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Introgression of desaturase suppressor gene(s) from Brassica napus L. to enhance oleic acid content in Brassica juncea L. Coss.

Gurpreet Kaur, S.K. Banga and S.S. Banga

Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana – 141004, Punjab,
India Email surin11@rediffmail.com

Abstract

Desaturase suppressor gene(s) were introgressed from B. napus cv GSC-5 into three low (<2%) erucic acid (C22:1) B. juncea genotypes by simple hybridization followed by two cycles of recurrent backcrossing with the recipient parents. Selfing and selection for high oleic acid (C18:1) was practiced after each backcross generation (BC1⇒BC1S1⇒BC2⇒BC2S1). Identified high C18:1 plants were used for next cycle of backcrossing and selection. All the six evaluated BC1S1 families had higher C18:1 means than the recurrent parent, whereas excepting one, all the BC2S1 families were at par with corresponding BC1S1. Family CRL 1359(IS)-2 was an exception, where consistent improvement in family mean was observed (from 39.7% to 43.3% to 50.6%). Some plants having C18:1 upto 62 per cent were selected and confirmed for stability of expression.

Media summary

Novel genetic stocks having high oleic acid (>60%) as against 15-20 per cent in conventional mustard cultivars and about 35-40 per cent in low C22:1 genotypes, were synthesized in B. juncea through introgression of desaturase suppressor gene(s) from B. napus. Stability of expression of introgressed variation was demonstrated by growing plants under varied environmental conditions.

Key words

Canola quality, oleic acid, interspecific hybridization, stability, influence of temperature.

Introduction

Brassica juncea (Indian mustard) is a predominant oilseed crop in India with an approximate coverage of nearly 85 per cent of the area under rapeseed-mustard. Although intensive breeding efforts have helped in the development of erucic acid (C22:1) free genotypes in mustard; these low C22:1 strains contain 35-40 per cent oleic acid (C18:1), 40-45 per cent linoleic acid (C18:2) and 15-20 per cent linolenic acid (C18:3). In contrast, the corresponding low C22:1 B. napus (AACC), having similar genetic block in chain elongation pathway, possesses higher level of C18:1 (55-65%) and lower levels of C18:2 (30-25%) and C18:3 (8-12%) due to the presence of strong desaturase suppressor gene(s) on C genome chromosomes. These gene(s) suppress the activity of desaturase enzymes mediating desaturation from C18:1 to C18:2 / C18:3, consequently resulting in increased C18:1 content. High C18:1 is desirable in edible oils as its intake tends to reduce the level of harmful low density lipoproteins in blood plasma (Ackman, 1990). Oils with higher level of C18:1 in combination with low C18:3 show higher oxidative stability and lower oxidation products without extensive hydrogenation (Hawrysh, 1990). This communication reports the successful integration of desaturase suppressor gene in B. juncea as was evident from increased oleic acid content.

Methods

Introgression of desaturase suppressor gene

Interspecific crosses were made between low C22:1 genotypes of B. juncea (CRL 1359 18-15-1, NDRC 190-8-1, YSRL 9-18-26) and B. napus (cv GSC 5 >65% C18:1). The resultant interspecific hybrids were used for recurrent backcrossing with B. juncea parents. Each cycle of backcrossing was followed by selfing (BC1⇒BC1S1⇒BC2⇒BC2S1) and selection for high oleic acid. Non-destructive half-seed method was used for estimating C18:1. Only putative plants having high C18:1 were transferred to field, to initiate next cycle of backcrossing and selection.

Analytical procedure

Half seed and whole seed samples were analysed for their fatty acid composition by gas liquid chromatography using ethyl ester preparation (Appleqvist, 1968).

Effect of temperature on fatty acid composition

To assess the stability of introgressed trait, and effect of temperature during seed ripening on relative amounts of C18:1, C18:2 and C18:3, low C18:1 (KLM 226), intermediate C18:1 (MHO 18-5) and high C18:1 (IHO-C3) genotypes were grown at temperature regimes of 15, 20 and 25C. Relative amounts of C18:1, C18:2, C18:3 were compared between the genotypes at each temperature regime.

Results

To introgress gene(s) for high C18:1 from B. napus to B. juncea, recurrent backcross strategy accompanied with selfing in between cycles of backcrossing was utilized. First cycle of selection for high C18:1 content was carried out in six BC1S1 families. The identified high C18:1 plants were used for the next cycle of recurrent backcrossing and selfing. Mean C18:1 content in all the BC1S1 families was significantly higher than the corresponding base C18:1 values of B. juncea recurrent parents (Table 1). The best results were available from the family CRL 1359(1S)-2, where a consistent improvement in C18:1 content was reflected over two backcrossings. Individual plants having 45-70 per cent C18:1 were, however, available in almost all the families.

Table 1. Response to selection for high C18:1 in backcross progenies derived from (B. juncea x B. napus) x B. juncea

Families

Base value (%)

BC1S1 (%)

 

BC2S1 (%)

Mean SE

Range

Mean SE

Range

CRL 1359(1S)-1

39.7

52.6 1.3

43.0-70.5

45.7 0.9

41.1-67.6

CRL 1359(1S)-2

39.7

43.3 1.3

35.1-53.8

50.6 1.9

41.2-65.1

NDRC 190(1S)-1

36.0

45.3 .71

39.9-48-7

47.1 1.1

40.2-54.1

NDRC 190(1S)-2

36.0

45.4 2.2

39.5-58.6

44.7 0.3

43.8-45.2

YSRL 9(1S)-1

40.7

44.8 1.9

46.0-57.1

45.4 3.0

41.0-57.6

YSRL 9(1S)-2

40.7

51.4 1.7

39.1-64.0

48.9 2.4

41.2-53.4

Significantly higher content of C18:1 in BC1S1 and BC2S1 B. juncea plants was reflective of the presence of desaturase suppressor gene(s) from C-genome of B. napus. The elevated C18:1 level could not be maintained or further enhanced in majority of the families, probably due to lack of introgression resulting from low frequency of homoeologous pairing between two well differentiated B and C genome chromosomes as demonstrated earlier (Attia and Rbbelen, 1986). This coupled with rapid elimination of intact C genome chromosomes during recurrent backcrossing with B. juncea did not permit frequent introgression of the desirable desaturase suppressor genes into B genome chromosomes. Further, low seed fertility in BC1S1 did not permit a large sample size for half seed analysis. Under these circumstances, the family CRL 1359(1S)-2 appeared especially promising where mean oleic acid increased to 50.6 per cent indicating desirable introgression of desaturase suppressor genes. It was possible to select some individual seeds having 65.1 to 67.6 per cent C18:1. Morphologically these selected BC2S1 plants resembled B. juncea parent and had improved pollen and seed fertility. The fatty acid composition of some elite high C18:1 plants at maturity is presented in table 2.

Table 2. Fatty acid composition in plants identified for high C18:1

Genotypes

Fatty acids (%)

 

C18:1

C18:2

C18:3

IHO C3-1

62.0

20.1

9.7

IHO C3-2

59.0

24.1

14.2

IHO F2

56.1

27.1

12.1

Effect of temperature on fatty acid composition

This experiment comprised of three genotypes, one of each type (low, intermediate and high C18:1) exposed to three temperature regimes (15C, 20C and 25C) during seed formation and ripening. No significant differences were recorded in desaturase activity at 15C or 20C as manifested by the same levels of three unsaturated C18 fatty acids. At 25C, however, there was significant increase in the amount of C18:1 (Fig. 1) in low and intermediate types indicating decreased oleate desaturase activity with increase in prevailing temperature. In contrast, the proportion of different C18 fatty acids remained nearly constant at all the temperature regimes in case of high oleic acid genotype (Fig. 1). This suggested a temperature neutral desaturase suppression similar to that observed in normal ‘0’ C22:1 (<2%) genotypes of B. napus (Tremolieres et al, 1982).

Fig. 1. Effect of temperature during ripening on low, intermediate and high C18:1 genotypes

Conclusion

Introgression of desaturase suppressor genes from B. napus proved successful in enhancing oleic acid content of ‘0’ C22:1 B. juncea to the canola standards. Stability of expression for high C18:1 was also demonstrated under varied temperature regimes.

Acknowledgments

This work was supported by funds provided under National Agricultural Technology project “Breeding Designer Brassicas”.

References

Ackman RG (1990). Canola fatty acids : an ideal mixture for health, nutrition and food use. In : Shahidi F (ed) Canola and Rapeseed : Production, chemistry, nutrition and processing technology. Van Nostrand Reinhold, New York. pp 84-98.

Appleqvist L (1968). Rapid methods of lipid extraction and fatty acid ester preparation for seed and leaf tissue with special remarks on preventing the accumulation of lipid contaminants. Ark Kenci 28, 351-370.

Attia T and Rbbelen G (1986). Cytogenetic relationship within cultivated Brassica analyzed in amphihaploids from the three diploid ancestors. Can J Genet Cytol 28, 323-329.

Hawrysh Z J (1990) Stability of canola oil. In : Shahidi F (ed) Canola and Rapeseed : Production, chemistry, nutrition and processing technology. Van Nostrand Reinhold, New York. pp 65-83.

Tremolieres A, Dubac QJP and Drapier D (1982). Unsaturated fatty acids in maturing seeds of sunflower and rape regulated by temperature and light intensity. Phytochemistry 21, 41-45.

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