ABSTRACT

The potentiality of rape as an forage crop was investigated on using 25 accessions belonging to 5 species of Brassica. Dry weight, fiber content, seed yield and seed protein contents affecting forage quality were determined during two successive years 1997 and 1998. Both Brassica species and accessions significantly affected dry weight. Brassica juncea accession No. 21 gave the highest significant dry weight/plant being 69.8 and 61.32 g in 1997 and 1998. While, Brassica carinata accession No.5, Brassica rapa (campestris) accession No.6 as well as Brassica napus accession No.17 had the highest significant mean crude fiber content/plant that were 39.29, 42.28 and 42.55% in 1997 and 41.54, 46.16 and 41.26% in 1998, respectively. The overall mean value for seed yield indicated that Brassica juncea accession No.23 revealed the highest seed yield being 35.32 g/plant in 1998. The statistical analysis revealed that Brassica nigra accession No.12 and Brassica juncea accession No.22 were the highest significant with respect to mean protein contents being 33.72 and 35.56% in 1997 and 36.20 and 34.75% in 1998, respectively. The electrophoretic pattern of buffer soluble protein extracts different species denoted the stability of different species towards crossing during the successive cultivation for four years.

INTRODUCTION

For centuries, Brassica species have exploited by man, domesticated and modified to meet changing needs. Rape has an important role, now in Egyptian agriculture, especially in new land of the North-west coastal region of deserts because it provides fresh herbage for livestock during autumn and winter because of their high protein content and high dry matter digestibility (Wilson et al., 1992 and Wiedenhoeft, 1993). Oilseed rape cultivation has increased tremendously during the last decade and, by now, oilseed rape is the second largest contributor to the world supply of vegetable oil.

The suitability of green rape as a forage for cattle was reported by several authors. In USSR, Ovsishcher and Bonderva (1983) indicated that rape contains 10.0 to 15.6% dry matter and 2.6 to 4.8% crude protein. In Egypt, Sharaan and Abdel-Gawad (1986) studied the influence of cultivars and seeding rate on forage yield and crude protein content of three cultivars of Brassica napus namely Bro, Brio and Orpal. Bro gave the highest total dry matter yield (3.5 t/fedan) and dry matter crude protein yield (0.36 t/feddan). Seed protein content was the subject of extensive studies. Liu (1990) determined seed protein content of 441 varieties of Brassica napus, Brassica campestris and Brassica juncea and reported that the protein content varied between 13 and 36%, of which 90% of the material had 20-30% protein, whereas Brassica juncea seed contained the highest protein content (over 33%).

Seed protein patterns obtained by electrophoresis had been successfully used to resolve taxonomic and evolutionary problems of several crop species (Ladizinsky and Hymowitz, 1979). Moreover, such protein patterns provided a promising tool for distinguishing cultivars of a particular crop species and are considered to be particularly reliable, as seed storage proteins are largely independent of environmental factors. The use of soluble protein and its fractions obtained by acrylamide gel electrophoresis for solving various taxonomic problems of different species of Brassica seeds was reported by Vaughan and Waite (1965), Yadova et al.(1979), Odintsova et al.(1987) as well as Raab et al. (1992). Cultivars could be also reliably distinguished from one another on the basis of water soluble proteins identified by polyacrylamide porosity gradient according to Gupta and Robbelen (1986). Further studies had indicated the applicability of using SDS-PAGE electrophoresis of water soluble proteins for the identification of Brassica species and cultivars (Oost and Relou, 1985). In Egypt, Brassica is still not widely cultivated as an oil crop, although there is a big shortage in oil production for human consumption. The aim of present research program is to determine the stability of 25 accessions of Brassica against crossing during cultivation for four successive years. This could be accomplished by following seed water soluble protein fractions obtained by gel electrophoresis.

MATERIALS AND METHODS

Materials

The twenty-five different accessions that belong five Brassica species, namely; Brassica carinata, Brassica rapa (campestris), Brassica nigra, Brassica napus and Brassica juncea were obtained from the Regional Plant Introduction Station, Iowa State University, A.R.S, U.S.D.A, as shown in Table 1. Each accession was planted for successive two years 1995 and 1996 for multiplying the number of seeds for future studies on field scale experiments.

Methods

Field evaluation: A randomized complete block experimental design, with four replicates, was used in both winter seasons 1997 and 1998.

The experimental field was mechanically plowed, harrowed, leveled and, then, divided into plots. Each plot consisted of three ridges, 3 m long and 60 cm apart. Accordingly, the area of each plot was 4.8 m² within ridges. Superphosphate (15.5% P₂O₅), at the rate of 150 kg/feddan (375 kg/hectare) was incorporated into the soil during land preparation. Nitrogen, in the form of urea, was applied once to the field at the rate of 120 kg N/feddan (300 kg N/hectare), thirty days after planting, according to the recommendations of the Egyptian Ministry of Agriculture. Before nitrogen application, plants in the experimental sites were thinned into two plants per hill. At heading time, only three plants were randomly chosen in each plot in order to be used for identification of seed protein through electrophoresis. Whereas, the remaining plants were left for open-pollination.

Crude protein content: Crude protein content was determined using the boric acid modification of Kjeldahl method (A.O.A.C., 1980). A duplicate sample of 0.5 g of the seed from each plot were analyzed. Samples were ground and dried to a constant weight. Percentage of protein was calculated by multiplying the nitrogen content by the factor 6.25.

Seed protein electrophoresis: Selfed seeds were milled, using a morter and pestile. After milling, seeds were defatted in soxhlet apparatus for eight hours, using hexane (60-80°C). Defatted meal was remilled by using a morter and pestile to a fine powder; whereas, hexane extracts of different accessions were used as a source of total lipids for determining the fatty acid composition. While, the defatted fine powder was used as a source of total proteins, as it was extracted with a buffer system (0.5 M Tris-HCl, pH 6.8). The mixture was stirred for twenty min, squeezed through muslin cloth and centrifuged (5000 x G) at 4°C for five min. The supernatant was decanted and saved for electrophoresis. Total protein was estimated by the method of Lowry et al.(1951), whereas fractionation of buffer soluble proteins was carried out using SDS-PAGE. Discontinuous system was performed, following the procedure of Laemmli (1970), using Slab’gels that contain 10% acrylamide. Protein samples were mixed with a loading buffer (1:2 v/v) which contained the following: 4.0 ml distilled water, 1.0 ml (0.5 M) Tris-HCl (pH 6.8), 1.0 ml glycerol, 0.8 ml SDS (10% w/v), 1.6 ml 2-mercaptoethanol and 0.4 ml bromophenol blue (0.05%). The low molecular weight protein Kit Sigma (from 14.400 to 94.000 cat. No. 11-B-036-07) was used as molecular-size standards. Unknown protein sample extracts (each of 10 μl) were loaded into a gel slab and were subjected to electrophoresis at 25 mA (80 voltage) constant current per 7.3x10.2x0.01 cm slab on Bio-Rad model No. mini-PROTEAN II Cell. The gels were run for one hour after the bromophenol blue dye had reached the bottom of the gel in the presence of a running buffer, which consisted of Tris base, 9.0 g, glycine, 43.2 g, SDS, 3 g dissolved in 600 ml distilled water and the pH was adjusted to 8.3.

Staining and destaining of gels: Proteins were stained for one hour with 0.1% Commassie Brilliant blue (R-250) in methanol:acetic acid:distilled water (40:20:40) v/v. Gels were destained with a solution containing methanol : acetic acid : distilled water (40:20:80) according to Gupta and Robbelen (1986).

RESULTS AND DISCUSSION

Forage potentiality characteristics

Dry weight per plant (g): The analysis of variance data, presented in Table 2, showed that Brassica accessions had highly significant effects on dry weight/ plant in the two successive seasons.

Mean values for this trait of different accessions are presented in Table 3. Data in this table indicated that Brassica juncea (accession No.21), in the two seasons, gave the highest dry weight per plant, being 69.2 and 61.32 g, respectively. These results explain the superiority of accession 21 (Brassica juncea) in fresh and dry weights/plant and thus could be used a good forage and range species for livestock in old or new land in Egypt. On the other hand, in 1997, Brassica nigra (accession No.11) and Brassica juncea (accession No.23) proved to have the lowest dry weight/plant, being about 14.2 and 12.9 g, respectively. In addition, in 1998, Brassica carinata (accession No.5), Brassica rapa (accession No.8), Brassica napus (accession No.17) and Brassica juncea (accession No.23) gave low dry weights/plant, ranging between about 14 and 15 g.

Similar results were obtained by Ahn et al.(1989) and Wiedenhoeft (1993). Jo and Kim (1988) and Kunelius and Sanderson (1990) reported that Brassica napus cultivars differed in dry matter yield and depended on the genetic variations among them. In the same time, Ahn et al.(1989) stated that the total dry matter yield was positively correlated with plant height and fresh weight. These results agreed with data of accession No.21 (Brassica juncea) for which their plant height (170.5 cm) was correlated with fresh weight/plant (383.9 g) and dry weight/plant (69.2 g).

Crude fiber content per plant (%): A highly significant effect of Brassica species and accessions on crude fiber content per plant is shown in Table 4.

The mean values for this character, as influenced by different accessions, are presented in Table 5. The data, herein, revealed that accession No.5 (Brassica carnata), accession No.6 (Brassica rapa) and accession No.17 (Brassica napus) had the highest significant mean values for crude fiber content/plant in both seasons. Their values were about 39.29, 42.28 and 42.55%, in 1997, and 41.54, 46.16 and 41.26%, in 1998, respectively. In the meantime, the accessions No.1 and 3 (Brassica carinata), 12 (Brassica nigra) and 22 and 25 (Brassica juncea) gave the lowest crude fiber content/plant in 1997. Their mean values were about 18.78, 17.34, 15.85, 17.61 and 18.32, respectively.

In 1998 season, the lowest accessions included Brassica carinata accession No.3 (16.18%), Brassica nigra accession No.11 (17.77%) and Brassica juncea accession No.22 (16.83%). Intermediate values for crude fiber content were obtained by the other accessions and ranged between about 17 to 34%. These results were similar to those obtained by Ovsishcher and Bonderva (1983), Ahn et al.(1989) and Kunelius and Sanderson (1990).

Crude protein content (%) in seed: The analysis of variance for protein content in seed, obtained in 1997 and 1998 seasons, is given in Table 6. Data indicated that Brassica accessions had a highly significant effect on protein content in seeds in the two growing seasons (1997 and 1998).

From the mean value data on crude protein content in seed, given in Table 7, it is clear that accessions No.12 (Brassica nigra) and No.22, which belongs to Brassica juncea had the highest significant protein contents in seeds with mean values of 33.72 and 35.56%, respectively, in the first season. Also, in the second season, the same accessions gave the highest significant mean contents of protein, their values being 36.20 and 34.75%, respectively. On the other hand, Brassica carinata accession No.5, Brassica rapa (campestris) accessions No.7 and 10, and Brassica napus accession No.17, gave the lowest significant protein contents in both seasons (1997 and 1998). Their mean values ranged from about 19.8 to 20.7%. The other accessions differed in their protein content in seeds and varied from 20 to 31%, respectively.

The present results were similar to those obtained by Ahn and Kwon (1989). They mentioned that the average content of crude protein in some cultivars of Brassica napus was about 22.5%. In the same time, Sharaan and Abdel-Gawad (1986) stated that the cultivars of Brassica napus were different in crude protein as a result of their genetic variation. Liu (1990) indicated that the seed protein content of Brassica napus, Brassica campestris and Brassica juncea ranged from 13 to 36% but Brassica juncea had the highest average content (over 33%). Also, Ovsishcher and Bondereva (1983) reported similar findings.

Fractionation of soluble proteins

The data of the prsent study was concerned with the differences in the number of protein bands of the buffer soluble protein extracts of examined monoploid, as well as amphidiploid species, instead of using the relative mobility and intensities of protein bands as a tool for differentiation between different accessions, as being stated by Batmanova et al.(1987).

The analysis of soluble protein fractions of different accessions and species showed distinguished variabilities in the number of bands between the monoploid and amphidiploid species (Table 8). For instance, the electrophoretic patterns of the monoploid, Brassica rapa (campestris) (AA) and Brassica nigra (BB) showed the presence of six bands versus eight bands in amphidiploid patterns, such as Brassica carinata (BBCC), Brassica napus (AACC) and Brassica juncea (AABB). The most pronounced difference, with regard to molecular weight (MW), between the monoploidy and the amphidiploidy species obtained in this study were observed from the presence of a protein band with a MW similar to the standard reference α-lactalbumin (14 kDa) in amphidiploid species and the absence of such band in monodiploid species. Whereas, the difference between the amphidiploid species, with regard to the number of bands and MW, was observed from the following:

1) The presence of three protein bands over each other in Brassica napus, where the faster one in mobility is a protein band with a MW similar to carbonic anhydrase (30 kDa) and the other two bands have MW of 33 and 35 kDa. On the other hand, these bands were characterized as two bands in Brassica juncea with MW of 33 and 35 kDa (Table 9 and Fig.1).

2) The presence of three protein bands over each other in Brassica carinata where the faster band in mobility is a band with a MW similar to trypsin inhibitor (20 kDa) and the other two bands revealed MW 22 and 24 kDa. On the other hand, in Brassica juncea, these above three bands in Brassica carinata are found to be two bands with MW 22 and 24 kDa (Table 9 and Fig.1).

The above findings are to be used as a criterion for the differentiation between monoploid and amphidiploid species. Buffer soluble protein patterns on polyacrylamide gel electrophoresis was used by Raab et al.(1992) as tools for the differentiation between different varieties of rapeseed. However, Yadova et al.(1979) were unable to identify a specific protein band of genome CC (Brassica oleracea) in the genomic combination, BBCC of Brassica carinata which is the natural amphidiploid derived from Brassica nigra (BB) and Brassica oleracea (CC). On the other hand, Vaughan and Waite (1965) found that monoploid species including Brassica campestris, Brassica oleracea and Brassica nigra, could be distinguished from each other by an unique band or bands in their seed protein profiles. Similarly, they reported that amphidiploids including Brassica carinata, Brassica juncea and Brassica napus, represented different combinations of protein bands of above monoploid species, that was taken as an additional evidence that the latter three Brassica species are of hybrid origin.

CONCLUSION

Brassica juncea accession No.21 gave the highest significant dry weight, while Brassica napus accession No.17 had the highest significant mean crude fiber content/plant. Statistical analysis revealed that Brassica nigra accession No.12 and Brassica juncea accession No.22 exhibited the highest significant mean protein contents than other accessions investigated. Buffer soluble proteins (Tris-HCl, pH 6.8) different Brassica species can be fractionated on SDS-PAGE to different proteins that can be used to differentiate between different species.

REFERENCES

1. Ahn, G.S. and Kown, B.S. (1989). J. Korean Anim. Sci. 31: 192-199.

2. Ahn, G.S., Kown, B.S. and Lee, J. (1989). J. Korean Crop Sci. 34: 335-340.

3. Association of Official Agricultural Chemists (A.O.A.C) (1980). 11^th ed., Washington, D.C., USA.

4. Batmanova, L.S., Odintsova, T.I., Egorov, T.S.A., Kononkov, P.F. and Sozinov, A.A. (1987). Sel’skokhozy aistvennykh-Nauk-Imeni V.1. Lenina 7: 16-19.

5. Gupta, S.K. and Robbelen, G. (1986). Zeitschrift Pflanzenzuchtung 96: 363-370.

6. Jo, M.H. and Kim, D.A. (1988). J. Korean Societ. Grassland Sci. 8: 33-39.

7. Kunelius, H.T. and Sanderson, J.B. (1990). App. Agric. Res. (USA) 5: 159-163.

8. Laemmli, U.K. (1970). Nature. 227: 680-685.

9. Ladizindsky, G. and Hymowitz, T. (1979). Theor. Appl. Genet. 54: 145-151.

10. Liu, X.Y. (1990). Crop Genet. Resour. 4: 21-22.

11. Lowry, O.M., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chem. 193: 265-275.

12. Odintsova, T.I., Batmanova, L.S. and Kononkov, P.F. (1987). Tez. Dokl. 133-134.

13. Oost, E.H. and Relou, J.P.M. (1985). Cruciferae Newsletter No.10, 26.

14. Ovsishcher, B. and Bonderva, N. (1983). Molochnoe-I-Myasnoe-Skotovodstvo. No.7, 24-27.

15. Raab, B., Leman, H., Schwenke, K.D. and Kozlowska, H. (1992). Nahrung 36: 239-247.

16. Sharaan, A.N. and Abdel-Gawad, K.I. (1986). Annals Agric. Sci., Moshtohor 24: 1857-1870.

17. Vaughan, J.G. and Waite, A. (1965). J. Exp. Botany 17: 332-343.

18. Wiedenhoeft, M.H. (1993). Agron. J. Miadison, Wis. 85: 549-553.

19. Wilson, A., Kwanyuen, F.P., Dewey, R.E. and Settlge, (1992). Seed oils for the future pp.116-135.

20. Yadova, J.S., Chaudhary, J.B., Kakar, S.N. and Nainawatee, H.S. (1979). Theor. Appl. Genet. 54: 89-91.

Table 1. Origin of studied Brassica accessions.

Serial Species No. and lines	Source country	Common name
Brassica carinata 1 331378 2 209023 3 194901 4 194253 5 193460 Brassica rapa (campestris) 6 162778 7 392052 8 426175 9 365643 10 458614 Brassica nigra 11 347621 12 368377 13 183020 14 254362 15 176881 Brassica napus 16 470063 17 531287 18 469767 19 469783 20 458935 Brassica juncea 21 426376 22 220511 23 340220 24 179183 25 478328	Poland Puerto Rico Ethiopia Ethiopia Ethiopia Argentina Canada Afghanistan Canada, Manitoba New Zealand India Yugoslavia India Delhi, India Turkey South Korea South Korea Germany South Korea Sweden Pakistan Afghanistan India Turkey China	NU 51639 - - NU 51467 - NABO CANASPAN K-1071 POLAR Green resistant IB 330 ELEV 900 M - - - Dwarf Essex Dong Hae 10 Santana Yonkoku Ban Brink K-569 - In. 48151 - O 63

Table 2. Analysis of variance for dry weight/plant (g) of Brassica accessions in 1997 and 1998 seasons.

Source of variation	D.F.	Mean squares
		1997	1998
Replications Accessions Error	3 24 72	21.917 1001.25** 7.331	13.354 487.63* 21.87
C.V. %		8.05	18.01

** : Significant effects at 0.01 level.

C.V. : Coefficient of variability.

Table 3. Mean values of dry weight/plant (g) of twenty-five Brassica accessions in 1997 and 1998 seasons.

Soeciees and accessions	Dry weight (g)
	1997	1998
Brassica carinata 1 2 3 4 5 Brassica rapa (campestris) 6 7 8 9 10 Brassica nigra 11 12 13 14 15 Brassica napus 16 17 18 19 20 Brassica juncea 21 22 23 24 25	44.80 e 29.57 h 42.63 e 42.82 e 17.52 kl 50.42 d 25.20 i 14.52 lm 18.97 jk 16.00 klm 14.20 lm 17.92 kl 34.75 f 51.42 cd 30.75 hg 46.22 e 24.55 i 21.83 ij 57.12 b 42.87 e 69.28 a 24.82 i 12.92 m 34.02 fg 54.90 bc	28.72 efg 20.70 hijk 35.22 cde 19.70 ijkl 15.20 kl 39.7 bc 21.35 hijk 14.95 kl 26.92 fgh 25.72 fghi 22.30 ghij 22.47 ghij 30.00 36.55 cd 19.20 ijkl 18.70 jkl 13.60 l 16.02 jkl 26.92 fgh 26.57 fgh 61.32 a 18.25 jkl 15.20 kl 45.47 b 28.22 fg
L.S.D (0.05)	3.81	6.59

Means with the same letter(s) are not significantly different at 0.05 level

of probability.

Table 4. Analysis of variance for crude fiber content/plant (%) as affected by 25 Brassica accessions in 1997 and 1998 seasons.

Source of variation	D.F.	Crude fiber content/plant (%)
		1997	1998
Replications Accessions Error	3 24 72	5.726 261.92** 5.709	3.892 286.27** 6.180
C.V. %		8.79	9.072

** : Significant effects at 0.01 level.

C.V. : Coefficient of variability.

Table 5. Mean values for crude fiber content/plant (%) of twenty-five Brassica accessions in 1997 and 1998 seasons.

Species and accessions	Crude fiber content/plant (%)
	1997	1998
Brassica carinata 1 2 3 4 5 Brassica rapa (campestris) 6 7 8 9 10 Brassica nigra 11 12 13 14 15 Brassica napus 16 17 18 19 20 Brassica juncea 21 22 23 24 25	18.78 ghi 21.06 g 17.34 hi 32.66 bc 39.29 a 42.28 a 32.48 bc 32.56 bc 24.47 f 31.30 cd 19.25 gh 15.85 i 20.30 gh 27.94 de 33.98 bc 34.80 b 42.55 a 33.43 bc 28.61 d 21.55 fg 20.08 gh 17.61 hi 28.38 d 24.69 ef 18.32 ghi	23.48 fgh 22.30 gh 16.18 i 34.66 c 41.54 b 46.16 a 34.76 c 29.21 de 21.60 ghi 30.19 d 17.77 jkl 18.20 ijkl 20.10 hijk 30.98 d 38.38 b 29.63 de 41.27 b 31.64 cd 26.28 ef 20.95 ghij 20.38 ghij 16.83 kl 28.45 de 23.78 fg 20.38 ghij
L.S.D (0.05)	3.36	3.5

Means with the same letter(s) are not significantly different at 0.05 level

of probability.

Table 6. Analysis of variance for crude protein content/plant (%) in seeds and whole plant as affected by 25 Brassica accessions in 1997 and 1998 seasons.

Source of variation	D.F.	Mean square of protein content (%) in seeds		Mean square of protein content (%) in whole plant
		1997	1998	1997	1998
Replications Accessions Error	3 24 72	2.82 92.081** 4.909	6.801 88.689** 3.089	5.6819 35.9212** 1.479	2.233 33.565** 1.997
C.V. %		8.3	6.69	9.06	10.855

** : Significant effects at 0.01 level.

C.V. : Coefficient of variability.

Table 7. Mean values for protein content (%) in seeds and whole plants as affected by 25 Brassica accessions in 1997 and 1998 seasons.

Species and accessions	Protein content in seeds (%)		Protein content in whole plant (%)
	1997	1998	1997	1998
Brassica carinata 1 2 3 4 5 Brassica rapa (campestris) 6 7 8 9 10 Brassica nigra 11 12 13 14 15 Brassica napus 16 17 18 19 20 Brassica juncea 21 22 23 24 25	28.27 fge 26.05 ghij 31.65 bcd 27.52 fgh 19.67 mn 19.07 n 21.08 lmn 21.90 klm 23.10 jkl 22.36 klm 32.08 bc 33.72 ab 31.66 bcd 29.04 cdefg 27.02 fghi 24.54 ijk 19.04 n 21.18 lmn 24.08 ijkl 28.58 defg 31.34 bcde 35.56 a 28.80 defg 29.59 cdef 29.07 cdefg	29.63 de 27.68 efg 32.30 bc 26.58 fgh 20.15 mn 21.45 lmn 20.73 mn 21.63 jklm 22.60 jklm 19.78 n 29.43 de 36.20 a 31.18 cd 27.35 efg 24.43 hij 23.85 ijkl 19.73 n 24.00 ijk 21.45 lmn 29.35 de 27.68 efg 34.75 ab 28.85 def 29.35 de 26.13 ghi	11.75 hij 13.54 efg 15.91 cd 14.38 def 10.10 jk 11.66 hij 10.73 ij 11.18 hij 12.67 fgh 10.89 ij 14.48 de 21.35 a 17.13 bc 11.61 hij 12.10 ghi 13.80 efg 8.81 k 10.25 jk 11.70 hij 13.64 efg 14.42 de 18.80 b 11.19 hij 16.77 c 16.39 c	11.08 hijklm 11.99 ghijk 16.37 c 12.81 efghi 11.76 ghijkl 14.20 def 9.99 lm 12.20 ghij 10.43 jklm 10.90 ijklm 14.70 cde 21.68 a 14.53 cde 12.35 fghij 11.10 hijklm 13.00 efgh 9.11 m 10.15 klm 11.21 hijkl 14.63 cde 13.55 defg 18.70 b 10.01 klm 15.39 cd 13.66 defg
L.S.D. (0.05)	3.120	2.4775	1.710	1.992

Means with the same letter(s) are not significantly different at 0.05 level of probability.

Table 8. Number of buffer soluble protein bands of different species and accessions of Brassica obtained on SDS-PAGE.

Species and accessions	Number of bands
	1	2	3	4	5	6	7	8	9	Total No. of bands
B. carinata (BBCC, 2n = 34) 1 2 3 4 5 B. napus (AACC, 2n = 38) 16 17 18 19 20 B. juncea (AABB, 2n = 36) 21 22 23 24 25 B. rapa (campestris) (AA, 2n = 20) 6 7 8 9 10 B. nigra (BB, 2n = 16) 11 12 13 14 15	+ + + + + + + + + + + + + + + + + + + + + + + + +	+ + + + + + + + + + + + + + + + + + + + + + + + +	+ + + + +	+ + + + + + + + + + + + + + + + + + + + + + + + +	+ + + + + + + + + + + + + + + + + + + + + + + + +	+ + + + + + + + + + + + + + + + + + + + + + + + +	+ + + + +	+ + + + + + + + + + + + + + +	+ + + + + + + + + + + + + + + + + + + + + + + + +	8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 6 6 6 6 6 6 6 6 6 6

Table 9. Molecular weight of buffer soluble protein of different species and accessions of Brassica obtained on SDS-PAGE stained by Commassie Brilliant blue..

Species and Accessions	Protein bands MW (kDa)
	1	2	3	4	5	6	7	8	9
B. carinata (BBCC, 2n = 34) 1 2 3 4 5 B. napus (AACC, 2n = 38) 16 17 18 19 20 B. juncea (AABB, 2n = 36) 21 22 23 24 25 B. rapa (campestris) (AA, 2n = 20) 6 7 8 9 10 B. nigra (BB, 2n = 16) 11 12 13 14 15	67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67 67	35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35	- - - - - 33 33 33 33 33 - - - - - - - - - - - - - - -	30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30	24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24	22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22	20 20 20 20 20 - - - - - - - - - - - - - - - - - - - -	14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 - - - - - - - - - -	10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Fig.1-A. SDS-PAGE seed protein from Brassica species (monoploids).

Fig.1-B. SDS-PAGE seed protein from Brassica species (amphidiploids).