Strawberry transformation with antisense cellulase and pectate lyase genes: effects on production and fruit firmness.

Abstract

Strawberry (Fragaria x ananassa Duch.) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, leaf discs of cv. Chandler were transformed with a non-oncogenic Agrobacterium tumefaciens strain carrying the binary vector pIA3 that contains the antisense sequence of a construct with cellulase-pectate lyase genes under the control of the 35S promoter. After 32 weeks of culture, a 78% of inoculated explants produced kanamycin resistant calli but did not regenerate shoots. These calli were transferred to different culture media in an attempt to regenerate transgenic shoots. When these calli were subcultured on Murashige and Skoog (MS) basal medium supplemented with 0.2 mg/l NAA and 0.3 mg/l TDZ, shoots were formed, obtaining a transformation frequency of 16.67%, based on kanamycin resistance. Molecular analysis was carried out by PCR to confirm gene integration. Fifteen independent transgenic lines (DC lines) were evaluated. In addition, one transgenic line with the pGUSINT and a non-transformed line were used as controls. Our results show that the simultaneous inhibition of both genes has a negative effect on the accumulated production. In most of the DC lines, the ripened fruits analyzed were firmer than control fruits. Strawberry (Fragaria x ananassa, Duch.) is considered one of the most important crops for exportation in Egypt, as well as for local fresh consumption and food processing. During ripening, the fruits soften greatly acquiring a melting texture. Softening of ripe strawberry fruits appears mainly due to middle lamella and cell wall degradation (Knee et al., 1977; Perkins-Veazie, 1995). Some hydrolytic enzymes, which catalyse the degradation of pectins or other cell wall polysaccharides, have been implicated in fruit softening (Manning, 1993). Recently, pectate lyase and cellulase genes isolated from ripe strawberry have been proposed as new candidates contributing to the lost of fruit firmness (Medina-Escobar et al., 1997; Manning, 1998). To assess the involvement of the putative pectate lyase gene in the strawberry ripening process, Jiménez-Bermúdez et al. (2002) obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Total yield was significantly reduced in most of the Apel lines. Ripened fruits of transgenic lines were significantly firmer than controls. These results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry. Woolley et al. (2001) obtained transgenic plants with reduced levels of the cellulase gene, cel1, by both antisense and sense transformation, and did not detect any effect on fruit firmness. Jiménez-Bermúdez et al. (2001) obtained similar results when they transformed plants with a different strawberry cellulase gene, cel2. In this study, we present results of transgenic strawberry plants transformed with antisense sequences of cellulase-pectate lyase genes to study the effect of simultaneous inhibition of both genes on fruit softening.