Transformation and Regeneration of Plants

In general, expression of (poly)peptides relies on stable integration of the encoding genes in the plant genome. An advantage of this approach is that identical offspring can be obtained by self-fertilization of the transgenic crops and consequently the stable inheritance of the trait. An alternative is production by transient expression in plants via genetically engineered viruses as CaMV (39), TMV (40-42), or CPMV (43-45).

Stable integration of genes generally consists of three phases: (a) introduction of foreign DNA into tissue amenable to transformation by methods as PEG-mediated transformation, particle bombardment, Whiskers, and Agrobacterium tumefaciens-mediated transformation; (b) selection of transformed material in the nontransgenic background by either selecting for tolerance to antibiotics, herbicides or another toxic chemical and/or by screening for expression of visual markers like luciferase, anthocyanin and GFP; (c) regeneration of shoots and roots after the successful selection of transgenic cells. This last phase may be quite cumbersome as cells amenable to transformation (competent cells) may not have the ability to regenerate.

Transformation of plants has developed over the last decades into a routine procedure for many crop species, including tobacco, tomato, potato, canola, soybean, alfalfa, sugar beet, lettuce, carrot, cotton, apple, rubber, rice, corn, wheat, and banana. Preference for Agrobacterium tumefaciens-mediated transformation exists as this generally leads to precise integration of only a limited number of copies of the gene of interest into the plant genome. However, cereals have appeared very recalcitrant to this method. Hence, the extensive use in the past of particle bombardment. With the recent development of a commercial transformation procedure for rice and the initial successes with the method in corn, wheat, and barley, it is likely that Agrobacterium tumefaciens will become the general vehicle for DNA delivery into plant species.

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