Transformation Efficiency and Quality of Transformants

Transformation efficiency is sometimes crucial for the success of research, because a great number of transformed plants, even for one gene construct, need to be created to obtain transformants that incorporate a single, highly expressing transgene and show little somaclonal variation. There is wide variation between transformants in expression level of a gene, due probably to the state of gene integration in the plant genome. Different promoters combined with a single gene may change the level, position, and timing of gene expression. The copy and locus numbers of a transgene may vary from one plant to another. Transformants may show some somaclonal variation, including low fertility and abnormal morphology.

The transformation efficiency is calculated as a percentage based on the ratio of the number of independent transformed plants to the total number of explants, calluses, or immature embryos infected by A. tumefaciens. Hiei et al.2 and Ishida et al.3 reported high percentages (18.5% and 15.1%) in rice and maize in experiments involving about a thousand explants. These high numbers explain why this technology is reliable. Efficiencies of 4.8% and 1.8% have been reported in barley4 and wheat,5 respectively.

The number of copies of an incorporated gene in a transformed plant is estimated by Southern blot analysis, and the number of loci is determined by the segregation ratio in its progeny. Hiei et al2 estimated that 35% of transformed rice plants had a single copy, 40% had two copies, and only 25% had more. They also observed single locus segregation in the progeny from 60% of original transformed plants and two locus transformation in 25%. In maize, Ishida et al.3 reported that about 70% of positive transformants, which is amazingly high, showed a single copy incorporation, and more than 90% possessed the expected length of transgenes. In wheat, Cheng et al5 observed that 35% of transformants possessed single copies of transgenes, which was significantly higher than with the gene gun method. These results are consistent with the introduction of a few copies of genes, showing a simple Mendelian inheritance, into the genome of dicotyledonous species. This is one of the advantages of this method over direct DNA delivery.

Agrobacterium-mediated transformation of rice is now being extensively used as an efficient method for biotechnological research in both japonica and indica varieties. In barley, relatively good efficiency of transformation has also been reported, but more effort is needed in practical application. Transformation should improve in wheat as experiments are repeated.

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