CA2385464A1 - Stable cytoplasmic male sterile brassica campestris plant which contain "polima" cytoplasm and method for obtaining such plants - Google Patents

Abstract

The present invention involves cytoplasmic male sterile Brassica campestris (syn. Brassica rapa) plants that contain "Polima" CMS cytoplasm. The plants remain completely male sterile in short duration Indian types as well as in "00" canola quality long duration material and are fully female fertile during the growing season. The plants of the present invention have been developed by traditional breeding techniques.

Description

TITLE
STABLE CYTOPLASMIC MALE STERILE BRASSICA CAwiPESTRIS PLANT
WHICH CONTAIN 'POLIMA' CYTOPLASM AND METHOD FOR-OBTAINING
SUCH PLANTS.
FIELD OF THE INVENTION
The present invention relates to stable cytoplasmic male sterile Brassica campestris plants containing 'Polima' CMS cytoplasm and to a method of producing the said plants which are completely male sterile, exhibit normal growth and are fully female fertile.
BACKGROUND OF THE INVENTION
Brassica crops are known to be source of vegetable oil food and condiment. In recent years the demand for the aforesaid products from Brassica plants has increased sharply, necessitating increasing the productivity. In order to increase productivity, plant breeders develop F1 hybrids by crossing two parental lines having desirable characteristics such as higher yield potential, faster growth rate, disease resistance etc.. In case the parental lines involved are self-pollinating then measures are taken to prevent self-pollination in one of the parents in order to obtain pure hybrid seeds. A method known for preventing self-pollination is by using male sterility, which is selectively- introduced in the female parent used for the eventual hybrid seed production. Male sterile plants allow the breeders to produce- hybrid seeds more economically by eliminating self fertilization and allowing cross pollination to occur effectively. Male sterility can be either nuclear or cytoplasmic. Cytoplasmic male sterility (CMS) is the most widly used means-of producing hybrids in crop-plants. It is a maternally inherited pheno-menon, which manifests itself as the inability to produce functionally viable pollen grains the male reproductive unit in a plant. Factors controlling CMS reside in the cytoplasm, particularly in the cytoglasmic organelle, WO 01/22805 CA 02385464 2002-03-20 pCTdN99/00054 the mitochondria. It can be transmitted through conventional breeding and is passed from CMS mother to the progeny.
CMS can also be transferred through sexual crosses from a CMS plant to a pollen donor plant. To effect complete transfer of CMS in the above mentioned type of crosses, usually 6-8 generations of repeated back-crossing is done with the pollen donor parent. The first filian generation (F1) in these types of crosses are male sterile as the CMS character is contributed by the mother but the nucleus is constituted of equal contribution from both CMS and pollen donor plant. By repeated back-crossing to the pollen donor parent the nuclear contents of the CMS parent is gradually eliminate and -~s completely substituted by the nuclear-content of the pollen donor 'parent. This results in the development of a CMS line in the pollen donor parent.
A number of CMS systems are known in Brassica species, of which 'Polima' CMS has been. used most extensively to produce CMS lines in oilseed B. napus (Barsby et al. 1987, Plant Science, 53: 243-248; Sodhi et a~l. 1993, Plant Bree-ding 110: 334-337). One significant problem with the expression of cytoplasmic male sterility by the 'Polima' cytoplasm is that it is unstable under high temperature in the field (Fan et al. 1985, Can. J. Plant Sci. 66:
221-227)-. However, PCT publication WO 97/09873 d-iscloses - 25 a cytop-lasmic male sterile Brassica oleracea plant that contains 'Polima' CMS cytoplasm. In distinction to above, the present invention involves production of stable Bras-sica campestris (syn. Brassica rapa) plants that contain 'Polima' CMS cytoplasm. The plant material of the present invention -remains completely male sterile and are fully female fertile. -Brassica campestris is one of the edible oil seed producing Bra-ssica 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 accomodate other longer duration Brassica crops. In- India, three distinct types of Brassica campestris, namely brown sarson, yellow sarson and toria are grown in northern and eastern India. A number of CMS systems such as Diplotaxis muralis (Hinata and Ronno 1979, Japan J. Breed. 29: 305311), Oxy (Prakash and Chopra 1988, Plant Breeding 101: 25:3-255). Tour (unpub lished results) and Eruca sativa (Matsuzawa et al. 1999, Plant Breeding 118: 82-84) have been earlier identified in B. campestris. However, none of these have been put to any practical use to the best of our knowledge, due to certain disadvantages inherent in these CMS systems.
OBJECTS OF THE INVENTION
An object of the present invention is to propose stable _cytoplasmic male sterile Brassica campestris (syn. Brassica rapa) plants which contain 'Polima' CMS cytoplasm.
Another object of this invention is to propose cytoplasmic male sterile Brassica campestris plants that contain 'Polima' CMS cytoplasm which are fully male Sterile and fully female fertile even at high field temperatures.
Yet another object of this invention is to propose a process for producing cytoplasmic male sterile Brassica campestris plants that contain 'Polima' CMS cytoplasm.
SUMMARY OF THE INVENTION
According to this invention there, is provided a process for producing a stable male sterile line for 'Polima' CMS in Brassica campestris (syn. Brassica rapa which comprises in the steps of t i) Crossing 'Polima' Brassica napus ISN 706 with B. campes-tris to produce Fl generation;
ii) Subjecting the Fl generation to successive steps of back crossing with B. campestris and at least upto BC3 to produce a stable male sterile line for 'Polima' CMS.
Further in accordance with this invention there is provided Brassica campestris modified with 'Polima' CMS.

Briefly, the purpose of the present invention relates to a stable cytoplasmic male sterile Brassica _campestris (syn. Brassica raga) plant which has 'Polima' CMS cytoplasm and nucleus of a cultivated B. campestris variety and is completely female fertile. The Brassica campestris plants of the present invention can be produced by conven-tional breeding methods. Different Brassica varieties can then be developed into stable CMS lines for 'Polima' cytoplasm by further crossing and back-crossings.
To obtain .the Brassica campestris plants of this invention by breeding methods, an inter-specific cross was made between Brassica napus var. ISN 706 containing 'Polima' CMS cytoplasm (Sodhi et al. 1993, Plant Breeding 110:
334-337) and Brassica campestris. The plants produced as a result of the first inter-specific cross were repea-tediy back-crossed with B. campestris such as B. campestris ar. 'Pusa kalyani'. Subsequent-ly diversification of the said CMS was done into different B. campestris varieties.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawing .
Fig.l is a schematic flow chart showing the breeding metho-dology for the transfer of~'Polima' CMS cytoplasm from B. napus to B. campestris and its subsequent diversifica-.
tion to other B. campestris varieties. It comprises the preparation of F1 hybrid by inter-specific cross between 'Polima' CMS B. napus var. ISN 706 and B. campestris var.
'Pusa kalyani' followed by serial back-crosses of the said F1 hybrid to B. campestris var. 'Pusa kalyani' and subsequent diversification to other B. campestris varieties namely, 'robin' and 'Pant toria'.
DETAILED DESCRIPTION OF THE INVENTION
Cytoplasmic male sterile Brassica napus plants containing 'Polima' cytoplasm are known to be unstable under cer-tain temperature conditions. However, when transferred to an Indian B. napus var. ISN 706 this CMS was shown to be stable under varied temperature and photoperiodiG conditions (Sodhi et al. 1993, Plant Breeding 110: 334-337).
The present invention -involves CMS B. campestris plants that contain the 'Polima' cytoplasm. The male sterility is stable and the CMS plants exhibit full female fertility.
It also relates to the development of different CMS B.
campestris types by employing further crossings and back-crossings.
Thus according to this invention there is provided a bree-ding 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 F1 generation. Normal fertile B. napus ISN 706 is a synthetic Brassica napus the seeds of which are obtained from Indian Agricultural Research Institute, New Delhi, India. 'Polima' CMS was introduced in B. napus ISN 706 from an unknown French cultivar containing 'Polima' cytoplasm (Sodhi et al. 1993, Plant Breeding 110: 334-337). After crossing, siliqua (seeds) developed. The siliqua were collected, examined for the seeds and the seeds were planted.
The resulting F1 plants are completely male sterile and partially female sterile and hence, upon back-crossing to B. campestris produce a few seeds. The seeds are planted to raise BC1 generation.
The BC1 generation is subjected to successive steps of back-crossing with B. cainpestris and- at least upto BC3 to produce a stable 'Polima' CMS B. campestris.-Preferably, but without implying any limitation, Fls of B. napus ISN 706 are back-crossed with B. campestris till BC6 generation:- Specifically, B. campestris, by way of example may be B. campestris var. 'Pusa kalyani'. B. cam-pestris var. 'Pusa -kalyani' -is a released cultivar in India. It .is self incompatible, brown sarson type and S

has high contents of erucic acid (7 50$ ) and glucosi:~~ Lates (~ 70 umo les/g deoiled meal ) . The seeds of this :variety were obtained fro~r~ Indian Agricultural Research Institute, New Delhi, India.
From BC3 generation anwards the progeny plants have the appearance of 'Puss kalyani' and are completely male sterile, show floral features typical of 'Polima' CMS
ie. 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 assessing the pollen viability. The staining procedure was adopted according to the protocol described by Sodhi et al. (1994, Plant Breeding 112: 223-227). In all the subsequent back cross generations the 'Polima' CMS in 'Puss kalyani' remain completely stable.
'Polima' CMS 8. campestris var. 'Puss kalyani' BC3 is crossed with two other lines of B. campest~is namely, 'robin' and 'Pant toria'. The F1 plants of 'Polima' 'Puss kalyani' x 'robin' and 'Polima' 'Puss kalyani' x 'Pant toria' are completely male sterile. These are back-crossed to 'robin', a long duration '00' variety and 'Pant toria', a short duration, heat tolerant line respectively. Study of the two back cross generations show that 'Polima' CMS
in 'robin' and 'Pant toria' impart complete male sterility.
These CMS lines also exhibit floral features of '~Polima' CMS ie. crinkled petals- and short stamens bearing small conical anthers. The pollen grains are shriveled and do not stain with FDA: 'robin' is a Canadian 'Canola' quality cultivar having low erucic acid and low glucosinolate, wheras 'Pant toria' is an Indian toria type cultivar with high erucic acid and- high glucosinolate contents. The seeds of 'robin' were obtained from Agriculture Canada, Saskatoon, .Canada and 'Pant toria' seeds were from G.B.
Pant Agriculture University, U.P., INdia.
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 thereof 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.
The following examples are provided to further illustrate the present invention and are not to be confused as limi ting thereof.
cvrunr o i 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 H-imalayas (Leh, J&K). The plants were grown by sowing seeds in 3m long rows with 40cm spacing between rows and l5cm 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. ISN 706 and B. campestris. Ten inflorescence on 'Polima' CMS B. napus~var. ISN 706 were randomly selected and 10-12 buds from each of these inflorescence were emas-culated and pollinated with B. campestris var. 'Pusa kal-yani'. 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 F1 seeds were harvested.
F1-seeds were planted in the next growing-season. Germina-tion 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 WO 01/22805 CA 02385464 2002-03-20 pCT/~99/00054 and partially female fertile. Flowers had short stamens bearing conical whitish anthers, which were devoid of pollen grains. The said F1 plants were back-crossed with B. campestris var. 'Pusa kalyani'. About 100 crosses were made each cross comprising about 10-12 buds. Siliqua deve-loped in situ. The -development was, however, retarded as compared to that of normal siliqua and only 2-3 siliqua developed per cross (1.5-2.5cm having 2-3 seeds as against around S.Ocm and 16-18 seeds in normal-case).
The BC1 -seeds were harvested and sown in the next growing season. Plants 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 having 10-12 buds. Female fertility and siliqua development showed discrete improvement over F1.
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 2~ more perceptible in this generation and majority of them.
came to flowering within -60d ays of sowing. Flowers were completely male sterile and female fertility was normal.
About 15 crosses were made each having 10-12 buds. Sili qua 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 e. campes tris var. 'Pusa kalyani' and were about 5cm long. The number of seeds per pod varied between 12-15/pod.
The BC4 seeds were grown in next season. The plants resem-bled 'Pusa -kalyani' fully and showed setting was normal on open-pollination proper female fertility and complete male sterility. 6-8 cross were made to obtain BC5 seeds.
BC5 seeds were harvested and sown in the next generation.
All the plants were morphologically uniform resembling 'Pusa kalyani' and male sterile. Six to 6 crosses were made with 'Pusa kalyani' with 10-12 buds per cross. Seed set was normal and BC6 seeds were harvested.
BC6 seeds were sown in the next growing season and all the plants were~fully male sterile and female fertile.

Diversification of 'Polima' CMS in B. campestris varieties:
The BC3 plants of example 1 were also crossed to 'robin' and 'Pant toria' for diversifying 'Polima' CMS in different B~ campestris backgrounds. Six to 8 crosses were made each having 10-12--buds per cross. The pod development in these two crosses was normal and about 15-18 seeds per pod were formed.
The F1 seeds were harvested and sown in the next growing season. The plants derived from both the crosses were intermediate between the-respective parents, with reference to plant type and flowering duration: All the plants were male sterile. When the F1 flowers were back-crossed to respective pollen donor parents namely 'robin' and 'Pant toria'wormal-pod development and seed set were obtained.
The BC1 seeds were grown in the next growing season. The plants -were completely male sterile and female fertile.
The BC1 plants segregated for plant types and flowering duration. Plants having close morphological resemblance W~ 01/22805 CA 02385464 2002-03-20 to pollen donor parent were selected for farther back-crossing and crossing was confined to 2-3 plants. Similar-procedure was adopted to raise BC2 generation where all the plants were again found to be male sterile and com-pletely female fertile. Plants having close morphological resemblance to pollen donor parent were selected for fur-ther crossing to obtain BC3 seeds.

Claims (12)

WE CLAIM:

1. A process for producing a stable male sterile line for 'Polima' CMS in Brassica campestris (syn. Brassica rapa) which comprises in the steps of :
i) crossing 'Polima' B. napus ISN 706 with B. campestris to produce F1 generation, ii) subjecting the Fl generation to successive steps of back-crossing with B. campestris and at least upto BC3 to produce a stable male sterile line for 'Polima' CMS.

2. A process as claimed in claim 1 wherein B. campestris is 'Pusa'kaiyani'.

3. A process as claimed in claim 1 or 2 wherein the F1 generation is subjected to successive steps of back-cros-sing with B. campestris to produce BC6.

4. A process as claimed in claim 1 or 2 wherein F1 gene-ration is subjected to the step of back-crossing with B. campestris to produce BC1.

5. A process as claimed in claim 4 wherein BC1 is subjec-ted to the step of back-crossing with B. campestris to produce BC2.

6. A process as claimed in claim 5 wherein 8C2 is subjec-ted to the step of back-crossing with B. campestris to produce BC3.

7. A process as claimed in claim 6 wherein BC3 is crossed with a long duration canola quality 'Tobin' or a short duration 'Pant toria' to produce F1 plants which are back-crossed to produce stable CMS lines of 'Polima' CMS in 'Tobin' and 'Pant toria'.

8. A process as claimed in claim 3 wherein BC6 is crossed 'Tobin' or 'Pant toria' to produce F1 plants which are then back-crossed to produce-stable CMS lines of 'Polima' CMS.

9. Brassica campestris modified with 'Polima' CMS.

10. Modified Brassica campestris as claimed in claim 9 wherein Brassica campestris is B. campestris 'Pusa kalyani'

11. Modified Brassica campestris as claimed in claim 9 wherein Brassica campestris is 'Tobin' or 'Pant toria'.

12. Modified Brassica campestris as claimed in claim 9 wherein 'Polima' CMS is 'Polima' B. napus ISN 706.