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An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis. [ABSTRACT FROM AUTHOR]

The successful application of plant biotechnology to Alstroemeria improvement will largely depend on the availability of an efficient regeneration/transformation system. Regeneration in Alstroemeria is accomplished from nodular embryogenic callus initiated from zygotic embryos. Histological studies of embryogenic callus initiation from 4-weeks old cultured ovules revealed that the outermost layers of the protoderm of the embryogenic nodules divided to form either a new nodule or aproembryo. Transient gene expression after particle bombardment of nodular embryogenic callus was optimized using DNA of pAHC25. The highest β-glucuronidase expression was found when the GUS gene was under control of the maize ubiquitin promoter, the target tissue was placed 5 cm below the microcarrier launch assembly and when the rupture disc-breakage point was between 650–900 psi. Kanamycin blocked regeneration of somatic embryos, however, did not block growth of nodular embryogenic callus. With phosphinothricin both callus growth and regeneration were blocked. Bombardment of nodular embryogenic callus with DNA of pAHC25 combined with selection on medium containing phosphinothricin resulted in putative transgenic chimeric. Friable calli were selected from nodular embryogenic callus and used to initiate suspensions. These cell suspensions were subjected to transformation by particle bombardment using DNA of pAHC25 and resulted in a stable transformed friable callus line after selection based on luciferase activity. Even after 2 years of maintenance this callus line was luciferase positive and the Polymerase Chain Reaction analysis demonstrated the presence of the introduced gene in this friable callus line. [ABSTRACT FROM AUTHOR]

Three amylose-free genetically modified potato clones were used both as male and female parents in a breeding program with non-GMO potato clones. Segregation data on the expression of the inserted antisense gene construct in tubers of progeny plants were in agreement with previous molecular analysis of the transgenic clones. The inheritance of the inserted genes was according to Mendelian segregation. Therefore, these clones can be very useful in a breeding program for large scale introduction of amylose free potato cultivars into agriculture. Because of varying number and expression levels of inserts in the GMO-clones, but also because of the varying strength of the endogenous GBSS-alleles of the non-GMO-clones, a segregation into a range of amylose contents occurred. The segregation of the starch colour after iodine staining of pollen of transgenic clones did not follow the obtained segregation in the progeny and was, therefore, not useful in predicting the breeding result. [ABSTRACT FROM AUTHOR]

A prerequisite for the development of a successful transformation system is the availability of efficient regeneration systems. Up to 1995 the only available regeneration system in cassava was an organized type of somatic embryogenesis. Transformation of these organized somatic embryogenic cultures with particle bombardment or Agrobacterium tumefaciens resulted in chimeric transformed embryos. However, the transformed sector was lost after repeated cycles of secondary somatic embryogenesis. After 1995 a less organized system of somatic embryogenesis was developed, so called friable embryogenic callus (FEC) and a system of adventitious shoot regeneration. The FEC regeneration system was combined successfully with particle bombardment. Selection of transgenic plants was based on either luciferase activity, or resistance to the aminoglycoside paromomycin or the herbicide phosphinothricin. Furthermore, protoplasts of FEC are able to regenerate into plants and can be transformed by electroporation. The adventitious shoot regeneration system was combined successfully with Agrobacterium tumefaciens. For this mature somatic embryos were cocultivated with Agrobacterium and cultured for adventitious shoot development. After selection based on the aminoglycoside geneticin or on hygromycin transgenic plants were formed. [ABSTRACT FROM AUTHOR]

The genes coding for the main enzymes involved in cassava starch biosynthesis have been cloned and characterised. Molecular analysis of these genes revealed high amino acid sequence homology with other cloned genes from starch forming plant species. Use of these genes to modify the pathway of starch biosynthesis in cassava has become possible with the advent of a reproducible transformation and regeneration protocol for cassava. This would enable the development of new cassava cultivars able to produce starches with different physico-chemical properties and uses. [ABSTRACT FROM AUTHOR]