46. RFLP-analysis of regenerated rice plants

The aim of this study is to investigate the occurrence and extent of somaclonal variation in rice at the molecular level. The phenomenon of somaclonal variation has previously been reported to occur among regenerants from in-vitro cultures of many different plant species (Larkin and Scowcroft, 1981).

The Indica lines IR40, IR54 and others were used to establish in-vitro cultures. Immature embryos were taken as explants and cultured on modified CC and MS media for the induction of callus growth and plant regeneration. For RFLP (Restriction fragment length polymorphism) analyses, total genomic DNA was isolated from direct regenerants (R\0\-plants) and digested with the restriction endonucleases HindIII and DpnI. The digestion experiments were started using HindIII, which recognises the hexanucleotide sequence AAGCTT. However, when the inner adenine is methylated in the N-6 position, restriction is reduced. Therefore, even though N⁶-methyl adenine is regarded as occurring only infrequently in plants, the same genomic DNA was digested with the restriction enzyme DpnI which restrict only in the presence of N⁶-methyl adenine. The restriction fragment pattern was analysed by 1% agarose gel electrophoresis and hybridization with the soybean actin gene (Fig. 1).

Fig. 1. RFLP in rice plants regnerated from a single immature embryo. HindIII digestion of genomic DNA. Probe: 3 kb fragment of the actin gene of soybean.

Fig. 2. DpnI digestion of genomic rice DNA. Probe: 3 kb fragment of the actin gene from soybean.

The control plants (tracks 1-4) were grown from seeds in the glasshouse and no restriction polymorphisms were detectable among them. However, when genomic DNA from regenerants was restricted with HindIII (tracks 5-18) considerable variation in the restriction fragment pattern was observed.

Variation was seen in plants which appeared phenotypically normal, and molecular differences were observed both between plants regenerated from different callus cultures and also between those regenerated from a single callus culture. To determine whether these changes were due to alterations in the methylation status ofthe adenine residues, the experiment was repeated using the DpnI enzyme. The results (Fig. 2) show a close relation between those plants showing RFLP changes with HindIII and methylation changes with DpnI, indicating that tissue culture is responsible for alterations in the level of N⁶-methylation of adenine. (This work is supported by the Rockefeller Foundation grant RF86059 #52).

Larkin, P.J. and W.R. Scowcroft, 1981. Somaclonal variation-A novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60: 197-214.