In vitro screening of guava plantlets transformed with endochitinase gene against Fusarium oxysporum f.sp. psidii

Abstract

Guava wilt disease is a severe threat to guava growers all over the world. It is caused by the soil-borne fungus Fusarium oxysporum f.sp. psidii. To control the disease, the Trichoderma-endochitinase gene was first introgressed into guava (Psidium guajava L.). The transgenic plantlets were screened in vitro for resistance against the wilt pathogen. Six-months-old genetically transformed plants raised in cocopeat under in vitro conditions were inoculated with a 7-days old culture of F. oxysporum f.sp. psidii. The presence of the pathogen in the cocopeat medium was confirmed by cultural as well as PCR analysis using species-specific primers. The roots of transgenic plants were wounded to facilitate the entry of the pathogen. The histopathological analysis revealed the presence of mycelium in vascular bundles. However, none of the plants showed symptoms of wilt disease during the investigation. In vitro pathogen inhibition assay and subsequently spore germination assay revealed that the crude leaf extract of transformed plants inhibited the germination of fungal conidia. The leaf tissue studied for expression of endochitinase revealed that two transgenic plants showed very high activity of N-acetyl-d-glucosamine (0.741 and 0.738 µM/min/µg of protein, respectively) which clearly indicated that transgenic plants could not develop any symptoms of wilt disease due to overexpression of endochitinase.

Keywords

• chitinase
• genetic transformation
• pathogen inhibition assay
• quantification of chitinase
• wilt disease