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VOL. 4, NO. 1, JANUARY 2009 ISSN 1990-6145

ARPN Journal of Agricultural and Biological Science

©2006-2009 Asian Research Publishing Network (ARPN). All rights reserved.

www.arpnjournals.com




DEVELOPMENT OF ETHIOPIAN MUSTARD (Brassica carinata)

WITH IMPROVED QUALITY TRAITS THROUGH INTERSPECIFIC HYBRIDIZATION WITH ELITE LINES OF



Brassica napus AND Brassica juncea
F. A. SheikhPPP1PPP, Shashi BangaPPP2PPP, S. S. BangaPPP2PPP, ­S. NajeebPPP1PPP, B. A. LonePPP2PPP, Asif B. ShikariPPP1PPP and A. G. RatherPPP1PPP

PPP1PPPRice Research and Regional Station, SKUAST-K, Khudwani, Anantnag, India

PPP2PPPDepartment of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana, India

E-Mail: TUTUTUsfaroq4@rediffmail.comUUUTTT


ABSTRACT

The present study was undertaken to develop agronomically superior genotypes of Brassica carinata (BBCC 2n = 34) having improved oil and meal quality. Interspecific hybridization was used to enhance the spectrum of genetic variability for desired oil and meal quality traits using elite genotypes of two related species viz. B. napus (AACC 2n = 38) and B. juncea (AABB 2n = 36) as donor parents. Genes from B. napus and B. juncea were successfully introgressed into B. carinata cv. PC 5. Individual plants with low erucic acid (11.8% in BNC 2 vs. 45.5% in PC 5), high oleic acid (26.5% in BNC2 vs 11.2% in PC 5) and high oil content (41.5% in BJC1 vs. 34.5% in PC 5) could be isolated.


Keywords: mustard, B. carinata, hybridization, gene introgression, variability, fatty acid, oil content.


INTRODUCTION

Ethiopian mustard (B. carinata) is the most neglected Brassica digenomic species of U triangle in terms of crop improvement despite the fact that the species is an excellent repository of genes for tolerance to various biotic and abiotic stresses (Malik, 1990; Getinet et al., 1996). Among the quality traits the naturally high levels of erucic acid and very low levels of oil content are the major limiting factors for a wide usage of this species. The restricted amount of genetic variability available in natural B. carinata for these traits require utilization of new sources of variability for broadening the genetic base of source population. Inter specific hybridization has a great potential and is widely used to expand gene sources and to introduce exogenous genes in crop Brassica species (Inomata 1992). Thus present study was conducted to introgress useful variability for quality traits into B. carinata from elite lines of two related digenomic species of B. napus and B. juncea through inter specific hybridization.


MATERIALS AND METHODS

Brassica carinata cv. PC5 (BBCC) was crossed as a male with different elite cultivars of B. juncea and B. napus (Table-1) with the objective of substituting B and C genome of B. carinata with B and C genome of B. juncea and B. napus, respectively in addition to cytoplasmic substitution. The FBBB1BBB plants of both interspecific crosses were backcrossed to B. carinata with the objective to eliminate unwanted A genome chromosomes and improve fertility and seed set. The interspecific nature of hybrids was confirmed on the basis of morphological and cytological basis (Figures 1 and 2). The flower buds were fixed around 6:00 to 8:00 a.m. in Carnoy’s solution II (Ethanol: Chloroform: Acetic acid; 6:3:1), containing a few drops of ferric acetate (a filtered solution of saturated ferric acetate made by adding ferric chloride to glacial acetic acid). After 48 hours of fixing, the young anthers were crushed in two percent acetocarmine on a slide and observed under inverted microscope to study the chromosome number and pairing behaviour of chromosomes. The photographs of the best cells were taken with digital camera and downloaded in the computer using the programme Camedia master. Selected backcross plants (BCBBB1)BBB from both interspecific crosses on the basis of cytology and having 34 chromosomes backcrossed to B. carinata to produce BCBBB2BBB plants. These advance backcross plants were further subjected to cytological analysis and the pollen grain stainability to confirm their genomic stability akin to B. carinata. These advance stable progenies of B. carinata were subjected to fatty acid analysis as per the standard procedure of ethyl ester preparation (Appleqvist, 1968) followed by gas liquid chromatography (GLC), oil content estimation using nuclear magnetic resonance and meal glucosinolate estimation using the method developed by Kumar et al., (2004) a modification of the previous common method of Thies, (1982).


RESULTS AND DISCUSSIONS
B. napus x B. carinata

The cytological studies on the pollen mother cells (PMCs) of B. napus x B. carinata hybrid (ABCC) revealed a somatic chromosome number of 2n = 36 with occurrence of upto 12 bivalents and 10 univalents coupled with one trivalent and one quadrivalent in most of the cells (Figure-1). The BCBBB1BBB plants of B. napus x B. carinata with B. carinata as the recurrent parent exhibited varying number of chromosomes. The somatic chromosome number varied from 29 to 41. In BCBBB2BBB Cytological studies revealed variable chromosome number (2n = 34-35) in different plants, with 17II being the predominant meiotic configuration (Figure-1).



The fatty acid profile BCBBB2 BBB progenies from (B. napus x B. carinata) x B. carinata cross (Table-2) revealed a significant increase in mean oleic acid and linoleic content over the check PC5. Besides increase in the mean value, range of variability also enlarged with different lines and individual plants containing up to 26.5 per cent oleic acid and 30.4 per cent linoleic acid (BNC2) in comparison to 11.2 per cent and 18.2 per cent respectively of check variety (PC5). The eicosenoic acid content of the introgression lines was either at par or was marginally higher than the check (PC5). The mean erucic acid content of all the lines showed a significant decrease as compared to PC5, revealing successful introgression of genes from B. napus parent for low erucic acid content. Individual plants having a recorded decrease up to 11.8 per cent erucic acid were identified. Erucic acid content in B. napus is controlled by two genes (Harvey and Downey, 1964) suggesting the presence of one gene in each of it’s A and C genomes which act in additive manner and display no dominance. Interspecific hybrid between high erucic acid B. carinata parent (BPPP+PPPBPPP+PPPCPPP+PPPCPPP+PPP) and zero erucic acid B. napus parent (APPP-PPPAPPP-PPPCPPP-PPPCPPP-PPP) would be of the genome constitution (APPP-PPPBPPP+PPPCPPP+PPPCPPP-PPP). Normally A and B genomes do not show allosyndetic pairing in haploid state (Mizushima 1950, Olsson 1960) when such hybrids are selfed after one backcross, BCBBB1BBBFBBB2BBB plants of constitution BPPP+PPPBPPP+PPPCPPP+PPPCPPP-PPP, BPPP+PPPBPPP+PPPCPPP-PPPCPPP-PPP with C genome free of erucic acid are possible. Fernandez-Martinez et al., (2001) also reported genetic stock 25 x -1 of Ethiopian mustard characterized by seed oil with essentially no erucic acid from interspecific crosses of selected lines of Ethiopian mustard, rapeseed and Indian mustard. The oil content of the progenies BNC5 and BNC2 showed an increase in the mean value over the check (PC5) with individual plants in these lines showing significantly high oil content of 41.0 per cent and 38.6 per cent, respectively. This could be due to gene introgression from B. napus parent which contains high oil content (43.4%). Mean glucosinolate content in the progeny BNC 4 (93.5 ± 10.0 µ moles/g defatted meal) was lower than the check (107.5 µ mole per g of defatted meal). However, individual plants giving very low glucosinolate content (70.5 µ moles per g of defatted meal) have been identified in the line BNC3. Previously successful introgression of desirable alleles for low glucosinolate in mustard from B. rapa was made through hybridization followed by backcross and selfing (Banga, 1996)
B. juncea x B. carinata

In B. juncea x B. carinata the cytological analysis hybrid (ABBC) confirmed the expected somatic chromosome number of 2n = 35 with upto 12 bivalents (Figure-2). The BCBBB1BBB plants exhibited varying number of chromosomes from 28 to 35. Upto 17 IIs were observed in BCBBB1BBB of B. juncea x B. carinata whereas 12II +1III + 3I were the predominant meiotic configuration. Meiotic analysis of the representative plants of each BCBBB2BBB progeny revealed chromosome no. 2n = 34 and high (16.88) mean bivalent frequency (Figure-2).

The fatty acid profile of BCBBB2BBB progenies of (B. juncea x B. carinata) x B. carinata revealed a significant increase of oleic acid content in all the lines over the check, PC5 (Table-3). Individual plants containing oleic acid content up to 23.9 per cent were identified in the progeny line BJC8. Eicosenoic acid also showed a slight decrease in majority of the progenies whereas mean erucic acid content was lower than PC5 in all the progenies. The individual plants containing as low as 17.0 per cent erucic acid (BJC8) against 45.9 per cent in PC5 indicated successful and stable transfer of genes governing erucic acid from canola mustard parent (NUDH-YJ-4). This clearly indicated stable expression of introgressed genes for low erucic acid content from NUDH-YJ-4. Brassica carinata when crossed with zero erucic acid lines of B. juncea followed by selfing and selection for lower erucic acid content led to reduced erucic acid content (Fernandez and Alonso, 1988)

Kirk and Hurlstone, (1983) reported that erucic acid content in B. juncea is controlled by two independent genes which act in an additive manner and display no dominance and concluded that one gene could be present in each of A and B genomes. The interspecific hybrid between high erucic acid B. carinata (BPPP+PPPBPPP+PPPCPPP+PPPCPPP+PPP) and zero erucic acid B. juncea (APPP-PPPAPPP-PPPBPPP-PPPBPPP-PPP) would be of the genomic constitution (BPPP+PPPBPPP-PPPCPPP+PPPAPPP-PPP). The A and C genomes in the interspecific hybrid are in the haploid state and may, therefore, pair and subsequently exchange genetic material (Attia and Roebbelen, 1986). This is expected to result in the formation of zero erucic acid BPPP-PPPCPPP-PPP gametes in the interspecific hybrid. When such BPPP-PPPCPPP- PPPgametes are fertilized with BPPP+PPPCPPP+PPP gametes of high erucic acid backcross parent, then double heterozygous BCBBB1BBB plants (BPPP+PPPBPPP-PPPCPPP+PPPCPPP-PPP) are expected to segregate into different types following selfing with some of them free of erucic acid. Previously Getinet et al., (1994) also reported a significant decrease in erucic acid content in B. carinata in BCBBB4BBB FBBB3BBB generation from a cross of B. carinata x B. juncea (zero erucic acid). The mean oil content showed a significant increase in the line BJC1 (39.1 ± 0.9%) over the check (34.3%). This line also depicted excellent variability with some plants showing as high as 41.5 per cent oil content, indicating a clear effect of gene introgression. Glucosinolate content recorded a significant decrease in most of the lines over the check (PC5). The excellent variability for low glucosinolate was present in the lines BJC30, BJC24 and BJC8 with 60.2-95.0, 73.2-127.3 and 79.5-90.3 µ moles per g of defatted meal, respectively, showing the effect of introgressed genes from B. juncea parent (NUDH-YJ-4). Earlier Getinet et al., (1997) were successful in identifying plants that contained only 20 µ mole of 2 propenyl glucosinolate from (B. carinata x B. juncea) x B. carinata combination. This constituted an 85% reduction compared with levels in B. carinata parent.


CONCLUSIONS

The present study was successful in developing diverse B. carinata types having desired variability introgressed from elite, related species for fatty acid composition, oil content and meal quality. The use of these in lines in breeding programme may result in the development of canola quality cultivars of B. carinata having high oil content and desired morphotypes.



REFERENCES
Appleqvist LA. 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, Roebbelen G. 1986. Cytogenetic relationship within cultivated Brassica analyzed in amphidiploids from the three diploid ancestors. Can. J. Genet. Cytol. 28: 323-329.
Banga S. K. 1996. Breeding for oil and meal quality. In: Chopra V L and Prakash S (eds) Oilseed and Vegetable Brassicas: Indian Perspective. pp. 234-249. IBH Publishing Co. Pvt. Ltd, New Delhi.
Fernandez-Martinez JM, Rio M Del, Velasco L, Dominguez J, Haro A De. 2001. Registration of zero erucic acid Ethiopian mustard genetic stock 25x-1. Crop Sci. 41: 282.
Fernandez-Serrano O, Alonso LC. 1988. Breeding Brassica carinata for low erucic acid contents. Cruciferae Newslett. 13: 32-33.
Getinet A, Rakow G, Downey RK. 1996. Agronomic performance and seed quality of Ethiopian mustard in saskatchewan. Can.J. Pl. Sci. 76: 387-392.
Getinet A, Rakow G, Raney JP, Downey RK. 1994. Development of zero erucic acid Ethiopian mustard through an interspecific cross with zero erucic acid Oriental mustard. Can. J. Pl. Sci. 74: 793-795.
Getinet A, Rakow G, Raney J P, Downey R K. 1997. Glucosinolate content in interspecific crosses of Brassica carinata with B. Juncea and B. napus. Plant Breed. 116: 39-46.
Harvey BL, Downey RK. 1964. The inheritance of erucic acid content in rapeseed (Brassica napus). Can. J. Pl. Sci. 44: 104-111.
Inomata N. 1992. Embryo rescue techniques for wide hybridization.In. Labana KS, Banga SS, Banga SK(eds).Breeding oilseed Brassicas. Narosa Publishing House, New Delhi, India. pp. 94-107.
Kirk JTO, Hurlstone HD. 1983. Variation and inheritance of erucic acid in Brassica juncea. Z. Pflan. zenzuchtg. 90: 331-338.
Kumar S, Yadav SK, Chauhan JS, Singh AK, Khan NA, Kumar PR. 2004. Total glucosinolate estimation by complex formation between glucosinolates and tetrachloropalladate (II) using ELISA Reader. J. Food Sci. Technol. 41: 63-65.
Malik RS. 1990. Prospects for Brassica carinata as an oilseed crop in India. Exp. Agric. 26: 125-129.
Mizushima U. 1950. Karyogenetic studies of species and genus hybrids in the tribe Brassiceae of Cruciferae. Tohoku. J. Agr. Res. 1: 1-14.
Olsson G. 1960. Species cross within the genus Brassica I. Artificial Brassica juncea Coss. Hereditas. 46: 171-220.
Thies W. 1982. Complex-formation between glucosinolates and tetrachloropalladate (II) and its utilisation in plant breeding. Fette. Seifen. Anstrichm. 84: 338-342.
Zou N., Dart P.J. and Marcar N.E. 1995. Interaction of salinity and rhizobial strain on growth and NBBB2BBB-fixation by Acacia ampliceps. Soil Biology and Biochemistry. 27: 409-413.
Table-1. Genotypes of B. napus, B. juncea, B. carinata and B. nigra used in the interspecific hybridization.


Species

Genotypes

Particulars

B. napus

NHO 7-10

‘0’ C22:1




NHO 7-12

‘0’ C22:1




NHO 7-13

‘0’ C22:1




NHO 7-14

‘0’ C22:1




NHO 7-15

‘0’ C22:1




NHO 7-16

‘0’ C22:1




MHO 18-1-21

Canola




MHO 18-1-26

Canola




MHO 18-1-35

Canola




MHO 18-1-36

Canola




MHO 18-1-184

Canola




GSC 5

Canola

B. juncea

NUDH YJ-4

Canola




NJHO 3-10A

‘0’ C22:1




NJHO 3-25

‘0’ C22:1

B. nigra

Mozambique

High C22:1




FRG 1

High C22:1




Indian nigra

High C22:1

B. carinata

PC 5

High C22:1



Table-2. Mean and range of fatty acid composition, oil and glucosinolate content in (B. napus x B. carinata) BC2.


Entries

Fatty acid composition (%)

Oil content

(%)


Glucosinolate (µ moles/g defatted meal)

Palmitic acid


Stearic

acid

Oleic

acid

Linoleic acid

Linolenic acid


Eicosenoic acid


Erucic acid

BNC 1

4.8±0.3

0.8±0.3

16.3±0.4

21.0±0.4

13.9±0.6

12.6±0.3

30.5±0.4

35.1±0.8

99.0±9.0



(4.2-5.9)

(0 – 1.9)

(15.2-17.4)

(19.9-22.8)

(11.6-15.6)

(11.9-13.5)

(30.0-31.3)

(32.9-37.9)

(84.2-115.2)

BNC 2

5.6±0.4

0.8± 0.2

20.4±1.2

25.8±1.0

12.5±0.4

10.8±0.3

24.0±1.4

35.3±0.3

109.7±7.0



(3.5-6.8)

(0-2.0)

(11.9-26.5)

(19.3-30.4)

9.8-14.4)

(8.9-12.6)

(11.8-32.0)

(33.8-38.6)

(80.5-118.9)

BNC 3

4.8±0.3

0.7±0.2

16.4±0.8

23.7±1.1

14.7±0.5)

10.0±0.3

29.5±0.3

31.9±0.5

117.7±7.9



(3.9-6.3)

(0-1.0)

(13.2-19.1)

(20.9-25.6)

(12.2-16.2)

(9.2-11.4)

(28.5-31.0)

(29.9-34.7)

(70.5-130.7)

BNC 4

6.3±0.4

0.7±0.3

20.5±1.7

24.0±1.0

13.0±0.9

11.5±0.4

24.0±2.8

34.4±0.5

93.5±10.0



(5.2-7.3)

(0.1-1.7)

(16-25.7)

(21.5-27.8)

(9.7-14.5)

(10.4-12.4)

(17.4-32.2)

(33.2-36.0)

(73.6-105.6)

BNC 5

7.0±0.4

1.6±0.2

22.3±1.1

21.7±0.4

11.9±0.5

12.7±0.3

22.7±1.1

39.2±0.7

124.6±6.0



(6.2-7.8)

(0.8-1.9)

(21.0-23.9)

(19.7-23.5)

(9.8-14.1)

(11.6-13.8)

(17.3-32.3)

(37.4-41.0)

(118.3-134.2)

PC5 (check)

3.5

0.6

11.2

18.1

11.1

9.6

45.9

34.3

107.5

NHO 7-10

5.1

1.3

67.5

18.3

6.6

0.3

0.6

43.4

80.0

Figures in parenthesis indicate range



Table-3. Mean and range of variability for fatty acid composition, oil and glucosinolate content in BCBBB2BBB progenies of the cross, B. juncea x B. carinata.


Progenies

Fatty acid composition (%)

Oil content

(%)


Glucosinolate

(µ moles/g



defatted meal)

Palmitic
acid


Stearic acid

Oleic
acid


Linoleic acid

Linolenic acid

Eicosenoic acid

Erucic
acid


BJC 1

5.2±0.2

0.4±0.1

17.4±1.4

24.8±0.5

16.4±0.8

8.6±0.3

27.2±1.2

39.1±0.9

92±3.7




(4.9 - 5.7)

(0 -0.7)

(14-20.1)

(23.5-26.9)

(14.9-18.7)

(7.9-9.2)

(25.7-30.8)

(37.2-41.5)

(84.4-97.4)

BJC 8

7.0±0.2

1.2±0.4

20.3±1.8

28.7±0.7

12.6±0.9

9.8±0.8

20.4±1.7

35.5±0.2

83.4±3.4




(6.5 - 7.5)

(0 -1.9)

(15.8-23.9)

(26.7-30.4)

(11.2-14.5)

(7.0-11.3)

(17.0-24.9)

(34.9-36.0)

(79.5-90.3)

BJC 9

5.4±0.5

0.3±0.2

12.8±0.4

23.5±0.2

14.3±0.5

8.8±0.9

34.9±1.3

35.4±1.3

97.1±3.7




(4.2 - 6.5)

(0-0.8)

(11.3-14.2)

(21.4-25.5)

(13.3-15.5)

(6.9-10.9)

(31.9-37.8)

(31.8-38.7)

(89.7-101.8)

BJC 21

4.7±0.3

0.2±0.2

18.2±0.6

28.2±1.1

13.9±0.4

9.0±0.3

25.8±0.8

30.4±0.8

93.9±1.2




(4.3-5.3)

(0 -0.6)

(16.6-19.9)

(26.4-29.6)

(12.5-15.2)

(7.7-9.9)

(25-26.6)

(28.6-32.9)

(90.8-97.1)

BJC 23

4.0±0.1

0.3±0.2

15.5±1.4

25.7±0.5

12.9 ±0.4

8.6±0.3

33.0±2.5

34.0±1.3

103.4±19.7




(3.7-4.5 )

(0 -0.7)

(12.8-18.0)

(24.4-26.9)

(11.2-14.4)

(8.0-9.2)

(29.4-37.9)

(31.0-37.1)

(80.7-126.2)

BJC 24

5.4±0.2

0.4±0.1

16.8±0.7

23.9±1.3

14.3±0.5

9.3±0.4

29.9±1.3

35.0±0.7

94.6±9.4




(4.6-6.8)

(0 -1.2)

(12.5-20.7)

(19.1-30.6)

(10.0-15.6)

(7.7-10.7)

(26.4-39.2)

(32.7-40.7)

(73.2-127.3)

BJC 28

5.6±0.3

0.6±0.2

15.7±1.8

22.3±0.5

13.9±0.4

11.0±0.6

30.6±1.1

35.6±0.6

107.7±6.5




(4.7-6.3)

(0 -1.3)

(12.9-22.5)

(20.5-25.1)

(12.9-15.9)

(9.5-13.2)

(27.9-34.3)

(33.7-37.8)

(82.7-131.1)

BJC 29

4.8±0.5

0.4±0.1

13.7±0.5

22.4±0.8

14.4±0.3

9.0±0.6

35.3±0.9

32.9±0.6

100.0±6.9




(3.7-6.8)

(0 -1.2)

(9.3-22.5)

(19.3-25.2)

(13.5-15.4)

(6.7-11.0)

(32.8-40.3)

(29.4-35.1)

(83.4-125.2)

BJC 30

3.8±0.3

0.3±0.1

18.0±2.5

23.2±0.6

13.4±0.5

11.6±0.3

29.7±2.4

33.9±1.0

81.4±7.4




(3.2-4.8)

(0 - 0.7)

(13.6-22.3)

(21.9-25.2)

(12.7-14.3)

(10.0-12.9)

(27.8-35.6)

(31.6-37.4)

(60.2-95.0)

BJC 31

6.4±0.3

0.6±0.4

18.9±1.2

25.7±1.5

13.5±0.3

11.1±1.2

23.8±2.9

36.0±0.8

95.5±8.6




(5.7-6.7)

(0.5-1.5)

(17.0-21.2)

(22.4-27.5)

(12.2-14.9)

(8.8-13.2)

(18.2-27.8)

(34.4-38.2)

(82.2-108.3)

PC5 (Check)

3.5

0.6

11.2

18.1

11.1

9.6

45.9

34.3

107.6

NUDH-YJ-4

5.2

1.5

44.3

34.9

10.7

2.8

0.2

45.1

28.3

Figures in parenthesis indicate range








A B C









D E F






G H I


Figure-1. Meiotic configuration in the PMCs of B. napus x B. carinata hybrid (A-B), BCBBB1 BBB(C-F) and BCBBB2 BBB(G-I) generations. A. 11II+ 1IV+10I; B.12II+1III+9I;BBB BBBC.10II+9I; D11II+13I; E.12II +17I; F.16-13 distribution at

anaphase; G.15II+5I; H.17II; I.18-16 distribution at anaphase.











A B C













D E F


G H I
Figure-2. Meiotic configuration in PMCs of Brassica juncea x Brassica carinata hybrid (A-B), BCBBB1 BBB(C-F) and BCBBB2

BBB BBB(G-I). A.11II+13I; B. 12II+1IV+7I; C. 12II+1III+3I; D.14II+1III+3I; E.13II+1IV+4I; F. 17II; G.16II+2I;

H.17II; I.17-17 distribution at anaphase.







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