An example of genotype flexibility in tissue culture: switch from type 1 to type 2 embryogenic cultures in the inbred B79

In recent years, great attention has been paid to genotypes of maize capable of inducing in vitro friable highly embryogenic calli, termed type 2 calli. These cultures have been developed as a more promising source of material for maize genetic manipulation than the first callus cultures, which were hard and morphogenic, typically derived from most of the maize genotypes, known as type 1 cultures. So far, embryogenic friable type 2 calli have been established mostly from the inbred A188, A188 X B73 and reciprocal, under the conditions first described by C.E. Green (in: A. Fujiwara, ed., Plant Tissue Culture, Maruzen, Tokyo, 1982), essentially on N6 medium supplemented with l-proline. This genotype X culture condition appeared to be highly specific in obtaining type 2 callus cultures (D.T. Tomes and O.S. Smith, Theor. Appl. Genet. 70:505, 1985: C.L. Armstrong and C.E. Green, Planta 164:207, 1985). By using the same cultural conditions, we were capable of inducing type 1 calli from the inbred B79 and its behaviour in culture has been characterized in comparison with the most extensively studied inbred W64A. Evaluations have been made on cultures derived and propagated on Murashige and Skoog (1962) basic medium without l-proline (ZM) and N6 basic medium as recommended by Armstrong and Green (1985). Callus induction was on 2 mg/l 2,4-D and propagation on 1 mg/l 2,4-D in the light (3000 lux, day/night 16/8 hrs). Results obtained in the establishment of the primary cultures are summarized in Table 1. Only in B79-N6 derived cultures, calli essentially of type 1 displayed a more pronounced friability, thus being indicated as type 1a. In no case were type 2 calli derived.

Primary callus cultures lasted 4-8 months on ZM medium, and 7-12 months on N6 medium, thus confirming that N6 medium may be considered a superior medium also for type 1 callus culture. Light was essential for propagation. Regenerative capability obtained on MS hormone-free medium of the type 1 cultures declined along subcultures ranging from 12-15 plantlets regenerated per gram of fresh weight tissue in the first three months, to the complete loss of regenerative capability in the subsequent 5-6 months of life. A slightly longer callus life and regenerative potential were detected on N6-propagated callus cultures.

However, during regeneration on hormone-free medium, in the case of B79-derived calli, developing plantlets underwent embryogenic callus proliferation at the coleoptilar node, a phenomenon strictly reminiscent of the recurrent or secondary somatic embryogenesis described for a few plant species not belonging to the Gramineae (alfalfa, clover, papaya, cassava). The secondary somatic embryogenesis in B79 allowed the establishment and propagation of true type 2 embryogenic calli, as secondary cultures, on N6 medium, completely comparable to the callus cultures obtained in A188 and its derivatives. The most dramatic effect of the switch from type 1 to type 2 callus in B79 cultures was mainly reflected by two important features: a) cultures became long-term embryogenic cultures and b) the regenerative potential, calculated on the basis of frequency of regeneration per gram of fresh weight tissue, increased dramatically. All the cell lineages which underwent secondary somatic embryogenesis originating type 2 callus cultures could be propagated on N6 medium in dim light without losing their peculiar morphology. To date they have been stably propagated for almost two years. The regenerative potential of the secondary cultures is as high as four fold over the one recorded in young (3 months old) primary cultures of B79 and did not decline with time. Evaluations made recently at the 20th month of life confirmed the high regenerative capability still present in these cultures (Table 2). The peculiarity of these data assumes particular relevance since B79 is one of the most currently utilized genotypes in maize breeding programmes.

Table 1 - Evaluations on primary and secondary cultures of W64A and B79.

Table 2 - Regeneration efficiency in primary and secondary embryogenic cultures of the inbreds W64A and B79.

E. Lupotto and M.C. Lusardi


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