Cyclic somatic embryogenesis and plant transformation in cassava
1993
Raemakers, C.J.J.M. | Jacobsen, E. | Visser, R.G.F.
With a four step procedure embryos and plants were obtained with a number of clones originating from Africa, South America and Indonesia. The culture procedure consisted of four steps. In step one embryos were induced on young leaf lobes (1-6 mm) on a Murashige and Skoog (1962) based medium supplemented with sucrose and 2,4-D. The optimal 2,4-D concentration varied for each clone. In step two embryos were germinated on an MS medium with BAP. Isolated embryos can be used to start a new cycle (step 1-- step 2) or cultured for shoot development in step three. In step four shoots were rooted on a hormone free medium. Somatic embryos were used as starting material for transformation experiments using Agrobacterium tumefaciens as the DNA mediating vector. For primary embryogenesis it was found that the growing conditions of the plants of which the lobes were taken, especially the light intensity during growth, were crucial to the response of the leaf lobes and the subsequent formation of embryos. Once embryos were formed it was no problem to obtain more embryos by, going through a number of embryogenic cycles. The response of somatic embryos was much better than that of leaf lobes. After one year of culture (equaling 10 successive cycles) the production of embryos and the conversion into shoots was comparable with that of 2nd cycle embryos. The use of liquid medium and fragmentating of the embryos enhanced the production of new embryos. Infection with Agrobacterium tumefaciens was integrated in the cyclic somatic embryogenesis system at the beginning of step 1 and at the beginning of step 3. With the first approach partly transformed embryos were obtained and with the second approach partly transformed shoots were obtained.
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