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Pest-resistant cocoa via in vitro technology and genetic transformation: 4. Refinement of a genetic transformation system

Project leader : Douglas Furtek, Ph.D

Research Background of the Project

The objectives of the project are:

• To improve foreign gene transfer into cells of cocoa primary somatic embryos using Agrobacterium tumefaciens
• To induce transgenic cells to form secondary somatic embryos
• To regenerate plants from transgenic secondary embryos
• To molecularly and biochemically characterize transgenic plants

This project is the continuation of Pest-resistant cocoa via in vitro technology and genetic transformation 2. Development a genetic transformation system (IRPA No. 01-04-07-0304)

Genetic engineering: A strategy that can safely and inexpensively control insect pests is to genetically engineer plants for resistance. Boulter et al. (1989) list seven advantages of genetic engineering for insect resistance over other methods: 1) season-long protection, 2) protection independent of weather, 3) protection of plant tissues difficult to reach with sprays, 4) insects are affected at most sensitive stage, 5) only crop-eating insects are exposed, 6) protection confined to plant tissues, and 7) active factor is biodegradable and usually non-toxic to man and animals. Genetic engineering is the approach we have chosen to control CPB.

Despite the great potential for improving cocoa through genetic engineering, only one article on introducing foreign DNA into cocoa cells has been reported: Sain, S.L.; Oduro, K.K.; Furtek, D.B. (1994) Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens. Plant Cell Tiss. Organ Cult. 37, 243-251.

Genetic engineering of cocoa: In our proposal to develop a genetic transformation for cocoa written three years ago, we said “Creating transgenic cocoa is likely to be the most difficult part of a project to genetically engineer the tree for CPB resistance and is the theme of this proposal.” This has indeed been difficult. But we have made notable accomplishments – especially considering that only one other laboratory in the world (in the US) has a transgenic cocoa plant:

1. We have created partially transgenic cocoa somatic embryos as determined by staining for the reporter gene -glucuronidase (GUS). Some of these embryos have been placed onto media to convert them into plantlets. Others have been placed onto media to stimulate the production of secondary somatic embryos.
2. We have identified primary somatic embryos as the tissue most responsive to transformation by Agrobacterium tumefaciens.
3. We are able to stimulate secondary somatic embryo production by exposing primary somatic embryos to the cytokinin 2iP.
4. We have identified KKM-9 as a clone that is highly responsive to transformation.
5. We have determined that paromomycin is a much better antibiotic than kanamycin for selecting transgenic calli and somatic embryos.
6. We have determined that the super-avirulent A. tumefaciens strain AGL1 is superior to strain EHA105.
7. We have accumulated a large (>25) collection of transformation vectors and have inserted several of them into AGL1.

Transgenic somatic embryos still must be converted into rooted plantlets. This has been to most difficult step in the plant regeneration process. We have recently begun to dip tap roots into the auxin IBA in order to induce secondary roots. After secondary roots appear, embryos are much more apt to convert into plantlets by producing true leaves. To date, three plants have been potted and placed outdoors.

At the 13th International Cocoa Research Conference in Kota Kinabalu, October 2000, Dr. Laurence Alemanno of CIRAD in France reported that primary somatic embryos are derived from multiple cells, whereas secondary somatic embryos are derived from single cells. Some of our transformation experiments confirm the multicellular origin of primary embryos. When calli that have not yet begun to differentiate somatic embryos were co-cultivated with agrobacteria, the transgenic embryos that were subsequently produced were invariably chimeric, i.e. a mixture of transgenic and normal cells. We have placed some of these chimeric embryos onto secondary somatic embryogenesis media with antibiotics in order to obtain completely transgenic somatic embryos.

Dr. Alemanno also reported that secondary somatic embryos are more normal appearing than primary embryos and are more apt to convert into plantlets. Our experiments substantiate the more normal appearance of secondary somatic embryos. We cannot yet confirm whether they are more apt to convert into plantlets, however.