Immature embryos of the inbred line A188 were excised from milk stage kernels and cultured on 2,4-D (2 mg/l) containing medium for the induction of embryogenic callus. Approximately 3 months after culture initiation embryogenic callus parts were subsequently transferred to media with lower 2,4-D concentrations (1 mg/l, 0.5 mg/l) and without 2,4-D for plant regeneration. Our main interest with regards to the direct regenerants (R1-generation) and the following generations is to determine whether any form of variation is transmitted to the selfed progeny. The discovery of variants amongst the offspring is being combined with molecular analyses of these altered plants in order to examine whether changes at the molecular level can be correlated to the occurrence of somaclonal variants.
Among 117 plants regenerated from embryogenic callus
cultures and grown to maturity in the glasshouse (R1-generation), the following
phenotypic variations were observed:
|all plants||reduced height|
|1 plant||longitudinal yellow leaf stripes that persisted to maturity|
|3 plant||no ear development|
|9 plant||no tassel development|
|6 plant||feminization of the male flower|
Of the 117 regenerated plants, 99 could be self-pollinated; 91 set seed. The number of seeds per cob ranged from 2 to over 200.
To test the R2-seeds, samples of 20 seeds from, up to now, 40 R2-lines were potted and their development was followed. With different frequencies the following alterations were observed:
- lack of germination
- 'early death' of seedlings on the coleoptile stage or on a later seedling stage
- 'late death'; 5-6 week old plants suddenly turned brown and died
- leaf stripes
- curling of leaves
- sterile plants, either lacking tassels or ears or both
Phenotypically 'normal' looking plants were self-pollinated for the production of the R3-generation. In a few lines this R3-generation has already been tested as well, and similar distortions as described above were found. Further characterization of the R2- and R3-generations is in progress.
To find possible molecular origins of the described variation it is being determined whether gene methylation is altered as a consequence of the tissue culture pathway and whether somaclonal variation can be correlated with any changes in the methylation state. Total genomic DNA from over 100 R2-plants has been extracted and digested with the isoschizomer restriction endonucleases MspI and HpaII. These digestions showed that the majority of the DNA's (85%) restricted with MspI, and the remainder showed restriction differences indicative of gross changes in the degree of DNA methylation. Further experiments include the attempt to determine whether particular gene sequences are also changed in their methylation state by probing MspI digested DNA's from phenotypically normal and disturbed plants with 32P-labelled known gene sequences. Work is also being undertaken to map the region of known gene sequences for different somaclones by the process of restriction fragment length polymorphism. In conjunction with the methylation studies, this will allow us to determine whether tissue culture induced variation is a result of methylation changes alone, or occurs as a consequence of mutations in the genome.
Elke Gobel, Peter T.H. Brown, Horst Lorz
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