Doubled haploid cells, embryos and plants
a technology of haploid cells and embryos, applied in the field of doubled haploid cells, embryos and plants, can solve the problems of inefficient use of labor and time resources
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example 1
Transformation and Regeneration of Transgenic Maize Plants
[0062]Seeds from any maize genotype are planted and the resulting plants are used as female parent plants (pollen receivers). Seeds from haploid inducer lines, such as Stock 6, RWS, KEMS, KMS or ZMS, are planted and the resulting plants are used as male parent plants (pollen donors). The ears of the female parent plants are shoot-bagged before silk emergence. The silks of the ears on the plants of the female parent plants are pollinated with viable pollen grains collected from the anthers of the male parent plants (haploid inducer plants). This pollination is controlled by the method used regularly in maize breeding program to avoid any foreign pollen contamination. The pollination method results in the production of a frequency of about 5-12% of haploid embryos in each ear. At 4 to 20 days, preferred at 6-15 days and more preferred at 8-13 days and most preferred at 9-11 days after pollination, the immature ears are harveste...
example 2
Agrobacterium-mediated Transformation of Maize
[0073]For Agrobacterium-mediated transformation of maize the method of Zhao is employed essentially as described in U.S. Pat. No. 5,981,840, the contents of which are hereby incorporated by reference. Haploid embryos (preferably about 7-13 days after pollination) are isolated from maize and the embryos are contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the nucleotide sequence(s) of interest to at least one cell of at least one of the embryos. In this step the embryos are typically immersed in an Agrobacterium suspension for the initiation of inoculation. Preferably, the Agrobacterium suspension contains 100 μM acetosyringone. The embryos are co-cultured for a time with the Agrobacterium.
[0074]Generally the embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step lasting 6-7 days is contemplated. In this resting step, t...
example 3
Transformation of Meristematic Tissue from Haploid Seeds
[0079]Transformation of meristematic tissue is disclosed in U.S. Pat. No. 5,736,369. The method comprises particle bombardment of meristem tissue at a very early stage of development and the selective enhancement of transgenic sectors toward genetic homogeneity in cell layers that contribute to germline transmission. Embryos are obtained as described in Example 1 and incubated on maturation medium. The apical dome of several embryos is disrupted prior to bombardment to force the meristem to reorganize and form new meristematic areas. Mechanical disruption is performed by means of micromanipulation needles. Embryos are maintained in the dark at 28° C. for seven days on maturation medium and then transferred to 272K medium containing 150 mg / L Tobramycin sulfate. The embryos will have multiple meristem formation with elongated cotyledons. The embryos are then incubated in light at 28° C. A chromosome doubling agent is used at the ...
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Abstract
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