Characterization And Genetic Study Of The Male Abortion Induced By A Chemical Hybridizing Agent Hua-Sha-Ling In Brassica Napus L.

Rapeseed is one of the world’s major oil crops, the acreage and total production in Chinese ranks first in the world. Heterosis is a universal biological phenomenon, which has important theoretical value and broad application prospects. On heterosis utilization of rapessed in different ways, the easiest theoretically to breeding objectives is chemical hybridizing agent (CHA) inducing male sterility. In this study, as the material around the CHA Hua-Sha-Ling inducing male sterility trait, varieties (lines) 9313B, and R121, etc., in Brassica napus L. would be searched. The main findings are as follows:1.Main characteristics of male Abortion induced by the chemical hybridizing agent (CHA) Hua-Sha-Ling in Brassica napus L.Four B. napus lines (R121,9313A,9313B, Leyou-5) were sprayed with 5 different concentrations of CHA Hua-Sha-Ling on leaves of the rapeseed plants at the microspore development stage. Activities of SOD, POD and CAT in the leaves and flower buds, glutathione S-transferase (GSTs) activity in the leaves, and chlorophyll contents in the leaves were determined. Male fertility and morphological characteristics of the flowers, and main economic characters of the rapeseed materials were investigated. Cytological observations were made under microscope with the paraffin sections of aborted anthers for R121(Tr)and 9313B(Tr).The results showed that:(1) growth and development of the plants were restrained in various degrees, and stamens were degradated with empty, flat and aborted anthers. (2) The rates of male abortion in the four lines were 95-100% by two times of spraying, the optimal concentrations were 0.07 g/L-0.21 g/L,8-10 ml/plant.(3) SOD, CAT Activities in the leaves of the 4 lines continued to rise; POD activity maintained at a higher level but lower compared with the control. SOD, CAT activities in the bud continued to decline, but POD activity was higher than the control; all lines showed a rise first and then a decline except for R121. In all, mismatch of the three kinds of enzymes in the process of active oxygen metabolism is an important characteristics of the male abortion. (4) The activity of GSTs showed a declining trend, the relative activity of GSTs in leaf and buds was less than 1, and the range of change in 9313B was smaller than in R121.The difference in metabolic capacity of GSTs is another important reason for the difference in CHA sensitivity.(5) The contents of chlorophyll declined in varying degrees. There was a comparatively large difference among the 4 lines and the degree of decline was directly related to the sensitivity. (6) In the observations of paraffin sections, a variety of differences appeared between the treated lines and the controls in the period of pollen mother cell development. The male abortion appeared to happen due primarily to the pollen mother cell dysplasia, the abnormal tapetal disintegration and the anomalies in microspore development. The main abortion periods were in the pollen mother cell development stage for R121 (Tr), and in the microspore stage for 9313B (Tr).2. Bioassay of sensitivity to CHA Hua-Sha-Ling in Brassica napus L.Thirty-six B.napus lines were taken as study materials and treated with different concentrations of CHA "Hua-Sha-Ling". The influences on primary root length and main characters of seedling in pots, and main characters of plants in the field were investigated. The main results in this experiment included:(1) It was preliminarily found that 3 lines were weakly sensitive,6 lines were strongly sensitive, and 27 lines were moderately sensitive to Hua-Sha-Ling. (2) Seeds of the lines were treated with Hua-Sha-Ling solution after germination12 hours, and primary root lengths were measured after 72 hours. It was observed that the primary root lengths could reflect the different sensitivity to Hua-Sha-Ling by sensitive index and IC50 value in the lines. (3) Very significant positive correlations were observed between phytotoxicity inhibition index, rate of leaf reduction, rate of leaf angle increase, rate of fresh weight inhibition after 14th days, These can be used as evaluation indicators of the sensitivity in seedling stage. (4) There was a highly significant positive correlation between the reduction in rate of chlorophyll content on the 4th day,8th day and 12th day and the reduction in rate of fresh weight after the 14th day. So the reduction in rate of chlorophyll content on the 4th day can be used as an early evaluation index of the sensitivity to Hua-Sha-Ling. (5) The sensitivity of rape to Hua-Sha-Ling can be evaluated and selected in three growth periods including the germination period, the seedling period and the flowering period. The indices including half maximal inhibitory concentration of substances (IC50) on primary root length after germination, half maximal inhibition rate of fresh weight (ID50) at the 5th-leaf stage, and rates of male sterility and female fertility at flowering stage can be used for conventional indices to evaluate the sensitivity of rapeseed lines. The reduction in chlorophyll content in seedling stage can be used as a supplementary index to the conventional evaluation system.3. Inheritance of sensitivity induced by CHA Hua-Sha-LingSix genetic generations were prepared using a Hua-Sha-Ling sensitive pure line R121 (P1) as the female parent and an insensitive pure line 9313B (P2) as the male parent, and their reciprocal hybrid F1 (RF1) was also made. Morphological characters of the floral organs in combination with the pollen quantity under microscope were used as the standards for abortion grading of flowers. Sterility of the first 30 flowers was investigated and the male sterility grade of each line was evaluated. Genetic analyses were made using methods for both the qualitative trait and the "major gene+polygene"-or quantitative trait Genetic models.It was observed in this study that:(1) the character of male abortion induced by CHA was controlled by nuclear gene without any effect of cytoplasm, (2) However, the degree of male sterility belonged to a quantitative treat, which was controlled by a genetic model of "two pairs of additive-dominant-epistatic major genes+additive-dominant-epistatic polygenes", This character is mainly controlled by the two major genes, but the polygenes also exert certain roles, bringing about minor affection from the environment. The inheritance rates of major genes are much higher than that of polygenes. The major genes will have a great influence on the male abortion performance of the offspring generations.4. Association of ALS and MF6 genes with the sensitivity to chemical hybridization agent Hua-Sha-LingThe highly sensitive line R121, weakly sensitive line 9313B, and moderately sensitive line Zhongshuang No.4 (ZS-4), were used as study materials to clone and investigate the sequences of ALS1, ALS3, MF6 genes. Based on the differences in gene sequences, corresponding amino acid sequences and protein structures and functions were speculated and analyzed.The study results showed that:(1) In all the 3 lines,9313 B, ZS-4 and R121, the sequences of ALS3 gene were similar. The predicted amino acid sequence, protein sequence and structure were same. In R121, the sequence of ALS1 gene missed part of nucleotides, and at the 833th locus a nucleotide G was inserted, forming a termination code TAG at the 835th to 837th nucleotide loci. This alteration may be an important factor for the strong sensitivity of line R121. (2)Two copies of MF6 genes, named MF6.1 and MF6.2, were cloned in the 3 study lines,9313B, ZS-4 and R121. The sequences of MF6.1 were similar, and the predicted amino acid sequences and protein structures were same using the Prediction software in NCBI. In 9313B, the 372th nucleotide in MF6.2 sequence was changed into A, forming a termination codon TGA with the former two nucleotides. The nucleotide at the same locus in the other 2 lines was G, forming with the former two nucleotides a codon TGG for tryptophan (Trp).