FIELD OF THE INVENTION
The present invention relates to a fertility restorer (Rf) gene for 'Polima' cytoplasmic male sterility (CMS) in Brassica campestris and to a method of producing restorer lines for hybrid seed production.
BACKGROUND OF THE INVENTION
Breeding for yield improvement in the Brassica crops can be done by utilizing the phenomenon of hybrid vigour or heterosis. When two disparate parents having desirable agronomic characters, such as high yield potential, disease resistance, etc. are combined by traditional breeding methods, the Fl hybrid plante show higher yield man either of the parents. One of the most economical and convenient means for producing uniform heterotic population of Fl hybrids is to utilize cytoplasmic male sterility (CMS). A CMS plant is rendered incapable of producing pollen grains (the male reproductive unit in
plants) and therefore cannot get self-fertilized, thus ensuring cross pollination and the development of true hybrid seeds. Factors responsible for imparting CMS reside in the cytoplasmic organelle, the mitochondria and are transmitted maternally. Therefore, the Fl hybrid plant obtained by crossing a CMS female parent with a pollen donor parent is sterile and incapable of setting seeds. This is a highly undesirable feature in a crop where seed is the harvested product However, this problem is rectified by the presence of a genetic factor in the nucleus of the pollen donor parent This genetic factor when present in its active form, suppresses the expression of CMS and restores male fertility. This fertility restoration factor (RJ) is an essential component for the production of viable hybrid seeds at commercial scale where seed is the harvested product
In Brassica plants a number of CMS systems are available which are reported to have originated either from Brassica species itself or from related genera For example 'Polima', one of the well known CMS systems in Brassica has been shown to have its origin in Brassica napus. Another well worked out CMS system in Brassica is 'ogura' which derived its CMS cytoplasm from radish. Usually it has been observed that the restorer factors for the respective CMS systems come from the species that contribute the CMS inducing cytoplasm. The restorer for 'ogura' was transferred from radish to Brassica plants ( Delourme et al.1991, Proc 8th Int. Rapeseed Congr.5, 1506-1510). The restorer for 'Polima' was identified in B. napus itself
Brassica campestris (synonymus B. rapa) is one of the edible oil seed producing Brassica type plant and is grown in western Canada, parts of Sweden and Finland and north-west China where summer growing season is too short to accommodate other longer duration Brassica crops. In India, three distinct types of Brassica campestris,
namely brown Sanson, yellow Sanson and toria are grown in northern and eastern India. Although significant heterosis for yield in crosses between B. competing cultivars has been reported from India (Rai and Singh 1994, Indian J, genet, 54: 310-314; Varshney and Rao 1997, Indian J. Genet.57: 91-97) and Canada (Schuler et al. 1992, Can J. Plant Sci.72, 127-136; Falk et al. 1994, Can. J. Plant Sci. 74: 441-445), the development of hybrid cultivars has not been possible due to non-availablity of suitable CMS-restorer systems. A number of CMS systems such as Diplotoxis murolis (Hinata and Konno 1979, Japan J. Breed. 29: 305311), Oxy (Prakash and Chopra 1988, Plant Breeding 101: 253-255), Tour (unpublished results) and Eruca saliva (Matsuzawa et al. 1999, Plant Breeding 118: 82-84) have been earlier identified in B. campestris. However, none of these have been used for hybrid seed production either due to chlorosis,or lack of stability in CMS lines or lack of proper restorer lines.
OBJECTS OF INVENTION
An object of this invention is to propose a retorer (Rf) gene for the 'Polima' cytoplasmic male sterility in Brassica campestris plants.
Another object of this invention is to propose a process for introducing the identified restorer (Rf) gene for the 'Polima' cytoplasmic male sterility in Brassica
campestris.
SUMMARY OF THE INVENTION
According to this invention there is provided a process of producing restorer (Rf) gene for 'Polima' CMS in Brassica campestris (sya B. rapa) plant which comprises in the steps of:
i) Crossing 'Polima' CMS B. napus with B. campestris to produce Fl generation,
ii) Subjecting the Fl generation to successive steps of backcrassing with B.
campestris and atleast upto BC3 to produce a stable male sterile line for 'Polima'
CMS. iii) Crossing said BC3 plant with 'Candle' or 'ATC 94211' to produce Fl plants
which are male fertile.
Further according to this invention there is provided the identification of a restorer factor for 'Polima' CMS in Brassica campestris. The process also provides that the fertility restoration is governed by a single Mendelian locus. In accordance with this invention 'Polima' CMS was transferred from B. napus var. ISN 706 to 5. campestris. To effect this transfer, inter-specific cross was made between 'Polima' CMS B. napus var. ISN 706 with B. campestris plants. The resultant Fl hybrid plant was repeatedly back-crossed to normal B. campestris (used as pollen donor) till at least BC3.
The BC3 generation B. campestris containing 'Polima' CMS cytoplasm was subsequently crossed with other B. campeatris varieties such as long duration canola quality 'Candle' or accession 'ATC 94211'. The Fl plants segregated for male fertility and male sterility. The fertile Fl plants were self-pollinated. The resultant F2 progeny plants were scored for the segregation of male fertile and male sterile plants in order to establish the inheritance pattern of the fertility restorer factor.
BRIEF DESCRIPTION OF THE DRAWING
The accompanying drawing:
Fig. 1 is a schematic flow diagram showing the breeding methodology for the identification of restorer lines for 'Polima' CMS B. campestris. it comprises the preparation of Fl hybrid by making inter-specific cross between 'Polima' CMS B. napus var. ISN 706 and B. campestris var. 'Pusa kaiyani' followed by three back-crosses of the saidFl hybrid to B. campestris var. Tusa kaiyani/ Subsequently, BC3 'Polima' CMS B. campestris var. 'Pusa kaiyani' was crossed with 'Candle' and 'ATC 94211'. Fertile Fl plants were self pollinated to raise F2 progeny.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a plant of the species B. campestris, characterized in that it contains a Brassica nucleus and the cytoplasm having 'Polima' CMS organelles. Example of Brassica campestris plant usable in the present method include any plant line or cultivar that includes the Rf factor for the 'Polima' CMS.
According to the present invention the 'Polima' CMS B. campestris is initially developed through conventional breeding method by crossing 'Polima CMS B. napus var. ISN 706 with a B. campestris plant
Thus according to this invention there is provided a breeding process for producing a stable 'Polima' CMS line in B. campestris which comprises in the steps of:
Making an inter-specific cross between 'Polima' B. napus var, ISN 706 with B. campestris to produce Fl generation. Normal fertile B. napus ISN 706 is a synthetic
Brassica napus the seeds of which were obtained from Indian Agicaitaral Research Institute, New Delhi, India Tolima' CMS was introduced in B. napus ISN 706 from a unknown French variety containing 'Polima' cytoplasm (Sodhi et al.1993, Plant Breeding 110: 334-337). After crossing, siliqua (seeds) developed. The siltqua were collected, examined for the seeds and the seeds were planted.
Hie resulting Fl plants were male sterile and partially female fertile and hence, upon back-crossing to B. campestris produced a few seeds. The seeds were planted to raise BC1 generation.
Subjecting the BC1 generation to successive steps of back-crossing with B. campestris and at least up to BC3 to produce a stable 'Polima' CMS B. campestris.
Preferably, but without implying any limitation, Fls of B. napus ISN 706 are back-crossed wim B. campestris till BC6 generation. Specifically, B. campestris, variety used in the present invention is, for example, B. campestris var. 'Pusa kalyani'. B. campestris var. 'Pusa kalyani' is a released cultivar in India It is self incompatible, brown sarson type, and has high contents of erucic acid (>50%) and glucosinolates (> 70 µmoles/g deoiled meal). The seeds of this variety were obtained from Indian Agricultural Research Institute, New Delhi, India
From BC3 generation onwards the progeny plants have the appearance of 'Pusa kalyani' and are completely male sterile, show floral features typical of 'Polima' CMS i.e crinkled petals and short stamens bearing small conical anthers. The pollen grains are shriveled and do not stain with fluorescein diacetate (FDA), a specific stain for accessing the pollen viability. The staining procedure is according to the protocol described by
Sadhi et al (1994, Plant Breeding 112: 223-227). In all the subsequeat back-cross generations the 'Polima' CMS in 'Pusa kalyani' remain completely stable.
'Polima' CMS B. canpestris var. 'Pusa kalyani' BC3 is crossed with two other lines of fl. campestris namely, 'Candle', and 'ATC 94211'. The Fl plants of 'Polima' 'Pusa kalyani* x 'Candle* and 'Polima' 'Pusa kalyani' x 'ATC 94211' segregated for male fertility and male sterility. The fertile Fl plants had normal floral features with respect to flower opening and floral organ development. The pollen viability of the male fertile Fl plants of these two crosses are 99% and the production of the viable pollen are similar to that of normal B.campestris var. 'Pusa kalyani' (Table 1). The Fl plants showed normal seed Bet under open pollination.
Fertile Fl plants of the crosses mentioned above were self pollinated to obtain F2 seeds. The seeds were planted to raise F2 generation. Segregation for male fertility and sterility was also observed in the F2 generation of these crosses. Segregation pattern for fertility and sterility in Fl and F2 generations indicate that the fertility restoration is controlled by a single dominant gene and the parental population of 'candle' and 'ATC94211' are heterozygous for the fertility restorer gene (Table 1). The fertile restored F2 plants in both these crosses have normal floral features. The sterile segregants exhibit floral features typical of 'Polima' CMS i.e. crinkled petals and short stamens bearing small conical anthers. The pollen grains were shriveled and did not stain with FDA 'Candle' is a Canadian 'Canola' quality cultivar having low erucic acid and low glucosinolate. The seeds of'candle' were obtained from Agriculture Canada, Saskatoon, Canada 'ATC94211' is an Australian low erucic acid line and seeds were obtained from Brassica germplasm bank from Australia.
Although the invention has been described primarily in connection with the special and preferred embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses or adaptations of the invention, following, in general, the principles there of and including such departures from the presented disclosure as come within known or customary practice in the field to which the invention pertains, or as are obvious to persons skilled in the field.
Table 1. Pollen production and viability* and inheritance of fertility restorer gene in 'Polima' CMS B. campestris
(Table Removed)
Control Tusa kaiynnr showed polien viability of 97% and pollen production mean 10.2 ±3.1
The following examples are provided to further illustrate the present invention and are not to be confused as limiting thereof
EXAMPLE 1
The present invention involves conventional field breeding techniques. The breeding experiments were conducted twice in a year, utilizing the winter growing season (October -April) in the plains (Delhi) and summer growing season (May-September) in the higher altitude areas of Northern Himalayas (Leh, J&K). The plants were grown by sowing seeds in 3 m long rows with 40cm spacing between rows and 15 cm spacing between plants within the rows.
Transfer of 'Polima' CMS from B. napus to B. campestris:
An inter-specific cross was made between 'Polima' CMS B. napus var. ISN706 and B. campestris. Ten inflorescence on 'Polima' CMS B. napus var. ISN706 were randomly selected and 10-12 buds from each of these inflorescence were emasculated and pollinated with B. campestris var. Pusa kalyani'. Pod development was normal but the number of seeds per pod was less, around 8-10/pod as against 18-20 in normal cases. About 100 Fl seeds were harvested.
Fl seeds were planted in the next growing seasoa Germination occurred within 5-7 days. Standard intercultural practices were followed Plants were intermediate between B. napus and B. campestris in their morphology and were uniform in appearance. The plants flowered after 70 days and flowering was uniform. All the plants were male sterile and partially female fertile. Flowers had short stamens bearing conical whitish anthers, which were devoid of pollen grains. The said Fl plants were back-crossed with B. campestris var 'Pusa kalyani'. About 100 crosses ware made, each cross comprising about 10-12 buds. Siliqua developed in situ. The development was however
retarded as compared to that of normal siliqua and only 2-3 siliqua developed per cross (l.S- 2.5cm having 2-3 seeds as against around 5.0 cm and 16-18 seeds in normal case).
The BC1 seeds were harvested and sown in the next growing season. Plante showed variation in their morphological features. Some were intermediate between B. napus and B. campestris and some resembled B. campestris more closely. These plants segregated for initiation of flowering. The plants having closer resemblance with B. campestris flowered early. All the plants were male sterile. Three of the BC1 plants having closer resemblance with B. campestris were selected for further back crossing. About 20 crosses were made each havinglO-12 buds. Female fertility and siliqua development showed discrete improvement over Fl. Number of seeds was around 6-8/ siliqua, which was still low as compared to the normal pods.
BC2 seeds were harvested and sown in the next growing season. Resemblance of the plants to B. campestris was more perceptible in this generation and majority of them came to flowering within 60 days of sowing. Flowers were completely male sterile and female fertility was normal. About 15 crosses were made each having 10-12 buds. Siliqua development was almost normal, with each siliqua having on an average 12 seeds.
The BC3 seeds were harvested and sown during next growing season. The plants completely resembled B. campestris var. 'Pusa kalyani' phenotypically and came to flowering within 60 days. The flowers were completely male sterile with crinkled petals, short stamens and small whitish conical anthers. The female fertility was normal and the siliqua developed normally upon back-crossing to B. campestris var. 'Pusa kalyani' and were about 5 cm long. The number of seeds per pod varied between 12-15/pod. Seed setting was normal on open-pollination.
EXAMPLE 2
Identification of restorer gene for Polima CMS in B. campestrt and its mode of
inheritance :
Inter-varietal crosses were inside between 'Polima' CMS B. campestrtts var. 'Pusa
kalyani' with two other varieties namely 'Candle' and 'ATC 94211'. Siliqua
development and seed net were normal and 16-18 seeds per pod were obtained The Fl
seeds were harveeted.
The Fl seeds were apace planted in eight rows at a distance of approxinaately
25cm between plants for each of the crosses. Fl plants derived from both 'Puaa kalyani' x 'Candle' and 'Puaa kalyani' x 'A IV 94211' displayed plant paeaotype and floral morphology intermediate between the respective parents. The plants showed 1:1 segregation for male fertility and sterility. The pollen viability of the nude fertile Fl plants of these two crosses are 99% and the production of the viable pollen are similar to that of normal B. campestns var. Pusa kalyani' (Table 1). The Fl plants showed normal seed set under open pollination. The fertile Fl plants were selfed Prior to the opening of the buds the inflorescence were covered securely with butter paper baga to prevent cross-pollination. The siliqua were allowed to develop within the confine of the bag till the grain filling stage. On an average, 12 weeds per siliqua were obtained The selfed seeds were harvested
F2 seeds of each cross were space planted with a distance of about 20cm between plants, in 22 rows each. Segregation was observed for plant morphology and flowering
duration. At the time of flowering plants were scored in the field for male fertility and male sterility. The segregation pattern was recorded as J male fertile: 1 male sterile.
Plants were female fertile since teed netting was normal on open pollination.

We claim:
1. A process of producing restorer (Rf) gene for Polima' CMS in B. campestris plant
which comprises in the steps of:
i) crossing 'Polima' CMS with B. campestris to produce Fl generation,
ii) subjecting the Fl generation to successive steps of back-crossing with B.
campestris and at least up to BC3 to produce a stable male sterile line for
Tolima' CMS. iii) crossing said BC3 plant with B. campestris plants other than that subjected
under step (ii) to produce Fl plants which are male fertile, iv) self pollinating the fertile Fl plants of the above said crosses to produce F2
plants segregating 3 male fertile : 1 male sterile.
2. A process as claimed in claim 1 wherein B. campestris of step (ii) is selected from B.
campestris 'Pusa kalyani'.
i
3. A process as claimed in claim 1 wherein step (iii) comprises crossing said BC3 plant with B. campestris selected from 'Candle' or 'ATC94211 ;
4. A process as claimed in claim 1 or 2 wherein the Fl generation is subjected to successive steps of back-crossing with B. campestris to produce BC6.
5. A process as claimed in claim 1 or 2 wherein Fl generation is subjected to the step of back-crossing with B. campestris to produce BC1.
6. A process as claimed in cliam 5 wherein BCl is subjected to the step of back-crossing with B. campestris to produce BC2.
7. A process as claimed in claim 6 wherein BC2 is subjected to the step of back-crossing with B. campestris to produce BC3.
8. A process as claimed in 1 wherein 'Polima' CMS is 'Polima' CMS B. napus var. ESN 706.
9. A fertility restorer (Rf) gene for polima cytoplasmic male sterility in B. campestris comprising B. campestris modified with 'Polima* CMS by interspecific crossing followed by intervarietal crossing.
10. A fertility restorer (Rf) gene as claimed in claim 9 wherein said 'Polima* CMS is
'Polima' B. napus ISN 706.
11. A fertility restorer (Rf) as claimed in claim 9 wherein said B. campestris modified by interspecific breeding is B, campestris 'Pusakalyani'.
12. A fertility restorer (Rf) as claimed in claim 9 wherein said variety of B. campestris for intervarietal crossing is selected from 'Candle' and 'ATC 94211".