A novel principle for selection of transgenic plant cells is presented. In contrast to traditional selection where the transgenic cells acquire the ability to survive on selective media while the non-transgenic cells are killed (negative selection), this selection method actively favours regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed. Therefore, this selection strategy is termed 'positive selection'. TheE. coli β-glucuronidase gene was used as selectable (as well as screenable) gene and a glucuronide derivative of the cytokinin benzyladenine as selective agent which is inactive as cytokinin but, upon hydrolysis by GUS, active cytokinin is released stimulating the transformed cells to regenerate. Selection ofAgrobacterium tumefaciens inoculated of tobacco leaf discs on benzyladenine N-3-glucuronide (7.5-15 mg/l) resulted in 1.7-2.9 fold higher transformation frequencies compared to kanamycin selection. A significant advantage of this selection procedure is the elimination of the need for herbicide and antibiotic resistance genes.

Two yellow‐seeded white‐petalled Brassica napus F7 inbred lines, developed from interspecific crosses, containing 26–28% emcic acid and more than 40 μmol glucosinolates (GLS)/g seed were crossed with two black/dark brown seeded B. napus varieties of double low quality and 287 doubled haploid (DH) lines were produced. The segregation in the DH lines indicated that three to four gene loci are involved in the determination of seed colour, and yellow seeds are formed when all alleles in all loci are in the homozygous recessive state. A dominant gene governed white petal colour and is linked with an erucic acid allele that, in the homozygous condition, produces 26–28% erucic acid. Four gene loci are involved in the control of total GLS content where low GLS was due to the presence of recessive alleles in the homozygous condition in all loci. From the DH breeding population a yellow‐seeded, yellow‐petalled, zero erucic acid line was obtained. This line was further crossed with conventional B. napus varieties of double low quality and, following pedigree selection, a yellow seeded B. napus of double low quality was obtained. The yellow seeds had higher oil plus protein content and lower fibre content than black seeds. A reduction of the concentration of chromogenic substances was found in the transparent seed coat of the yellow‐seeded B. napus.

Joersbo, M, and Brunstedt, J, 1992, Sonication: A new method for gene transfer to plants. -Physiol. Plant. 85: 230-234.Sonieation is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects, are reviewed. The state of the art of acotistic transformation is presented and possible mechanisms are discussed.

The mannose selection system employs the phosphomannose isomerase (PMI) gene as selectable gene and mannose, converted to mannose‐6‐phosphate by endogenous hexokinase, as selective agent. The transgenic PMI‐expressing cells have acquired the ability to convert mannose‐6‐phosphate to fructose‐6‐phosphate, while the non‐transgenic cells accumulate mannose‐6‐phosphate with a concomitant consumption of the intracellular pools of phosphate and ATP. Thus, certain steps of mannose selection depend on the cells’ own metabolism which may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens‐mediated gene transfer to sugar beet cotyledonary explants. Four frequently employed saccharides (sucrose, glucose, fructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being able to counteract nearly 100% of an almost complete mannose‐induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly the mannose‐induced growth inhibition, but were 4‐, 5‐, and 7‐fold less potent than glucose, respectively (calculated as hexose equivalents). The transformation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highest transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non‐transgenic escapes were found over a range of concentrations. The effect of the light intensity was also investigated and the transformation frequencies were positively correlated to light intensity, although the relative impact of light on growth in the presence of mannose appeared not to be dependent on the mannose concentration. Additional phosphate in the selection media had a strong positive effect on the transformation frequencies, suggesting phosphate limitation during selection. The mannose selection system was found to be relatively genotype‐independent, provided a slight optimization of the mannose concentrations during selection. Analysis of F1‐offspring showed that all studied primary transformants resulted in PMI‐expressing plantlets and that the segregational patterns were in accordance with expectations in at least 50% of the transformants, confirming the stable and active inheritance of the PMI‐gene.

Production of transgenic plants started more than a decade ago, but it is still a time-consuming operation. One of the critical points is the selection procedure used for the recovery of transgenic shoots after transformation. Moreover, as more transgenic traits are to be incorporated into crops that already have been transformed, it is clear that there is a need for new methods with higher efficiencies. In this article, recently developed selection systems are reviewed. They differ from conventional selection techniques as they are based on supplementing the transgenic cells with a metabolic advantage rather than killing the non-transgenic cells. In many cases, these new selection systems have been found to be superior to conventional methods.