ABSTRACT

Differences were analyzed among the CMS line L17A and its maintainer line L17B, the sterile plant L18A and the fertile plant L18B of the GMS line L18AB and the genic sterile plant GCDMS-HA and cytoplasmic sterile plant GCDMS-BA of GCDA-1, a genic and cytoplasmic double-MS (GCDMS) line in their enzymatic activity and isoenzyme patterns. CAT activity decreased significantly with bud development in all the three types of male sterility, while POD activity remained little changed. POD activity in GCDMS-HA was similar to that in L18A, being highly significantly stronger than that in L17A and L17B and significantly higher than that in GCDMS-BA and L18B. Meanwhile, CAT activity in L17A was same as that in GCDMS-HA, extremely stronger than that in L17B and L18A and stronger than that in GCDMS-BA and L18B. In the early period of bud development, no significant difference was observed between CMS and GCDMS in their isoenzyme patterns though POD activity appeared low in GCDMS-HA and GCDMS-BA as compared with that in CMS and GMS lines. In the late period of bud development, the isoenzyme patterns and activity of POD become quite similar in all genotypes. The isoenzyme patterns of EST of the CMS lines were markedly distinguishable from those of the GMS and GCDMS lines, the former having three slowly moving bands in the small bud while the latter having none. A band with medium moving speed was noticed in the EST isoenzyme pattern of GMS, which had a higher activity than that in CMS or GCDMS lines. However, more similarity appeared as the bud grew, and their differences were manifested only in migration rate. The expressions of POD and EST enzymatic activity and isoenzyme patterns of CMS, GMS and GCDMS indicate that interactions exist between the GMS and CMS sterile gene systems.

INTRODUCTION

Usually, the male sterility of plant is divided to genic male sterility (GMS) system and cytoplasmic male sterility (CMS) system. For the production of rapeseed (Brassica napus L.) hybrids in China, several GMS lines as well as CMS lines have been utilized. Although the GMS lines are completely sterile and the degree of sterility is almost 100%, no marker characters have been found tightly linked with the sterility of GMS lines. Thus in hybrid seed-production half the fertile plants must be eliminated prior to flowering of the GMS lines, this increases seed-production cost and hybrid purity is difficult to ensure. However, in CMS lines the number of sterile plants could reach 100%, but the sterility is greatly affected by environmental factors especially temperature. The degree of sterility in individual plants varies from 70% to 90% in different years (Fan and Stefansson 1986, Burns et al. 1991, Tang et al. 1994, 1997, and Li et al. 1995a).

In order to combine the advantages of the GMS and CMS system and overcome their weaknesses, the sterile gene of recessive GMS line L18AB was transferred to pol CMS line L17A through seven generations. A new male sterile system -- genic and cytoplasmic double-MS (GCDMS) system in addition to its maintainer and restorer was successfully established in 1994 (Li et al. 1995b). In GCDMS lines, half the plants maintain the features of the pol CMS line, while the other half behaves like the GMS line, thus the sterility degree of the entire line reaches 85% to 95%. The GMS genes are correctly expressed in the pol cytoplasm, but there is little interaction between the GMS and CMS genetic systems.

In present study, the enzymatic activity and isoenzyme patterns in bud of above three types of male sterile systems were studied.

MATERIALS AND METHODS

Materials: Genic sterile plant GCDA-HA and cytoplasmic sterile plant GCDA-BA of GCDMS line GCDA-1, as well as CMS sterile line L17A and its maintain line L17B, sterile plant L18A and fertile plant L18B of GMS line L18AB of rapeseed (Brassica napus L.) were used as materials. In the early flowering stage, the buds of each material were sampled and divided to two types: small bud (less than 3 mm long) and big bud (more than 4 mm long).

Enzymatic activity test: Each sample was separated into two half. One half was used to determine enzymatic activity. Activity of peroxidase (POD) was tested by means of guajacolum while Catalase (CAT) by iodometric method (Shandong Agricultural College and Northwest Agricultural College 1980).

Electrophoretic analysis: The other half of sample was grinded with buffer solution of 0.1 mol/L pH 8.2 Tris-HCl in ice store-house, and were centrifugated in frost centrifugal machine with speed 3000 rpm and then 10000 rpm. Then, the supernatant fluids were the samples of isoenzyme analysis and were stored in refrigerator. Isoenzyme patterns and activity of peroxidase (POD) and esterase (EST) were detected by polyacrylamide gel electrophoresis with unequal concentration gradient. The electrophoresis was proceeded in refrigerator. The density of separation gel was 7% and concentration gel 4%. Benzidine acetate staining method was used in POD isoenzyme stain, and color staining solution of EST isoenzyme was composed with 0.1 g 1-Naphthyl acetate, 0.1 g Fast blue RR salt, 10 ml acetone and 100 ml pH 6.4 NaH₂PO₄-Na₂HPO₄ buffer solution (Hu and Wan 1985).

RESULTS

Enzymatic activity between small and big bud: The POD enzymatic activity of small bud was similar to big one, being 173.54 and 173.37 μμ mol / g FWMin respectively. The enzymatic activity of CAT in small bud was stronger than that in big one, being 36.46 and 19.97 mg / g FWMin respectively, and the differences existed in all materials and reached 0.01 significant level. These results indicated that the POD enzymatic activity of all three types of male sterility was stability and the CAT decreased significantly as the bud development.

Enzymatic activity between different materials: Between the CMS line L17A and its maintainer line L17B, there was no obvious difference in the POD activity. The CAT activity of the former was much stronger than that of the latter, which showed that the CAT activity was related to the cytoplasmic sterility gene but the POD not in CMS system. In GMS system, the activity of POD and CAT was not different between the sterility plant L18A and the fertile plant L18B (see Table 1).

Among the different sterility system, POD activity in GCDMS-HA was similar to that in L18A, being highly significantly stronger than that in L17A and L17B and significantly higher than that in GCDMS-BA and L18B. Meanwhile, CAT activity in L17A was same as that in GCDMS-HA, extremely stronger than that in L17A and L18A and stronger than that in GCDMS-BA and L18B (see Table 1).

Table 1 also showed that the activity of POD and CAT in sterility lines was stronger than that in fertile lines. In both small and big buds, the activity of POD enzyme in GCDMS-HA was high, that in L17B and L17A low, and that in L18A, L18B and GCDMS-BA middle. In CAT enzyme, the activity in L17A and GCDMS-HA was high, that in L18A and GCDMS-BA middle, L17B and L18B low.

Table 1 The difference of enzymatic activity in bud

* lowercases – 0.05 significant level, capitals – 0.01 significant level

POD isoenzyme: In small bud, the isoenzyme patterns and activity of POD had no significant differences between L17A and L17B, between L18A and L18B and between GCDA-BA and GCDA-HA. However, in big bud, L18A had a move quickly band than L18B, so as GCDA-HA than GCDA-BA.

Comparing the three types male sterility system, in small bud, the isoenzyme patterns of POD had little significant differences between CMS and GCDMS lines, although the POD isoenzyme activity of GCDMS-HA and GCDMS-BA was weaker than that of CMS lines. However, near the positive terminal, the GMS lines had a quickly moved band whose migration rate was 0.468, whereas other materials lacked this band. The migration speeds of isoenzyme were different each other for most bands of these systems. For the big bud, the patterns and activity of POD were similar to each other. Compared with GCDMS and GMS lines, the sterile lines of CMS had a weaken band that moved slowly so that near the negative terminal. Meanwhile, at the position terminal, GCDA-HA and L18B had a band whose Rf (relative flow rate) was 0.336 but GCDA-BA and L18A not.

EST isoenzyme: The patterns of EST isoenzyme were similar between L17A and L17B in small and big bud, but the isoenzyme activity of move-slowly band of L17A in big bud was stronger than that of L17B. For GMS line, L18A was same as L18B in isoenzyme pattern of big bud except that L18B was more one band whose Rf = 0.541 than L18A.

At EST isoenzyme patterns, when bud was small, the CMS lines were distinguishable from the GMS and GCDMS lines because the former had three slowly moved bands and the latter had not. A band with medium moving speed was noticed in the EST isoenzyme pattern of GMS, which had a higher activity than that in CMS or GCDMS lines. However, as bud grew, the patterns and activity of EST became identification among the six materials, and the differences were merely in migration rate.

DISCUSSION

In order to overcome the weakness of GMS and CMS lines of rapeseed (Brassica napus L.), the genic and cytoplasmic male sterility system was first establishing in China. These lines were the best materials for studying the mechanism of male sterility of rapeseed (Brassica napus L.) and could use in production of hybrid seed.

In all three types of male sterility systems, the POD enzymatic activity was stability and held in high level, and the CAT decreased significantly as the bud development. The expresses of male sterility of rapeseed were related to the changes of the activity of CAT enzyme in bud. The POD and CAT were the key enzymes which catalysis clearing free radical of oxygen, so the male sterility formation was perhaps associated with the accumulation of free radical of oxygen.

The POD activity of GCDMS line formed transferring the sterility gene of GMS line into CMS line increased comparing with GMS and CMS lines, and the CAT activity was similar to that of CMS line in small bud and same as that of GMS line in big bud. Electrophoretic analysis also showed that the POD isoenzyme activity of GCDMS-HA and GCDMS-BA was different from that of CMS and GMS lines in the early period of bud development. At EST isoenzyme patterns, the CMS lines had three slowly moved bands but the GMS and GCDMS lines had not in small bud. At the middle of the isoenzymes pattern figure, one band had much stronger activity in GMS line than that in CMS or GCDMS line. However, as bud grew, the patterns and activity of EST became identification among all materials. So in GCDMS lines, not only the GMS genes of morphological character were correctly expressed in the pol cytoplasm, but also the GMS genes of physiological property were partly expressed. Moreover, the expressions of POD, CAT and EST enzyme genes connected with sterility of CMS, GMS and GCDMS lines indicated that there were interactions between GMS sterile gene and CMS sterile gene.

REFERENCES

1. 1. Burns, D. R., R. Scarth, and P. B. E. McVetty, 1991: Temperature and genotypic effects on the expression of pol cytoplasmic male sterility in summer rape. Canada. Journal of plant Science. 71, 655-661.

2. 2. Fan Z., and B. R. Stefansson, 1986: Influence of temperature on sterility of two cytoplasmic male-sterility system in rape (Brassica napus L.). Canada Journal of plant Science. 66, 221-227.

3. 3. Hu N., and G. Wan, 1985: Technique of isoenzyme and its application. Hunan Science. and Technology. Press, Changsa.

4. 4. Li J, Z. Tang, L. Chen, and X. Zhang, 1995a: Study on the period and critical temperature in the fertility change of rape pol CMS lines. Journal of Southwest Agricultural University. 17, 391-395

5. 5. Li J, Z. Tang, X. Zhang, and L. Chen, 1995b: Breeding a genic and cytoplasmic double-MS line of rapeseed (Brassica napus L.). Plant Breed. 114, 552-554

6. 6. Tang Z, J. Li, L. Chen, and T. Zeng, 1994: Study on the effect of environmental factors on the fertility of CMS line L17A and its F₁. In: Exploration for Crop Breed. (Symp.), 249-252, Sichuan Science and Technology Press, Chengdu.

7. 7. Tang Z, J. Li, L. Chen, and X. Zhang, 1997: Temperature influence on sterility of CMS lines and their F₁ in rapeseed (Brassica napus). Journal of Southwest Agricultural University 19, 425-430

8. Shandong Agricultural College, and Northwest Agricultural College, 1980: Experimental guidebook of Phytophysiology. Shandong Science and Technology Press, Jinan.