SEEDLINK™ AND DOUBLED HAPLOIDY  OFFER SUCCESSFUL SYSTEM FOR THE DEVELOPMENT OF RESTORED HYBRIDS IN INDIAN MUSTARD  (BRASSICA JUNCEA)

N. GhoshDastidar *, N. S. Varma

Proagro Seed Co. Ltd. Dhumaspur Road, Badshahpur, Gurgaon, Haryana,

India-122001

ABSTRACT

Commercial hybrids of Brassica juncea has become a reality through Seedlink™ and Doubled Haploidy which so far  was not possible with the existing fertility control systems. The significant aspects of Seedlink™ are (1) stable and uniform male sterility, (2) complete restoration of fertility and (3) absence of any morphological disorder. Seedlink™ comprises of genetically engineered nuclear male sterile and fertility restorer lines carrying Barnase(Ms inducer) and Barstar(Rf inducer) genes respectively, both tagged to a selectable marker(glufosinate ammonium resistant) gene. During backcrossing, Ms & Rf plants are selected based on their resistant phenotype against glufosinate ammonium and Polymerase Chain Reaction analysis. Doubled Haploidy (microspore culture followed by colchichine treatment) is used to create variability between lines and also to attain homozygosity in restorer lines. Ten fully restored hybrids were thus developed and tested at four locations permitted by the Department of Biotechnology, Ministry of Science, Govt. Of India in winter 1997-’98. The heterosis for seed yield ranged from 9.94% to 34.53% over the best check. The hybrids were superior to the checks for important features like oil content and seed weight also. The hybrids are resistant to glufosinate ammonium too which helps in effective weed control. Data from toxicological and environmental safety studies show that the transgenics are not different from their non transgenic counterparts and their cultivation poses no harm to the environment.

KEYWORDS

Male sterile, fertility restorer, transgenics, lines, heterosis

INTRODUCTION

Development of Commercial Hybrids of B. juncea was not possible so far because of lack of any effective pollination control system. The existing cms systems have problems as follows:

            a. Unstable male sterility

            b. Lack of complete restoration of fertility

            c. Morphological disorders and yield penalty

SeedlinkÔ comes out as an effective pollination control system and together with doubled haploidy, helped Proagro Seed Co. Ltd. To develop and test the first ever fully restored hybrids of B. juncea.

MATERIALS AND METHODS

SeedlinkÔ comprises of genetically engineered nuclear male sterile line carrying Barnase gene(Ms inducer) and fertility restorer line carrying Barstar gene (Rf inducer); (Mariani et al 1990,      Mariani et al 1992). Both Barnase and Barstar genes are also tagged with a selectable marker(glufosinate ammonium resistant) gene.The Ms and Rf plants are identified based on their resistant phenotype against glufosinate ammonium during backcrossing of the genes to the elite parent lines. Glufosinate ammonium also helps to remove fertile and unwanted plants from A- lines and rogueing of  plants from R-lines during production of hybrids. The presence of MS & RF genes are confirmed by Polymerase chain reaction screening.

Doubled haploid (microspore culture followed by colchichine treatment) helps in creating variability between lines and attaining homozygosity at the same time. R lines homozygous for the Rf gene were developed using this technology.

In 1997-98, 9 hybrids developed using this system were tested against two checks in the Initial Hybrid Trial at 4 locations across the country permitted by the D.B.T. The plot size was 5m x 0.9m (3 rows / entry), number of entries were 11, no of replications were 2 , the design was R.B.D. The data is given in table 1.

RESULTS AND DISCUSSION

From 1994 onwards, experiments and breeding work has been carried out to transfer SeedlinkÔ into candidate parental lines at Gurgaon and Bangalore research stations under contained conditions as permitted by Dept. of Biotechnology, Ministry of science & information, Govt.of India. It has been observed during experiments from 1994-1998 that:

(I)   The male sterility is stable & 100 %

(ii)  The restoration of fertility is complete and perfect

(iii) There is neither any morphological disorder nor any yield penalty

Other experiments generated data on toxicological and environmental safeties which show that the transgenics are in no way different from the non-transgenics and their cultivation creates no hazard to the environment.

Table no.1 shows the results of I. H. T. at 4 locations in which most of the hybrids have been observed to give heterosis over the checks for seed yield. The range of heterosis varies from 9.94 % to 34.53 % for seed yield. Mustard Hybrid MT 97010 shows the highest heterosis of  34.53 % over the best check PRO-4001. The data of location 2 was not considered because of very high c.v. Most of the hybrids also showed better tolerance to biotic stresses eg. Alternaria blight & white rust diseases and abiotic stresses like lodging as compared to the checks.

The hybrids also showed heterosis over the checks for important features like oil content and 1000 seed wt.

The hybrids were all resistant to non- selective herbicide LibertyÒ (glufosinate ammonium) also. So, the hybrids offer a significant advantage over the non-transgenic varieties for weed control and thus for lower production cost & better yield potential under weedy conditions.

Table 1: Showing performance of transgenic mustard hybrids in 1997-’98

Entries: 11  Replications: Two  Design: R. B. D.  Plot size: 5m x 0.9m (3 rows/ entry)  Spacing: 30cm row to row, 10 cm plant to plant

	Seed yield as % of best check (best check mean is given in q/ha)					Oil Content	Days to Maturity	Plant height	1000 seed wt.
Entry	Location1	Location 2	Location 3	Location 4	Mean1(1,3,4)	Mean(%)	Mean	Mean(cm)	Mean(g)
MT97002	106.38	80.28	131.03	117.24	120.63	36.10	147	170	5.2
MT97003	125.53	80.99	141.38	100.00	126.80	36.06	148	175	4.4
MT97004	104.25	110.56	131.03	110.34	118.05	36.00	146	165	4.8
MT97005	87.23	103.52	127.59	118.62	112.98	36.13	147	180	5.0
MT97006	93.09	61.27	124.14	104.83	109.94	35.59	145	165	5.6
MT97007	127.66	85.92	134.48	87.59	121.45	35.70	146	175	5.4
MT97008	109.57	107.75	134.48	110.34	121.27	35.94	148	180	4.8
MT97009	114.89	100.00	117.24	94.48	111.97	35.92	148	170	4.4
MT97010	121.28	80.99	162.07	103.45	134.53	36.12	149	190	5.2
Varuna	97.87	85.92	100.00	86.21	96.96	35.81	141	165	4.4
PRO-4001	100.00	100.00	96.55	100.00	100.00	35.69	141	170	5.2
Best check(q/ha)	10.44	15.78	12.08	8.06	10.06
Grand Mean	11.28	14.30	15.38	8.30	11.65	35.913
C.V. (%)	11.41	28.94	17.99	13.76	16.15	2.123
L.S.D.	1.77	5.68	3.80	1.57	1.84	0.747

Mean: Data from Raniwara was not considered as it shows a very high C.V.

Bold sized numbers denote entries that outyield the best check by 10% or more

Locations:    Bangalore,   Raniwara,   Gurgaon,Niwarsi   Bangalore

CONCLUSION

The transgenic hybrids seem to offer many advantages alongwith higher seed yield over the non-transgenic varieties. The data from toxicological and environmental safety experiments of these transgenics poses no threat to the environment.

ACKNOWLEDGMENT

The authors are grateful to Dr. P. K. Ghosh , advisor, Dept. of Biotechnology for his valuable suggestions, cooperation, constructive criticism and encouragement to do things undone before. The authors are also obliged to Dr. Ellora Mubashir and Dr. D. S. Parihar for their contribution to the experiments and the testing programme. The authors acknowledge the helps offered by Vinod Kumar Parihar, Bhujang Rao, Dr.Sudesh Sharma and Dr. Surendra Kumar.

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