Characterization And Work Mechanism Of Chemical Hybridizing Agents For Plants

Although male sterility induced by chemical hybridizing agents provides a practicable approach for hybrid production, there are few good chemical hybridizing agents(CHAs)available for commercial utility. Attempt was made in the paper to select and evaluate chemical hybridizing agents from sulfonylurea and similar herbicides, which can inhibit acetolactate synthase (ALS), the key enzyme catalyzes the common reaction to synthesize leucine, valine, and isoleucine in plants. The characterization and mechanism of action of novel and effective CHAs founded in the paper were studied. The main results obtained are as following:1. Selection and evaluation of CHAs1.1 Among twenty-eight chemicals tested, twenty-two herbicides elicited male sterility in Brassica napus. The best CHAs for Brassica napus were Tribenuron-methyl and Amidosulfuron (China invention patent ZL200610042886.1). Double foliar application of them at the concentration of 0.2-0.4 mg/L and at 12days interval resulted in nearly 100% male sterility. They could also make three environment-sensitive male sterile lines Zhong 9A (Shaan 2A CMS), Y88A (Polima CMS), and Hy50S (recessive genic male sterility) to be completely sterile. The hybridity of seeds from two mimic hybrid production trails via CHAs and CHA-aided CMS were 98.4% and 99.9% respectively. Tribenuron-methyl and Amidosulfuron could also be applied on other Brassica and cruciferous species.1.2 Most of herbicides used in this experiment had not or very weak gametocidal effect on wheat (Triticum aestivum). Only Imazethapyr and Glyphosate could induce high male sterility but phytotocxicity of the later was very serious. Imazethapyr resulted in over 95% male sterility in 18 different cultivars at 7g/ha and 10 g/ha rate. The effect of CHA×cultivar interaction was found in some cultivars. Application of Imazethapyr also resulted in 100% male sterility in Barley (Hordeum vulgare) and Zea mays. Glyphosate had good gametocidal effect on cotton (Gossypium hirsutum)and pepper (Capsicum annuum).2. Studies of the target of CHAs2.1 The dose of Amidosulfuron was negatively correlated with hypocotyls growth and ALS activity of B.napus. After Amidosulfuron treatment, the in vitro activity of ALS was raised little and then went down, this suggested the expression of ALS gene was promoted firstly by feedback regulation but then the protein synthesis was inhibited. The in vivo activity of ALS of non-sensitive cultivar 82089 was somewhat higher and recovered fast than sensitive cultivar Qinyou3. The in vivo activity of ALS of flora buds was higher than mature leaf but decrease more quickly. This was the first evidence to interpreter the selection of CHAs among different cultivars and different tissues. Brassica napus and Triticum aestivum were treated with different ALS-inhibitors. The one with good gametocidal efficiency had stronger inhibiting ability than those with lower efficiency. These indicated that ALS was the target of these novel CHAs.2.3 ALS genes fragments was amplified against 16 different cultivars with 16 primers designed based on ALS genes of B.napus. Some polymorphic bands were found but the cluster results based them could not reflex the relationships among these cultivars. The sites in ALS genes determining the sensitivity to amidosulfuron was not found.2.4 The contents of total and individual free amino acid in the buds of B.napus from three treatments, i.e. amidosulfuron, amidosulfuron+ branched-chain amino acids, and 1,1-cyclopropanedicarboxylate(specific inhibitors of ketol-acid reductoisomerase), increased by one fold. This was the results of the turn over of protein. Branched-chain amino acids were supplemented to the treated B.napus from leaf and bud, but this treatment could not restore the fertility.3. Cytological characters of male sterility in Brassica napus induced by Amidosulfuron3.1 The number and structure of ovum and the papilla cells in the stigma of Amidosulfuron treatment were similar to the contrast. However, the number of pollen grains in the CHA-treatment decreased and most pollen grains had abnormal shape observed via a whole flower clearing technique.3.2 Light microscopy studies revealed that pollen abortion in CHA treated plants was associated with abnormal behavior of tapetum, endothecium and microspore. CHA treatment made the cytoplasm of pollen mother cell shrunk. The tetrads were malformed and thus most microspores derived from them were destructured. Some tapetal cells elongated radially, however, sometimes they failed to degenerate.3.3 Ultrastructural studies revealed the number of vacuole and organelle in the tapetal cells in Amidosulfuron-treated rapeseed decreased. The pollen mother cell are destroyed with the karyotheca and nucleolus damaged, and the cytoplasm degenerated. It is observed that the presence of plastid and mitochondria degenerated and phagocytosis in the microspore and tapetal cell in the anthers of amidosulfuron treated plants. The tapetal cells were damaged with tapetosomes absent and elaioplasts malformed. One significant feature was the plastids and chloroplasts in leaf, epidermis and endothecium were destroyed.4 Physiological and biochemical characters of male sterility induced by amidosulfuron4.1 Application of amidosulfuron influenced the activity of peroxidase and catalase, and the content of endotoxin hydrogen peroxide and malondialdehyde. The trend of these influences was different between amidosulfuron-sensitive cultivar Qinyou3 and non-sensitive cultivar 82089. The activity of glutathione S-transferase, which is an important detoxifying enzyme, decreased abruptly but then rose to surpass the normal level. Its activity in cultivar 82089 was higher than in Qinyou3. These differences in defense systems might be the reason for manipulating of amidosulfuron-sensitivity.4.2 Application of amidosulfuron resulted in decrease of content of pyruvate acid, soluble sugar, chlorophyll carotene soluble protein and DNA. The genomic DNA from aborted anther showed smear band by agarose gel electrophoresis. The decrease of photosynthesis of rapeseed was induced, but cultivar 82089 can maintain higher photosynthesis than sensitive cultivar qingyou3 and 656. The decrease of photosynthesis was also an important reason for male sterility.4.3 Supplementary of ascorbic acid, NaHSO₃, Na₂S₂O₃, hydrogen peroxide, salicylic acid,AgNO₃, Allantoin, and GA3, could not aggravate or relieve the effect of amidosulfuron. However, addition of ethephon shorten the flowering period, this was useful to keep complete male sterility during the whole flowering period. A slow-release formulation (Amidosulfuron+PV+CMCNa+adjuvants) could enhance the endurance to doubled dose and prolonged the gametocidal effect of Amidosulfuron on B.napus.In conclusion, the primary model of CHAs used in this paper could be established. CHAs including Amidosulfuron, Tribenuron-methyl, and Imazethapyr, inhibited the activity of their target—acetolactate synthase. The toxicity embodied from various aspects: imbalance of amino acid, abnormal cell split, disturbance of DNA and protein biosynthesis, destruction of chloroplasts and plastid, restriction of the photosynthesis, production of free radical etc. The development of microspore was suppressed because it was most sensitive to these changes.