Cisgenesis and intragenesis as new strategies for crop improvement

Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species

Quality and Agronomic Effects of Three High-Molecular-Weight Glutenin Subunit Transgenic Events in Winter Wheat

Quality and agronomic effects of three transgenic high molecular weight glutenin subunit (HMW-GS) events were characterized in advanced generation breeding lines of hard winter wheat (Triticum aestivum L.) in three Nebraska crop years. Two or the transgenic events studied, Dy10-E and B52a-6, ovenexpress HMW-GS IDY10, while the third event, Dx5 +Dy10-H, overexpresses HMW-GS IDx5 and to a much lesser extent. IDY10. In addition, novel proteins possessing solubility characteristics defined as HMW-GS were present in Dx5+Dy10-H and B52a-6. Average grain yield of lines derived from the three transgenic events was statistically lower than that of a group of control cultivars and advanced breeding lines. but not lower than the mean values of respective nontransgenic siblings. Grain hardness was influenced by one of the events. Dx5+Dy10-H produced harder kernels than controls. its nontransgenic siblings. and the two additional transgenic events. All three events produced doughs with unusual mixing properties. although not likely to be directly useful in commercial applications. As a consequence. loaf volumes were depressed to variable degrees by the three events. The results indicated that over-expression of HMW-GS could eventually lead to improved bread-making quality by optimizing the level of over-expression or by development and characterization of additional events

Stably expressed D-genome derived HMW glutenin subunit genes transformed into different durum wheat genotypes change dough mixing properties

Durum wheat (Triticum turgidum L. var. durum) is traditionally used for the production of numerous types of pasta, and significant amounts are also used for bread-making, particularly in southern Italy. The research reported here centres on the glutenin subunits 1Dx5 and 1Dy10 encoded by chromosome 1D, and whose presence in hexaploid wheats is positively correlated with higher dough strength. In order to study the effects of stable expression of the 1Dx5 and 1Dy10 glutenin subunits in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area ('Svevo', 'Creso', 'Varano' and 'Latino') were co-transformed, via particle bombardment of cultured immature embryos, with the two wheat genes Glu-D1-1d and Glu-D1-2b encoding the glutenin subunits, and a third plasmid containing the bar gene as a selectable marker. Protein gel analyses of T(1) generation seed extracts showed expression of one or both glutenin genes in four different transformed durum wheat plants. One of these transgenic lines, DC2-65, showed co-suppression of all HMW-GS, including the endogenous ones. Transgene stability in the transgenic lines has been studied over four generations (T(1)-T(4)). Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes from T(4) plants showed that the integration of transgenes occurred in both telomeric and centromeric regions. The three plasmids were found inserted at a single locus in two lines and in two loci on the same chromosome arm in one line. The fourth line had two transgenic loci on different chromosomes: one with both glutenin plasmids and a different one containing only the construct with the gene encoding the 1Dy10 glutenin subunit. Segregation of these two loci in subsequent generations allowed establishment of two sublines, one containing both 1Dx5 and 1Dy10 and the other containing only 1Dy10. Small-scale quality tests showed that accumulation of Dx5, Dy10 or both in transgenic durum wheat seeds resulted in doughs with stronger mixing characteristics