A STUDY ON INTERCROSSING BETWEEN TRANSGENIC B. JUNCEA AND OTHER RELATED SPECIES
N. GhoshDastidar*, N S. Varma
Proagro Seed Co. Ltd., Dhumaspur Road, Badshahpur, Gurgaon, Haryana
India- 122001
ABSTRACT
Transgenic Brassica juncea was crossed to different related species at Proagro-PGS India Ltd. In 1997-’98 winter at Gurgaon to study extent of intercrossing. Transgenic B. juncea was crossed to B. campestris var. toria, B. campestris var yellow sarson, B. campestris var. brown sarson, B. napus, B. carinata, B. nigra, B. tournefortii, Eruca sativa, B. oleracea var. botrytis, B. oleracea var. capitata , Sysembrium indicum(weed) and B. juncea using transgenic B. juncea as female and male both. It was observed that number of seeds formed in the intercrossings were very few (maximum 6.38% of seeds formed in B. juncea X B. juncea cross) and in most cases, especially with diploid species, were nil. Strong post fertilization barrier was observed in most cases. F1 seeds sown in next season, winter 1998-’99, were mostly viable, but both matromorphy and male sterility were observed in F1 plants in almost all cases. It can be concluded that cultivation of transgenic B. juncea will not encourage transgene escape to other related species and weeds.
KEYWORDS
Post fertilization barrier, matromorphy, male sterility, transgene
INTRODUCTION
An experiment on crossability of transgenic Indian Mustard (B. juncea) with other Brassica and related species found in India was carried out at Proagro-PGS India Ltd. Research Station, Badshahpur, Gurgaon in winter 1997-’98 to determine gene flow between transgenic Indian Mustard and other Brassica and related species found in India.
MATERIALS AND METHODS
Experiment 1 : A total of ten different Brassica and two related species were sown as 2 male rows with transgenic mustard carrying barnase (for male sterility) gene sown as 1 female row in separate sets (plot size 5m x 0.9m). The species used as male rows in the sets were I) Brassica campestris var. toria; II) B. campestris var. yellow sarson; III) B. campestris var. brown sarson; IV) B. napus (Gobhi sarson); V) B. carinata (Karan rai); VI) B. tournefortii; VII) Eruca sativa (Taramira); VIII) B. nigra (Benarasi rai); IX) B. oleracea var. botrytis (Cauliflower); X) B. oleracea var. Capitata (Cabbage); XI) Sysembrium indicum (closest weed); and XII) B. juncea . The male rows were sown on 24. 11. 97 in all sets except IX and X while mustard male sterile rows were sown on 24. 11. 97 in sets I-IV and XII and on 1. 12. 97 in the other seven sets (see figure 1) to facilitate synchronous flowering in both male and female rows. In case of sets IX and X, flowering plants of cauliflower and cabbage were transplanted on male rows as soon as female row came to flowering. Each set was covered with separate nylon net before flowering to prevent foreign pollen. Male sterile mustard rows were sprayed with Basta to kill all fertile plants, so all seeds formed on those rows will be due to the pollen from the non transgenic Brassica and related species sown as male rows inside the net.
Experiment 2: Manual cross pollinations (150 each) were attempted between each of the Brassica and related species and transgenic Indian Mustard lines carrying barstar (for restoration of fertility) gene using mustard as male and female parent both.
The data generated in the experiments will help us to determine crossability in various ways. In experiment (1) number of seeds formed on female rows will show extent of crossability naturally can take place when male sterile mustard is grown. In experiment (2) the number of seeds formed per pollination will show the extent of cross pollination that can occur between transgenic mustard hybrid and other Brassica and related species.
RESULTS AND DISCUSSION
Experiment 1 : It was observed that seeds were formed on male sterile mustard rows in sets I, IV, V and XII only. On the other hand, no seeds were formed on the female row (male sterile mustard row) in the other sets despite synchronous flowering between the male and female rows. The data has been summarized in table 1 and the extent of crossability under natural conditions has been determined by comparing amount of seeds formed on female rows of sets I, IV and V against that of set XII which is used as check (as in set XII the normal seed setting of mustard is obtained).
The data clearly shows that crossability of male sterile transgenic mustard is very less with other Brassica species, especially the diploid species and virtually nil with related species and weeds as also observed by Bing et al (1996).
Table 1: Showing amount of seeds formed on male sterile rows in different sets
|
Set |
Male row (Species) |
Seeds formed on female (Ms mustard) row |
% of Ms row of set XII (compared to normal) |
|
I |
B. campestris var.toria |
0.04 g (13 seeds) |
0.283 |
|
II |
B. campestris var.Y.sarson |
0 |
0 |
|
III |
B.campestris var. b.sarson |
0 |
0 |
|
IV |
B. napus |
0.1 g (35 seeds) |
0.709 |
|
V |
B. carinata |
0.9 g (282 seeds) |
6.382 |
|
VI |
B. tournefortii |
0 |
0 |
|
VII |
Eruca sativa |
0 |
0 |
|
VIII |
B. nigra |
0 |
0 |
|
IX |
B.oleracea var. botrytis |
0 |
0 |
|
X |
B.oleracea var. capitata |
0 |
0 |
|
XI |
Sysembrium indicum |
0 |
0 |
|
XII |
B. juncea |
14.1 g (3500 seeds approximately)
|
100 |
Experiment 2 : The average number of seeds formed per manual pollination among transgenic mustard and other Brassica and related species is given in table 2.
Table 2: Showing average number of seeds produced per flower pollinated manually
|
Species |
Transgenic Mustard used as female parent |
Transgenic Mustard used as male parent |
|
B. campestris var.toria |
0.293 |
0 |
|
B.campestris var.Y.sarson |
0 |
0 |
|
B.campestris var. b.sarson |
1 |
0.053 |
|
B. napus |
1.226 |
0.093 |
|
B. carinata |
0.746 |
0.04 |
|
B. tournefortii |
0 |
0 |
|
Eruca sativa |
0.086 |
0 |
|
B. nigra |
0 |
0 |
|
B.oleracea var. botrytis |
0 |
0 |
|
B.oleracea var. capitata |
0 |
0 |
|
Sysembrium indicum |
0 |
0 |
|
B. juncea |
14 |
14 |
Crossability of transgenic mustard with other related species as observed in terms of seed set was low and in many cases, was zero. Late abortion of siliquae was observed in most cases though pollination was successful, specially when mustard is used as male parent. It shows that strong post fertilization barriers exist in case of wide crosses involving transgenic mustard and it is even more when mustard is used as a male parent. Normal seed set in crossing with non-transgenic B. juncea indicates absence of any cytogenetic abnormality in transgenic B. juncea.
In 1998-’99, the F1 seeds were sown to see their viability. Most of the seeds in all cases were viable but very strong matromorphy and male sterility was observed in almost all cases. It shows that chance of transgene spread to related species and weeds in the next generations is further less. This is very well supported by similar findings of Scheffler and Dale (1994) in B. napus.
CONCLUSION
It can be concluded that crossability of transgenic Indian Mustard with other Brassica and related species and weeds found in India is very less and in most cases, especially with diploid species, virtually nil. Crossability is further lower when mustard is the pollen parent. So, cultivation of transgenic Indian Mustard will not encourage escape of transgenes from mustard to other Brassica and related species and weeds.
ACKNOWLEDGEMENT
The authors are grateful to Dr. P. K. Ghosh, advisor, Department of Biotechnology, Ministry of Science and Information, Govt. of India for his concern, valuable suggestions and encouragement for this programme. The authors are also thankful to Dr. Ellora Mubashir and Mr. Vinod Kumar Parihar for their kind cooperation.
REFERENCES
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