Indirect somatic embryogenesis in cassava for genetic modification purposes.

In cassava both direct and indirect somatic embryogenesis is described. Direct somatic embryogenesis starts with the culture of leaf explants on Murashige and Skoog (MS) medium supplemented with auxins. Somatic embryos undergo secondary somatic embryogenesis when cultured on the same medium. Indirect somatic embryogenesis is initiated by subculture of directly induced embryogenic tissue on auxin-supplemented medium with Gresshoff and Doy salts and vitamins. A very fine friable embryogenic callus (FEC) is formed after a few rounds of subculture and stringent selection. This FEC is maintained by subculture on auxin supplemented medium. Lowering of the auxin concentration allows the FEC to form mature somatic embryos that develop into plants when transferred to a cytokinin-supplemented medium.

Development of a highly efficient, repetitive system of organogenesis in soybean (Glycine max (L.) Merr).

A highly efficient, repetitive system of organogenesis was developed in soybean. Seeds of soybean cv. 'White hilum' pretreated with TDZ formed multiple bud tissue(s) (MBT) at the cotyledonary nodes. MBT initiation occurred only if the axillary buds were not removed from the cotyledonary node. The best MBT formation was achieved by pretreating the seeds for 1 week on medium supplemented with 0.1 mg/l TDZ, followed by culture of the cotyledonary node on medium supplemented with 0.5 mg/l BA for 4 weeks. Culture of the MBT on medium supplemented with 0.1 mg/l TDZ resulted in the proliferation of MBT. MBT was maintained in this way for 12 months. Three hundred thirty six shoots were obtained when 1 g of MBT was subcultured on medium supplemented with 0.5 mg/l BA. Plants were rooted on medium without growth regulators. The regenerated plants grew normally in the greenhouse. Unfortunately, they did not set seeds because of the long-day conditions during growth. This system was successfully applied in three other genotypes.

Regeneration of pea (Pisum sativum L.) by a cyclic organogenic system.

In a five-step procedure, plants were regenerated from meristematic tissue initiated from nodal tissue in four pea cultivars ('Espace', 'Classic', 'Solara', and 'Puget'). In step 1, stem tissue with one node (1-cm size) was subcultured on medium containing thidiazuron. As a result multiple shoots were produced, appearing normal or swollen at their bases. The multiple shoots were subcultured in the same medium, resulting in the formation of a green hyperhydric tissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)]. In step 2, BCT fragments were isolated and subcultured in the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. In step 3, subculture of BCT on medium supplemented with a combination of gibberelic acid, 6-benzyladenine and alpha-naphthalene acetic acid (NAA), resulted in the formation of shoots, which were rooted in step 4 on medium supplemented with 0.5 mg/l NAA, indole-3-acetic acid (IAA) or indole-3-butyric acid. In step 5, in vitro plants were transferred to the greenhouse for acclimatisation and further development. The four varieties tested were all able to produce meristematic tissue, suggesting that its production is genotype independent.

Development and application of transgenic technologies in cassava.

The capacity to integrate transgenes into the tropical root crop cassava (Manihot esculenta Crantz) is now established and being utilized to generate plants expressing traits of agronomic interest. The tissue culture and gene transfer systems currently employed to produce these transgenic cassava have improved significantly over the past 5 years and are assessed and compared in this review. Programs are underway to develop cassava with enhanced resistance to viral diseases and insects pests, improved nutritional content, modified and increased starch metabolism and reduced cyanogenic content of processed roots. Each of these is described individually for the underlying biology the molecular strategies being employed and progress achieved towards the desired product. Important advances have occurred, with transgenic plants from several laboratories being prepared for field trails.

Transgene organisation in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes.

Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transformed with a plasmid containing the selectable marker contained one and two additional non-selected genes, respectively. When gene cassettes were used in transformation, six out of eight plants were co-transformed. Expression analysis showed that 75-80% of the plants transformed with two transgenes expressed both of them, irrespective of the use of plasmids or gene cassettes. Thirty-eight plants containing the gusA reporter-gene and the nptII selectable-marker have been characterised with respect to the molecular organisation of the donor DNAs. Seventeen out of 49 (35%) gusA sites of integration contained one copy of the gene. Only 11 gusA sites (22%) were linked to the site of integration of the selectable marker. When one site of integration contained several copies of the transgene, a predominance of 3'-3' inverted re-arrangement repeats was observed.

A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop.

It is generally thought that transformation of plant cells using Agrobacterium tumefaciens occurs at a very low frequency. Therefore, selection marker genes are used to identify the rare plants that have taken up foreign DNA. Genes encoding antibiotic and herbicide resistance are widely used for this purpose in plant transformation. Over the past several years, consumer and environmental groups have expressed concern about the use of antibiotic- and herbicide-resistance genes from an ecological and food safety perspective. Although no scientific basis has been determined for these concerns, generating marker-free plants would certainly contribute to the public acceptance of transgenic crops. Several methods have been reported to create marker gene-free transformed plants, for example co-transformation, transposable elements, site-specific recombination, or intrachromosomal recombination. Not only are most of these systems time-consuming and inefficient, but they are also employed on the assumption that isolation of transformants without a selective marker gene is not feasible. Here we present a method that permits the identification of transgenic plants without the use of selectable markers. This strategy relies on the transformation of tissue explants or cells with a virulent A. tumefaciens strain and selection of transformed cells or shoots after PCR analysis. Incubation of potato explants with A. tumefaciens strain AGL0 resulted in transformed shoots at an efficiency of 1-5% of the harvested shoots, depending on the potato genotype used. Because this system does not require genetic segregation or site-specific DNA-deletion systems to remove marker genes, it may provide a reliable and efficient tool for generating transgenic plants for commercial use, especially in vegetatively propagated species like potato and cassava.