1. Field of the Invention
The present invention relates to a general process for regenerating corn and to plants produced by the process. More particularly, the present invention relates to the use of tissue and cell culture for the regeneration of corn plantlets from many varieties of corn.
2. Description of the Prior Art
Plant regeneration from cells in culture is essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation and for the use of genetic engineering in producing new varieties. Although plants can be regenerated from tissue culture of several varieties of corn, there are many varieties for which this has not been accomplished using similar techniques.
In recent years, plant cell culture successes have had a considerable influence on the respective roles of cell and organism in control of plant growth and development. This concept was supported when isolated plant cells were shown to be amenable to in vitro cultivation and complete plants could be regenerated from cultures derived from somatic tissues, either directly via somatic embryogenesis or indirectly via organogenesis. Generally the regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, especially growth regulators. Early investigations of certain plant species have suggested that exogenous auxin concentration is a major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of embryoid formation. In other species, exposure to a definite balance of auxin and cytokinin leads to the occurrance of organogenesis (shoots, then roots). Although several genotypes of corn have been regenerated using these techniques, no process is generally applicable to most genotypes of corn. Many genotypes remain extremely difficult if not impossible to culture using the prior processes.
The process which has become the standard system for corn tissue culture is described by Green et al., Crop Science 15, 417 (1975). In this process, immature embryos were plated onto a callus induction medium which comprises the MS mineral salts, Straus vitamins and amino acids (glycine, asparagine, niacin, thiamine, pyridoxine and pantothenic acid), 2% sucrose, 0.8% agar and a hormone selected from 2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid (PCA), alpha-naphthaleneacetic acid (NAA), 2-isopentyladedine (2-ip) or mixtures thereof. Plantlets were regenerated by subculturing the callus on medium containing reduced hormone concentrations. Hormone concentrations which were useful were 2 mg/l 2,4-D and a mixture of 1 mg/l 2,4-D, 4 mg/l NAA and 0.05 mg/l 2-ip. Regeneration was then accomplished on medium containing 0.25 mg/l 2,4-D or a mixture of 1 mg/l NAA and 0.05 mg/l 2-ip respectively. All culturing was conducted in a 16 hour light/8 hour dark cycle for 3-4 week intervals before transfer. This reference reports that callus induction did not occur in one of five genotypes tested.
Similar results have been reported by others. Freeling et al., Maydica 21, 97 (1976) obtained regeneration of corn by utilizing a sequence of callus induction on a RM medium containing 2 or 5 mg/l 2,4-D, 2% sucrose and no myo-inositol followed by regeneration on the same medium with 0-0.1 mg/l 2,4-D. Lu et al., Theor. Appl. Genet. 66, 285 (1983) obtained callus formation and shoot formation after 3 weeks of culturing when utilizing a MS medium containing 3-12% sucrose and 0.25-2.0 mg/l 2,4-D. High sucrose concentration was most favorable for embryogenic callus. Root formation was accomplished after transfer to (a) MS medium with 3% sucrose with or without 1 mg/l giberellic acid (GA.sub.3) or (b) 1/2 MS medium with 2% sucrose.
Edallo et al., Maydica 26, 39 (1981) obtained callus induction from immature corn embryos using the medium of Green et al., supra, with 2 mg/l 2,4-D. The culture could be maintained on the same medium with 30 day transfers. Regeneration was accomplished by using medium with no 2,4-D. Shoots were transferred to medium having a 1 mm overlayer of 5 mg/l NAA for root formation. Prior to transferring the plantlets to soil, they were cycled through media having 20%, 10% and finally 0% sucrose. Regeneration of corn plants using a similar sequence of callus induction with 2,4-D and regeneration with no or low 2,4-D has been shown by Lu et al., Theor. Appl. Genet. 62, 109 (1982); Hibberd et al., Proc. Natl. Acad. Sci. USA 79, 559 (1982); Gegenbach et al., Proc. Natl. Acad. Sci. USA 74, 5113 (1977); and Green at al., Crop Science 14, 54 (1974). The latter reference also demonstrates genotype affects on callus induction.
The prior art does not describe a process for the regeneration of most genotypes of corn Zea mays from tissue and cell culture. Examples of cultivars that cannot be regenerated or can only be regenerated with great difficulty at low frequency by prior art processes include B73, A632, A619, CM105, B37, B84, B14, Mo17 and R168. The present invention is the first instance of a broadly and generally applicable procedure for regenerating cultivars of corn with a high frequency and with a high growth rate.
Corn plants and seeds are produced by this process. The corn plants resulting from this process may differ from the starting plant material as a result of somaclonal variation. The pathway is also useful in that it will enable the use of various selection processes to provide further variation. The plants which are produced can be used in conventional breeding programs.