A large number of chemicals capable of selectively inhibiting pollen development have been identified as chemical hybridizing agents(CHA) for large-scale commercial production of hybrid seeds in various crops. Some commercial CHAs such as methyl arsenate (Gametocide No.1 and No.2) have been successfully used to produce commercial hybrid in oilseed rape in China. However, researchers of hybrid breeding are still attempt to develop novel efficient CHAs without environmental and toxic effects. In the present paper, the effect of a new CHA developed by the authors, named EXP, on inducing male sterility of oilseed rape and on the change of the content of pigments (chlorophyll and carotenoid) and soluble protein and the activity of peroxidase and esterase isozyme in treated plant was investigated. Plants ofBrassica napus breeding line L82089,Brassica juncea breeding line L638 andBrassica campestris variety Qinyou No.4 were treated with various concentrations of EXP in peduncle-growing period with 1 mm-2 mm long flower bud. All the treatments with EXP were found to be quite effective in inducing pollen sterility. The percentage of sterility and hybrids between treatment and control were significantly different for all doses. The treatments with 12－15 mL of 0.05 μg/mL and 0.25 μg/mL EXP per plant onBrassica napus breeding line L82089 caused about 92% and 97% sterile plants respectively. The plants treated with 0.25 μg/mL EXP were sterile steadily with 6.6% selfing rate (Table 1 and Table 2). So the treatment with 12－15 mL of 0.25 μg/mL EXP could be recommended for producing hybrid inBrassica napus. The development of vegetable and reproductive organs was all suppressed due to the spray of EXP. The induced sterile plants were distinctly characterized by fading leaf, shorter plant height, serried bud, later flowering, fading petal, and shriveling anther with no pollen. The morphology between male sterile and fertile flowers was so different that the treated and untreated plants could be easily discriminated. The content of chlorophyll and carotenoid in the flower of induced sterile plant was decreased (Fig.1). In treated stamen, the activity of peroxidase and esterase isozyme was diminished and the content of soluble protein was very low (Fig.2). Simultaneously, the treatment with 5 mL of 0.05 μg/mL EXP could make breeding line L638 (Brassica juncea) and variety Qinyou No.4 (Brassicacampestris) sterile, too. However, it was not suitable for producing hybrid because the period of male sterility of L638 was too short and the seedseting of Qinyou No.4 was poor (Table 3). The active ingredient of EXP belongs to sulfonylurea family that can inhibit acetolactate synthase (ALS, EC 4.1.3.18), the key enzyme that exist in the chloroplast of all green plants and catalyzes the common reaction to synthesize leucine, valine, and isoleucine. As a matter of fact, it have been found that other ALS-inhibiting chemicals could induce male sterility to a considerable level in oilseed rape and other Brassica family vegetables, too. A significance of this discovery may be the potential of a larger number of ALS-inhibitors to be used as the candidate of CHAs.

化学杂交剂EXP的主要成分是一种磺酰脲类化合物，作用靶标是植物的乙酰乳酸合成酶（acetolactate synthase，ALS），通过抑制酶活性，阻止支链氨基酸缬氨酸、亮氨酸和异亮氨酸的合成。在甘蓝型油菜自交系82089抽薹15～20 cm高，最大花蕾长度1～2 mm时期，单株用药量12～15 mL，用0.05 μg/mL、0.25 μg/mL EXP溶液处理可分别诱发92%和97%不育率。而0.25 μg/mL浓度处理不育株的育性较为稳定，自交结实率为6.6%，可以作为最佳用药浓度的参考值。EXP透导的不育株叶色变浅、株高降低、花蕾密集、花期推迟、花瓣褪色、花药干瘪无花粉。检测结果表明，不育株花蕾叶绿素和类胡萝卜素含量有所减少，处理雄蕊的过氧化物酶和酯酶活性降低，而水溶性蛋白质含量显著减少。同一时期用EXP处理芥菜型油菜和白菜型油菜也可诱导雄性不育，但芥菜型油菜不育持续时段较短，白菜型油菜结实性不好，不宜用于生产杂交种。本研究结果的意义在于，磺酰脲类ALS抑制剂种类非常多，有可能从中筛选到更高效、更持久的化学杂交剂。

Abstract

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure ofArabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.