of male sterility, crosses have been made involving Agrifound White A as female parent and other varieties (Arka Kirtiman,Arka Pitambar, Udaipur 102, Pusa White Round, Pusa White Flat, Punjab White, Gujarat Local, Phule Safed) as male parent. All these crosses were made in Rabi 2005 and seeds of these crosses were planted in Rabi 2006. Bulbs of these crosses were preserved in cold storage until planting in Rabi 2007. Results of these crosses are presented in Table 6.
Crosses involving Agrifound White A as female parent were made in order to study the nature and dominance of male sterility. Results of these crosses in Table 6 show male fertility of these hybrids. Interestingly, all the crosses irrespective of their cytoplasm and nuclear background, restored fertility in Agrifound White A. Analysis of sterility observed in Agrifound White A indicated that it is probably of GMS ( Genetic male sterility) type and the male fertility is dominant over male sterility and is a new source of male sterility in onion. Genetic male sterility is of very wide occurrence in flowering plants and as many as 60 genes for maize, 55 in tomato, 10 in cotton and 60 in rice are known (Horner and Palmer, 1995).
 Anonymous,1972, "Genetic vulnerability of major crops,"National Academy of Sciences, USA, Washington DC.
 Berninger, E., 1965, "Contribution a l'etude de la sterilite male de l'oignon (Allium cepa L.)," Ann Amelior Plant (Paris), 23, pp.183-199.
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G.G. Adsul, D.G. Patil, A.V. Dhake, U.B. Pandey, P.V. Sane and R.M. Kothari
Jain Hi-tech Agri. Institute (JHAI), Jalgaon, Maharashtra, 425 00, India
Table 1: Profile of genotypes and sterility/fertility of plants analyzed. Sr. No. Genotype No. of plants analyzed Male sterile/ Male fertile 1 Agrifound White 50 Male fertile 2 Phule Safed 50 Male fertile 3 Punjab White 50 Male fertile 4 Udaipur 102 50 Male fertile 5 Arka Kirtiman 50 Male fertile 6 Arka Pitambar 50 Male fertile 7 Pusa White Flat 50 Male fertile 8 Pusa White Round 50 Male fertile 9 V7 100 Male sterile 10 Gujarat Local 50 Male fertile 11 Agrifound White A 50 Male sterile 12 JISL5 50 35 Male fertile :15 Male sterile 13 Indam 50 Male sterile Table 2: Molecular markers used for classification of cytoplasms. Sr. Primer Sequence No. 1 PCR primers as per Sato (1998) Gene--cob S--specific 5'-GTCCAGTTCCTATAGAACCTATCACT-3' N- specific 5'-TCTAGATGTCGCATCAGTGGAATCC-3' Common primer 5'-CTTTTCTATGGTGACAACTCCTCTT-3' 2 PCR primers as per Engelke and Tatlioglu (2002) orfA 501 5'ATGGCTCGCCTTGAAAGAGAGC3' 5'CCAAGCATTTGGCGCTGAC3' Table 3 : PCR reaction mixture used for amplification Component Concentration Amount ([micro]l) Taq polymerase 5u/[micro]l 0.1 10 X PCR buffer -- 1.5 dNTP 0.1mM 1.2 Primer 0.5 [micro]M 0.6 [Mgcl.sub.2] 0.1mM 0.9 Template DNA 20 ng/[micro]l 1.0 Distilled water -- 9.7 Total volume -- 15 Table 4 : Amplification conditions for PCR Stage Temperature ([degrees]C) Duration (min) Initial denaturation 94.0 2 min. Denaturation 94.0 30 sec. Annealing 53.0 for primers as per 1 min. Sato (1998) and 60.0 for primers as per Engelke and Tatlioglu (2002) Extension 72.0 2 min. Final extension 72.0 7 min. Storage 04.0 -- Stage Cycle /s Initial denaturation 1 Denaturation 40 Annealing Extension Final extension 1 Storage Table 5: PCR based analyses of cytoplasm Sr. No. Genotype No. of N cytoplasm plants analyzed 1 Agrifound White 5 5 2 Phule Safed 5 5 3 Punjab White 5 5 4 Udaipur 102 5 5 5 Arka Kirtiman 5 - 6 Arka Pitambar 5 - 7 Pusa White Flat 5 5 8 Pusa White Round 5 5 9 V7 5 - 10 Gujarat Local 5 5 11 Agrifound White A 5 5 12 JISL5 5 - 13 Indam 5 - Sr. No. S cytoplasm T cytoplasm 1 -- -- 2 -- -- 3 -- -- 4 -- -- 5 5 -- 6 5 -- 7 -- -- 8 -- -- 9 5 -- 10 -- -- 11 -- -- 12 -- 5 13 5 -- Table 6: Male sterility/ male fertility analysis of crosses Sr. Name of cross No. of plants Male fertile / No. analyzed Male sterile 1 Agrifound White A 50 Male fertile X Gujarat Local 2 Agrifound White A 50 Male fertile X Phule Safed 3 Agrifound White A 50 Male fertile X Pusa White Flat 4 Agrifound White A 50 Male fertile X Punjab White 5 Agrifound White A 50 Male fertile X Udaipur 102 6 Agrifound White A 50 Male fertile X Pusa White Round 7 Agrifound White A 50 Male fertile X Arka Kirtiman 8 Agrifound White A 50 Male fertile X Arka Pitambar