Diagnóstico viral e limpeza clonal de matrizes de maracujazeiro (Passiflora edulis Sims) utilizadas na produção de sementes híbridas

Abstract

This work aimed to perform the viral diagnosis and cloanl cleaning of passion fruit (Passiflora edulis Sims) matrices used for hybrid seed production. To carry out the viral diagnosis, leaves of the genotypes CPMSC1, CPGA1, MR1, CPMGA2 and CPF1SSBR were collected and the RNA and DNA extracted and used for viral detection by RT-PCR and PCR, using primers that encode Cowpea aphid-brone mosaic virus (CABMV), Lettuce chlorosis virus (LCV) and Begomovirus genomic regions. In addition, the results obtained by using CABMV primers were hybridized by Southern blot for a more reliable diagnosis. The results revealed the presence of CABMV in 100% of the samples tested for all matrices. Furthermore, CPGA1 (17%) and CPMSC (11%) samples tested positive using the universal primers for begomoviruses. No sample tested positive for LCV. In a second experiment, a micropropagation protocol for the genotypes was developed, in order to perform a matrices multiplication arising from the clonal cleaning. Therefore, initially the best explant was evaluated for the in vitro establishment and multiplication phase. It was found that the development of pre-existing buds is only observed in the single node cuttings, compared to inoculation of isolated buds, generating shoots with an average size of 7 mm after 45 days of cultivation. Then, Only single node cuttings were used in another in vitro establishment experiment using MS medium supplemented with 4.43 µM of BA. In it, the shoots development was observed with at least 6 mm after 30 days of culture. The material arising from the single nodes cuttings was used for the other multiplication, elongation and rooting experiments. Finally, to test the efficiency of shoot tip culture in CABMV eliminating in previously diagnosed matrices, apical meristems (0.1 - 0.3 mm) were isolated and inoculated in MS medium supplemented with 0.44 µM NAA, 0.44 µM BA and 0.28 µM GA3. After the apical meristema regeneration, the explants were transferred to MS medium with 4.43 µM BA to induce shoot proliferation and multiplication. Shoot clusters from the shoot tip culture were used to index the plant material for the presence of CABMV by means of RT-PCR. The observed results showed a decrease in the bands intensity referring to CABMV in the genotypes CPGA1, MR1 and CPMGA2, an indication of decreased viral load. Finally, viral cleaning was observed in only one sample of the CPGA1 genotype.