The key factors generally involved in Agrobacterium-mediated transformation of cereal crops are the type and stage of recipient plant tissue, the strain and vectors of Agro-bacterium, and the cocultivation conditions. The tissue culture protocol for smooth regeneration of plants from transformed cells may also play a key role in production of normal, fertile transgenic plants. Every factor mentioned above has to be properly adjusted for successful transformation. This multiplicity may explain why it was initially so difficult to apply this technology to cereals.
Tissues showing active growth are suitable for transformation. In monocotyledon-ous species, such tissues are limited to shoot apex, root apex, scutellum of mature and immature embryos, and their calluses or suspension-cultured cells. Hiei et al2 reported that more than 90% of calluses induced from rice scutellum showed transient expression after cocultivation with A. tumefaciens for three days. They also observed that immature embryos are a suitable tissue for transformation of indica rice.6 Immature embryos are also used for Agrobacterium-mediated transformation of maize, barley, and wheat.
Transformation efficiency is highly dependent on the genotype of a germplasm to be transformed, particularly in recalcitrant cereal species. In rice, indica varieties are generally harder to transform than japonica varieties. The problem was solved by a slight modification of the cocultivation medium, and a high efficiency of transformation has been shown in a number of economically important indica varieties.6 Ishida et al3 succeeded in transforming a maize inbred, A188, at high efficiency. With the same protocol, however, they obtained no transgenic plants from another five inbreds, and only a small number of transformants of five hybrids parented by A188. Thus, choice of a suitable germplasm, for which a protocol for plant regeneration has been established, is a key factor for success.
A new vector system involving a super-binary vector has been developed.7 It is based on a Ti plasmid, pTiBo542, harbored by a supervirulent strain of A. tumefaciens, A281, which exhibits a wider host range and high transformation efficiency. In this system, a DNA fragment including a part of the virulence region of pTiBo542 is introduced into a small T-DNA-carrying plasmid in a binary vector system, in which the disarmed Ti plas-mid has its own full set of virulence genes. Super-binary vectors definitely showed very high efficiency in the transformation of rice and maize.2,3
Choice of vectors and strains of A. tumefaciens are important for transformation. Hiei et al2 tested the efficiency of four combinations of bacterial strains and vectors. The strains were an ordinary strain, LBA4404, and a supervirulent strain, EHA101. The vectors were an ordinary binary vector, pIG121Hm, and a super-binary vector, pTOK233. In rice transformation experiments, LBA4404(pTOK233) was definitely more effective than LBA4404 (pIG121Hm) or EHA101(pIG121 Hm). EHA101(pTOK233), which is the combination of a supervirulent strain with a super-binary vector, was not very effective. In maize, efficient transformation was possible only with super-binary vectors.3 Therefore, super-binary vectors proved to be more useful in transforming these cereal crops.
The conditions during cocultivation of plant tissues with Agrobacterium significantly affect the efficiency of transformation. Media containing acetosyringone and 2,4-dichlo-rophenoxyacetic acid and solidified with a gelling agent are suitable in rice and maize. In addition to acetosyringone, glucose and some surfactants were effective in enhancing transformation of wheat.5 In barley, however, Tingay et al.4 used immature embryos from which the embryonic axis was removed. The embryos were then shot with gold microprojectiles and cocultivated with Agrobacterium on medium without acetosyringone. Tingay et al observed an appreciable level of transformation. Because wounding the cultured barley embryos enhanced the recovery of transformed cells, they reported, such a pretreat-ment may release some phenolic compound, instead of added acetosyringone, which induces the vir genes of Agrobacterium. The bacterial concentration was adjusted to 1 to 5 x 109 cfu/ml in rice, maize, and barley, and cocultivation was done at 24-25°C for three days.
Selectable marker genes and their promoters often greatly affect the efficiency of transformation. A hygromycin resistance gene, hpt, driven by the CaMV 35S promoter was highly effective in rice.2 In maize3 and barley,4 a phosphinothricin resistance gene, bar, driven by promoters 35S (maize) and Ubil (barley) gave good results. In wheat5, the nptII gene driven by an enhanced 35S promoter was used, and selection was done on media containing G418 as the selectable agent.
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