THE USE OF TISSUE CULTURE TECHNIQUES TO STUDY PATHOGENICITY OF AND SELECT FOR RESISTANCE TO THREE MAJOR DISEASES (E. carotovora subspp, Pythium violae, Erysiphe heraclei) OF CARROT

Tez Türü: Doktora

Tezin Yürütüldüğü Kurum: The University of Bath, School of Biology and Biochemistry, School of Biology and Biochemistry, İngiltere

Tez Danışmanı: Richard M. Cooper,G.Gerald Henshaw

Tezin Onay Tarihi: 1996

Tezin Dili: İngilizce

Desteklendiği Program: Diğer

Özet:

ABSTRACT

Highly embryogenic callus and suspension cultures of carrot ([Daucus carota L.) were developed in order to generate, via somaclonal variation, disease resistance to three major pathogens of carrot in Europe for which there are no commercially used resistant cultivars i.e. Erwinia carotovora spp. (soft rot), Pythium violae (cavity spot) and Erysiphe heraclei (powdery mildew). Twenty seven percent of the regenerant seedlings screened from callus cultures expressed resistance to E. heraclei. Regenerant Somaclone-4 was significantly more resistant to powdery mildew than the original seed line NRI-92. However, this frequency of resistance among the regenerants was much higher than expected from somaclonal variation. It was concluded that variation may have come from the original seed used, because a high percentage of plants (ca. 40%) in the original seed line was free from powdery mildew. Nevertheless, it is clear that effective resistance already exists within the species. Scanning electron microscopy revealed no differences in terms of E. heraclei infection structures on leaf surfaces of resistant Somaclone-4 and susceptible NRI-92; resistance must be expressed after penetration. Six percent of roots of regenerants from embryogenic suspension cultures was resistant to P. violae and also the proportion of inoculated sites infected with P. violae was less than the original seed line cv. Morot Duke of which all roots developed cavity spot. Three percent of E. carotovora inoculated roots of regenerants was free from soft rot, in contrast to 18% of roots of cv. Morot Duke which remained symptomless after inoculation. These findings suggest that taproots showing resistance to E. carotovora may have arisen by one or a combination of: variation induced by tissue culture; variation resulting from segregation in the seed line used; “escapes” resulting from an imperfect inoculation procedure. Reinoculation of cloned putative resistant individuals is required to confirm these possibilities. Co-culture of carrot cells with both E. carotovora wild type and mutants and with P. violae was attempted with the aims of i) investigating determinants of pathogenicity and ii) regenerating putative, novel resistant plants from surviving embryogenic cells. Mycelial fragments of P. violae killed carrot cells very rapidly (< 24 hrs) such that this did not provide a suitable selection procedure. The pathogenicity determinants of P. violae are unknown, but cellulase, suberinase and pectin lyase were the major cell wall degrading enzymes, which may aid in penetration of the suberinized endodermis which surrounds carrot taproots. Carrot cells were killed rapidly (ca. 70% of the cell units and 80% of single cells in 24 hrs) with wild type E. carotovora (SCRI 193). Results in vitro and in vivo implied that the major pathogenicity factor of E. carotovora involved in maceration and killing of carrot cells was the extracellular cell wall degrading enzyme, pectate lyase (PGL). Thus pectate lyase killed carrot cells, enzyme deficient mutants (RJP 116 and RJP 243) had reduced pathogenicity and killed plant cells more slowly and to lower levels than wild type SCRI 193. However, in some cases cell killing in co-culture was initiated just before appearance of PGL, suggesting the involvement of another factor. The possible value of enzyme deficient mutants in screening carrot cells for disease resistance in vitro is discussed. Kanamycin at 25 pg/ml was used successfully for removing bacteria from co-culture whilst retaining viability of embryogenic cell units; however the antibiotic markedly reduced their regeneration capacity. No mature somatic embryo was regenerated from co-culture. However, it should be possible with a more suitable antibiotic to select and regenerate plants from this developed co-culture technique.