Methods for tissue culturing and transforming elite inbreds of Zea mays L.
a technology of zea mays and tissue culture, which is applied in the field of tissue culture and can solve the problems of short time taken to produce transgenic plants and do not allow efficient transformation of elite lines, and achieve the effects of enhancing the transformation of elite corn inbreds, enhancing the integration of foreign dna into corn tissue, and high frequency
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example 1
Improvement in the Induction Frequency of Type II Callus
[0040] Plants of the elite corn inbred Stine 963 were grown under controlled conditions, either in a grow room or in a greenhouse. Plants were exposed throughout growth to a 16 h photoperiod with a daytime temperature of around 30° C. and a nighttime temperature of around 25° C. Plants were selfed and after approximately 10 days ears were harvested. At this time the immature embryos were on average between 1.0 and 2.0 mm in length. Usually the ears were then placed at 4° for two days prior to immature embryo excision.
[0041] The response on standard N6A media (Table 1) for inducing Type II callus was variable and appeared to be dependent on the physiological state of the donor plant. The frequency of Type II callus ranged from 0% up to 95%, with an average of around 40%. Immature embryos were excised with a view to using them as targets for Agrobacterium-mediated DNA delivery. For this work a reproducible and high frequency re...
example 2
Transformation of an Elite Corn Inbred by Agrobacterium tumefaciens
[0046] Modifications to the protocol of Hei and Komari (1997) involving co-cultivation temperature, culture media, antibiotic concentrations and Agrobacterium source cultures.
[0047]Agrobacterium strain LBA 4404 harboring “super binary” vectors as described in U.S. patent Hei and Komari (1997) was used in corn transformation experiments. Vectors with a bar expression cassette from pBARGUS (Fromm et. al., 1990) were used to generate resistance to the herbicide bialaphos, and a gus expression cassette from plG221 (Ohta et al., 1990) was used to produce Gus expression for transient assays. The gus expression cassette contains an intron in the N-terminal region of the gus gene which prevents expression in bacteria, but upon expression in plant cells the intron is spliced out and Gus activity is achieved (Ohta et al., 1990; Ishida et al., 1996). Agrobacterium containing “super binary” vectors were stored in glycerol stoc...
example 3
Effect of Heat Shock Treatment on Integration of DNA
[0053] Use of a brief heat treatment induces a transient state of so-called ‘competence’ in bacteria, allowing them to take up and express DNA from a variety of sources (cited in Sambrook et al, 1989). Use of a heat shock treatment to improve transformation efficiencies in higher organisms has not been reported. It was decided to explore the possibility that a heat shock treatment could improve integration of DNA following uptake. This was investigated with Agrobacterium-mediated DNA delivery in the first instance. First, a heat shock treatment of 45° for 30 minutes was administered to immature embryos 21, 24, 27, and 30 hours after the initiation of co-cultivation with Agrobacterium on LSAS (Table 1). No enhancement of clone production was noted after 21 hours, but promising preliminary results were obtained with the longer time periods. Further experiments were then performed—the results are presented in Table 6.
TABLE 6Effect ...
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