申请人:Northwest Plant Breeding Company
公开号:US20020104128A1
公开(公告)日:2002-08-01
The present invention provides methods for generating doubled haploid and/or haploid plants from microspores. In a presently preferred embodiment of the methods of the present invention, plant material is selected that bears reproductive organs containing microspores at a developmental stage that is amenable to androgenic induction. The microspores are treated by contacting the selected plant material with water and subjecting the selected plant material to temperature stress, and optionally to nutrient stress. Preferably the selected plant material is contacted with an effective amount of a sporophytic development inducer and an effective amount of an auxin and/or a cell spindle inhibiting agent. Optionally, the selected plant material is contacted with an effective amount of a cytokinin and/or an effective amount of a gibberellin. The treated microspores are isolated, preferably by density centrifugation utilizing a solution of 0.3 M mannitol layered over a higher density solution of a sugar, preferably maltose. The isolated, treated microspores are then cultured in a liquid nutrient suspension medium supplemented with at least one plant ovary or with an aliquot of plant ovary conditioned medium, until the microspores develop into embryoids. The embryoids are transferred to a regeneration medium and incubated therein until the embryoids develop into plants. The resulting plants may be haploid or doubled haploid and may also be genetically transformed.
本发明提供了用小孢子产生加倍单倍体和/或单倍体植物的方法。在本发明方法的一个目前优选的实施方案中,所选植物材料具有含有小孢子的生殖器官,其发育阶段适于雄性诱导。处理小孢子的方法是将所选植物材料与水接触,并使所选植物材料受到温度胁迫和营养胁迫。最好将所选植物材料与有效量的孢子体发育诱导剂和有效量的辅助素和/或细胞纺锤体抑制剂接触。可选地,将所选植物材料与有效量的细胞分裂素和/或有效量的赤霉素接触。将处理过的小孢子分离出来,最好采用密度离心法,将 0.3 M 的甘露醇溶液分层覆盖在较高密度的糖(最好是麦芽糖)溶液上。然后将分离出的经过处理的小孢子放入液体营养悬浮培养基中培养,培养基中至少添加一个植物子房或等量的植物子房条件培养基,直到小孢子发育成胚胎。胚胎被转移到再生培养基中培养,直到胚胎发育成植株。生成的植物可以是单倍体或加倍单倍体,也可以进行基因转化。