Taking into account the statements above, we have carried out an investigation on maize.
Diploid embryos: The mature embryos of four maize lines (AT-1, UV-98, HPL-1 and HPL-52) were cultured on modified medium MS, containing different concentrations of 2,4-D and sucrose.
We ascertained that the spectrum of new formations, appearing in the process of culturing explants of different lines is not always identical. Practically all germinating embryos are able to form globular structures on the surface of the coleoptile, and sometimes on leaves. The development of globes can be realized in two directions: by way of formation of rhizogenic callus, or by differentiation of globes in embryo-like structures. The last ones form the new generation by gemmagenesis. As a result embryogenic complexes (EGC) are formed. EGCs, when extracted from test-tubes, disintegrate easily into pieces, including a great number of embryoids of the different origin. Some embryoids in EGC complement give rise to plantlets still on initial medium, or they germinate after passage on medium without 2,4-D, but with IAA and kinetin (1.0 mg/l). On fresh medium with 2.0 mg/l 2,4-D actively growing regenerable strains can be produced. It should be noticed that in the given conditions of culturing only embryos of AT-1 line are able to form EGC and give plantlets. The best results for this line were obtained on medium containing 1.0 mg/l 2,4-D and 4% sucrose and 3.0 mg/l 2,4D and 6% sucrose. Diploid embryos of the lines UV-98, HPL-1 and HPL-52 produce exclusively globes, giving rhizogenic callus.
Haploid embryos: From kernels of the line UV-98, produced with use of haploinducers, haploid embryos were isolated and cultured on medium containing 2.0 mg/l 2,4-D and 2.0% sucrose. In contrast to diploid embryos, haploid ones gave rise to EGC embryoids which developed in plantlets.
Unpollinated ovaries: Ovaries of 15 lines and hybrids, including sexual forms and forms with elements of apomixis were cultured on nutrient medium, containing also a different correlation of 2,4-D and sucrose.
Regenerants were produced only in line AT-1, having a predisposition to reduced parthenogenesis. The ovaries of other lines on all tested mediums degenerated after approximately 7 days from the beginning of culturing.
In ovaries, line AT-1 and its hybrids autonomously formed parthenogenetical proembryos with a great number of globular structures on their surface, which later transform into embryoids. As a result of this EGC appeared, like that for zygotic embryos of line AT-1.
Embryogenesis and regeneration can proceed on initial medium. The concentrations 2,4-D - 2.0 mg/l and 5-9% sucrose were most optimal for induction of the above-mentioned processes.
Consequently, maximal regeneration potential is characteristic of line AT-1. Differentiated embryos isolated from dry kernels and haploid parthenogenetical proembryos developing inside unpollinated ovaries, are able to produce practically countless numbers of plantlets of embryoid origin. The visible differences in regeneration ability of haploid and diploid embryos have not been established. In addition we were able to demonstrate an example of haploid embryos from line UV-98 and the possibility of producing in vitro regenerants from embryos originating from pollination of donor ears by haplo-inducing pollen.
Thus, two different technologies of
producing haploid strains and regenerants can be presented. The first combines
culturing of unpollinated ovaries with a genetically conditioned predisposition
to reduced parthenogenesis. The second combines culturing with producing
kernels with haploid embryos, obtained after pollination by a specially
created pollinator - haplo-inducer.
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