Transformation

Transformation techniques, using methods such as infection with Agrobacterium, electro-poration or biolistics, enable us to introduce 'engineered' genes into plant cells. The Agrobacterium co-cultivation technique is based on the infective activity of the bacteria to introduce the recombinant DNA into the plant cells and thus act as a natural vector. Agrobacterium co-cultivation was conventionally the transformation method used for dicots. Monocots were found not to be susceptible to Agrobacterium infection in early studies, but more recent studies have indicated that many are and that Agrobacterium can be successfully employed for pineapple transformation. Other transformation methods employed for monocots include the introduction of novel genes into plant protoplasts (Hauptmann et al., 1987; Rathus and Birch, 1992). However, protoplasts are easily damaged and cultures do not always grow into morphogenic callus, to produce trans-genic plant material. Another more successful transformation technique is biolistics, in which recombinant DNA coated on to gold or tungsten particles is propelled under gas pressure in a vacuum chamber into the plant cells. However, with this method multiple copies of the DNA can become incorporated and there is less control over the precise location of the inserted DNA in the plant genome.

The transformation techniques that have achieved the most success to date are the direct introduction of transgenes by microprojectile bombardment (biolistics) and the indirect method by co-cultivation using an Agrobacterium vector. Both methods have been applied to pineapple; however, commercialization and patenting issues have in the past prevented the results of the work from being published widely. Nan et al. (1996) reported the use of the biolistics technique on liquid embryogenic protocorm cultures, obtaining low levels of transgenic material. Graham et al. (1998) have successfully employed the biolistics technique to callus cultures, achieving an average of 1% transformation efficiency. On the other hand, Firoozabady and Gutterson (1998) have produced transgenic pineapples using the Agrobacterium-mediated technique on embryogenic callus cultures, while Isidron et al. (1998) have reported GUS positive activity using a similar system. Graham et al. (1998) have successfully employed Agrobacterium co-cultivation to 'Smooth Cayenne' pineapple leaf bases, resulting in the production of organogenic callus cultures and transgenic plants, with transformation efficiency levels of about 2%.

All methods require a good and reliable regeneration system (Rangan, 1982) to produce transgenic plants from transformed cells. Using Wakasa's (1989) recipe for 'Smooth Cayenne', Graham et al. (1998) initiated callus on 90% of excised leaf bases from well-developed micropropagated shoots within 3-4 weeks. Callus was maintained on the same medium for proliferation, producing different types of callus, very similar to those described by Taylor et al. (1992) for sugar cane. The majority of callus consisted of compact, hard, globular, yellow-green, smooth-surfaced structures. This type of callus was regenerated through organogenesis. To a much lesser extent the second (yellow and friable) and third type (soft, mucilaginous, grey-yellow) of callus was also produced. Only the first type of callus is believed to be morphogenic.

Traits that have been targeted for improvement in 'Smooth Cayenne' using genetic engineering include nematode resistance, pineapple mealy-bug wilt-virus resistance, flowering and fruit-ripening control and black heart resistance (Botella et al., 1998; Graham et al, 1998; Rohrbach et al, 1998; Sanewski et al, 1999).

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