Hybrid Rapeseed Research Center of Shaanxi Province, Dali, Shaanxi, P.R.China,715105
Studies are conducted on the rapeseed of apetalous line and its petalous near isogenic line selected through crossing between varieties of Brassica napus. The results show that the parts of apetalous floral organ are significantly larger than those of petalous near isogenic line. The sclerotiniose and virosis resistance of apetalous line is stronger than its near isogenic line and other conventional varieties. The apetalous line’s agronomic traits are not better except it has more effective branches, so they should be improved further in the future. The proteins in flower bud analyzed by reverse-phase high performance liquid chromatography (RP-HPLC) are different, and the esterasse and peroxidase isoenzyme in seedling stage are also different between petalous and its near isogenic line.
KEY WORDS: Brassica napus, apetalous line, protein, disease resistance, esterase, peroxidase.
Breeding of apetalous rapeseed started from 1980s (Buzza, 1983). Because of lack of petal, the light consuming by petal and its canopy was saved, resulted in high yield potentiality of apetalous line. The yellow canopy of the conventional rapeseed reflects 78% of photosynthetic active radiation (PAR), with only 22% of PAR reaching the lower layer, whereas 38% of PAR reach the lower layer in apetalous line (Fary. M.J, et. al, 1996). The harvest index is up to 40% in apetalous line, while that of conventional rapeseed is about 30%. The apetalous rapessed has a strong vigor in shoot, this makes the root vigorous, so it has a better resistance to drought (Mendham N.J, et.al, 1990). Sclerotiniose is a major disease in rapeseed, no immune source has been found till now. Petals are the medium of sclerotiniose disease. At least, the spread rate of this disease in apetalous rapeseed decreases 34.4% compared with the conventional rapeseed. We have selected the stable apetalous line through crossing between varieties in Brassica napus. In the process of selection, we also get a petalous line, because the apetalous line and petalous line have a same genetic background, we could assume they are near isogenic lines. This study makes a comparison between apetalous line and its near isogenic line in aspects of morphology and biochemistry, thus revealed the features and possible reasons for apetalous trait formation.
MATERIALS AND METHODS
Apetalous line and its near isogenic petalous line.
Measurement of floral organ
In the morning of initial flowering stage, 4 plants selected in each line, 5 flower bud that will bloom from terminal raceme sampled, and taken into room immediately to measure bud, pistil, and stamen.
Preparation of protein in flower bud
Flower buds were crushed in pH 8.3, 0.2mol/L Tris-HCL containing 0.2 mmol/L PMSF in ice bath. Centrifuged the extractor at 10,000 g for 10 min. Got 1 ml supernatant, added 3 ml of pre-cooled acetone in it. Centrifuged the mixture with Beckman J2-21 at 4℃ 15,000g. Discarded the supernatants and dissolved the protein in the bottom with 20 g/L SDS. Injected 20μl to HPLC analysis.
A Beckman 338 HPLC system with Sperisorb column(C18, 300 A pore size, 5 μm particle size, 20 cm x 4.6 mm ID) used for separation of bud protein as described previously(Chen S H, 1991).
Electrophoresis of esterase and peroxidase
Analysis as described previously. (Davis,1964. Li,1992).
The effect trait of apetalous rapeseed on the floral organ
The apetalous rapeseed had a big influence on the floral organ (Table.1). Compared with petalous
line, the weight of apetalous flower bud changed little, while the length increased. The weight of sepal increased 46.58%, and that of the pistil doubled the petalous line. The length and the width of pistil increased 51.79% and 26.43%, respectively. The weight of anthers increased 42.58%, this increase mainly attributed to the length extends. Because the petal disappeared, the nutrition that formed the petal was fed to pistil and anther, leading to fast growing of them, and resulting in big pistil and large amount of pollen. In the apetalous line, without any obstacle, the pollen was easy to float in the air. This is advanced for pollination. If apply the apetalous line in pollen parent in hybrid seed production field, it is possible to increase the hybrid seed yield. Pistil becoming larger offers a matter basement for pod growing in mature stage.
Table 1. Floral organ comparison between apetalous line and its isogenic petalous line n=20
The agronomic traits of Apetalous line
Table.2 The survey of the agronomic traits of apetalous line and its near isogenic line, n=10
In apetalous line, except that the number of first effective branches increased and the height of plant, length of terminal raceme decreased significantly, location of effective branches become higher, Grains number per pod reduced. These show that the agronomic traits need further improvement in breeding. In recent years, we have selected some elite lines of apetalous rapeseed. Although they are late and stems are soft, yield increase is large. It is possible to better the agronomic traits of apetalous rapeseed and achieve its high yield potentiality.
The disease resistance of apetalous line.
Compared with the petalous isogenic line, apetalous line ash a strong resistance to sclerotiniose and virosis (Table 3). The reason of good resistance to sclerotiniose was explained before ( Chen, 1991).
Table.3 Disease (Sclerotiniose and Virosis) investigation. Date: June 2nd, 1998
Note: Both apetalous and petalous lines were fully lodged because of heavy rain fall in 1998.
But its resistance to sclerotiniose was not as good as conventional variety Zhongyou 821, which has been a major cultivar in China for many years. The possible reason was that in the summer of 1998, there was much rainfall, and the stem of the apetalous line and its near isogenic petalous line were very soft, both of two lines were fully lodged. Why apetalous line has such good resistance to virosis is not clear, the reason needs to be studied.
The difference of protein profile in floral buds between apetalous and petalous lines
The bud protein is mainly composed by protein 2 and 3 (Figure 1). The apetalous line has a special protein 1, and quantity of protein 2 and 5 are higher than those of petalous line, while the quantity of the protein 3,4,6 are lower (Data not shown).
The difference of esterase and peroxidase isoenzyme in vegetable stage between apetalous line and its near isogenic rapeseed.
The profile of esterase and peroxidase isoenzyme is different at vegetable stage (Figure .2 and 3 ). As to the esterase, at sprout stage, petalous line has a specific band A. At two-leaf stage, petalous line has an additional band B and the apetalous line has an additional band C. At six-leaf stage, petalous line has an additional band D. As to the peroxidase isoenzyme, in two leaf stage, apetalous has a very strong E, petalous line has an additional F. In six-leaf stage, petalous line has an additional G. These differences show that the genes that control the apetalous trait have different expressions at different stage. According to these features, we can identify the apetalous line and petalous line.
Figure.2 Esterase isoenzyme profile. “A” is Apetalous, “P” is Petalous
Figure.3 Esterase isoenzyme profile. “A” is Apetalous, “P” is Petalous
Apetalous line has big pistil and anther that is advantageous to form big pods and pollinate, but it has some inferior agronomic traits. Others reported apetalous lines also had the problem (Buzza,1983; Fary.M.J. et.al.,1996; S.Z.Fu,1985). The linkage between apetalous trait and inferior agronomic traits may cause this problem. The apetalous line has very good resistance to sclerotiniose and virosis. The mechanisms of resistance to virosis need to be pursued.
The analysis of bud protein shows that not only the quantity of proteins is different between two lines, but also the apetalous line has an additional protein. The esterase isoenzyme and peroxidase isoenzymes are different between apetalous line and its isogenic petalous line in vegetable stage. All these differences are associated with the apetalous trait. This work will lay a foundation for research of formation of apetalous characteristic.
Thanks to Mr. Dong Huiqing for his translation and Mr. Fan Sanhong for his help in word process.
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