Transformation of microspores for generating doubled haploid transgenic wheat (Triticum aestivum L.).

摘要

Microspores can form homozygous doubled haploids (DH) in one generation by androgenesis (microspore culture). The goal of this study was to develop a microspore transformation method for the production of transgenic wheat (Triticum aestivum L.).; In the first part of this study, optimal conditions for generating DH wheat plants from microspores were identified. First, the chemical inducer formulations effectively triggered microspore embryogenesis. Second, large populations of isolated embryogenic microspores were cultured to form embryoids and green plants at optimal conditions, that required purification of embryogenic microspores, a liquid culture medium with an osmolality around 300 mOsmol Kg-1 H2O, and co-culture with ovaries. Third, conversion of albinos to green plants was obtained by nutrient addition during pretreatment. Fourth, spontaneous chromosome doubling was achieved in vitro by use of low toxic chemical caffeine.; In the second part of this study, microspores were transformed by co-cultivation with Agrobacterium tumefaciens strain AGL-1. Over 200 putative primary transformants were regenerated and 24 primary (T0) spontaneously DH transgenic lines were obtained. Polymerase chain reaction (PCR), DNA sequencing of the amplificate, Southern blot analyses and assay of the recombinant enzyme confirmed the presence of transgenes in T0 primary transformants and their stable inheritance in homozygous T1 DH progenies. Several factors for successful transformation were identified: (1) Co-cultivation with Agrobacterium for transfer of the plasmid T-DNA into microspores should take place at day 0 for 24 hours. Volume of the inoculated AGL-1 cells at OD600 = 1.0∼1.5 had to be 1% of the co-cultivation solution. (2) Killing of AGL-1 cells after co-cultivation was by filtration and addition of timentin at a concentration of 200--400 mg/L. (3) Selection of transformants should be carried out with bialaphos at a concentration of 2--4 mg/L. (4) Identification of transformants by PCR was carried out when regenerating seedlings were at 4--6 leaf stage.; This is the first report on successful transformation of microspores followed by regeneration of homozygous transgenic plants expressing a recombinant protein in wheat grains. The method described and conditions worked out in this study are likely to be applicable to other plant species.