Christian Schöpke

Improvement of somatic embryogenesis and plant recovery in cassava.

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures

Abstract A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.

Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz)

Disorganised embryogenic tissues have been utilised as target tissues for transgene insertion and transgenic plant regeneration in cassava (Manihot esculenta). The production of friable embryogenic callus in fourteen geographically diverse cassava cultivars, from which eleven were established as embryogenic suspension cultures, is reported. Embryogenic tissues were similar in nature in all cultivars tested although there was variation in the time required to generate friable callus and the growth rates of suspension cultures. Regeneration of plants has been achieved from eight cultivars but varied significantly in efficiency, with cv. TMS 60444and Line 2 from Zimbabwe being the most responsive. Tissues from the remaining eight cultivars became arrested at globular and torpedo stages of regeneration indicating that they most likely process an inherent ability to produce plants but require further research to allow this to be realised. Significant numbers of transgenic plants containing transgenes for putative resistance to important viral diseases of cassava in addition to visual marker genes have been regenerated. Transgenic plants from three the cultivars TMS 60444, Bonoua Rouge and M.Col 1505 were recovered after particle bombardment of embryogenic suspension cultures. Correlations have been made between abnormal leaf morphology and plant vigour with the use of embryogenic suspension cultures for transgene insertion. As an result friable embryogenic callus is now being successfully utilsed as the target tissue for genetic transformation and plant regeneration at ILTAB.

Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis

Tissue derived from embryogenic suspension cultures of cassava was bombarded with microparticles coated with a plasmid containing theuidA gene, which codes forβ-glucuronidase (GUS). After 3 days, the effect of different bombardment parameters was evaluated by comparing the numbers of blue spots that resulted from histological GUS assays. Counting of blue spots was performed using a system comprised of a black and white video camera, a stereoscope and a personal computer. A reproducible counting method was established by optimizing GUS assay conditions, preparation of tissue samples and acquisition of video images in view of attaining the highest possible contrast between the blue spots and the surrounding tissue. The effects of bombardment pressure, microparticle size, number of bombardments, and osmotic pretreatment on GUS expression were investigated. Optimal transient expression of theuidA gene was observed after bombardment at 1100 psi, with a particle size of 1 µm, an osmotic pretreatment and two bombardments per sample. The highest number of blue spots observed was 2400 per square centimeter of bombarded tissue.

An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.

Transient gene expression in cassava using high-velocity microprojectiles

The bacterial gene encoding β-glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter fromArabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3′ ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.

Transformation in Cassava (Manihot esculenta Crantz)

Cassava is cultivated throughout the tropics for its starchy, tuberous roots. In Africa it plays an important role as a subsistence crop, while in Latin America cassava is also an industrial crop that is processed into more than 300 products. In Asia it is mainly used for the production of animal feed and for exportation. In terms of caloric production it ranks fourth after rice, wheat, and maize in developing countries (FAO 1987; cited in De Bruijn and Fresco 1989). Compared to other crops, cassava has played a minor role in agricultural research. However, during the last decade, the interest in cassava has grown (Cock 1982; Cooke and Cock 1989) and it was recognized that biotechnology might be a useful tool for cassava improvement (Roca 1984, 1989; CIAT 1989; Bertram 1990). With the aim to concentrate and coordinate efforts using biotechnology for the improvement of cassava, in August 1992 the First International Scientific Meeting of the Cassava Biotechnology Network (CBN) was held in Cartagena, Colombia.

Genetic Biotechnologies and Cassava-Based Development

Cassava (Manihot esculenta Crantz) possesses a number of characteristics which make it an irreplaceable food security for smallholder farmers in certain areas of the tropics, where climate, soils, or societal stresses create particularly difficult conditions. Yet because of other characteristics, and especially historical factors, cassava’s value for improving the quality of life of farm families who depend on it is far less than it could be. One resuit is that cassava remains relatively more important in poor, unfavored areas where there are few crop alternatives — areas where smallholder farmers predominate. Appropriate research and development (R&D) to improve cassava’s productivity and value therefore has unusual and direct linkages to global development objectives: food security, poverty alleviation, equity, and environmental protection.