Biotechnology-assisted development of banana bunchy top virus resistance in banana by mutation breeding - phase2 multi-location field evaluation and characterization of banana bunchy top virus (BBTV)-resistant mutant lines

Thirty three mat/line selections from 14 BBTV resistant mutant lines generated from the Phase 1 of the project were further evaluated for stability of BBTV resistance in the field under high disease pressure and in the greenhouse using aphid inoculation of the virus. Based on BBTV incidence in the field, % BBTV-free fruiting plants, aphid preference, and host reaction to the virus, ten mutant lines namely lines 13-30-2, 7-29-1, 22-28-2, 23-28-7, 60-30-2, 9-28-2, 9-28-3, 9-29-1, 23-30-2, and 28-30-2 were selected. The selected lines, nine mutants and one somaclonal virant, showed low disease incidence in the field under natural infection, and/or obtained low disease incidence using artificial aphid inoculation of the virus compared with Lakatan control plants. The preference of the aphids (vector) to the mutant lines (host) was evaluated using free choice feeding method. The aphid colony count taken at the peak of colonization showed that lines 13-30-2, 9-28-2, 9-28-3, 6-30-2 and 23-28-28-7 were significantly less preferred by the aphids compared with Lakatan control plants, while lines 7-29-1 and 9-29-1 and Lakatan control plants were equally preferred by the vector. The reaction of mutant lines to artificial inoculation of the virus using viruliferous aphids was likewise evaluated. Seven lines consistently showed low BBTV incidence compared with the Lakatan control plants. Line 28-30-2 showed lowest BBTV incidence (19.6%) followed by lines 13-30-2 (25.0%), 9-29-1 (32.8%) and 22-28-2 (38.4%). Likewise lines 28-30-2 and 13-30-1 had the longest incubation period of up to 6 weeks. Other lines including the Lakatan control had 4-5 weeks incubation period. The symptoms observed on infected plants ranged from bunchy top, rosette growth, and marginal chlorosis. Results of the vector-virus-host relationship study using the free choice or no choice feeding of the vector showed that BBTV resistance in some mutant lines (e.g. 28-30-2, 13-30-2, 22-28-2, and 23-28-7) was due to less preference of the vector to the host plant. While lines 6-30-2, 13-30-2, 23-28-7, 28-30-2, which showed low BBTV incidence (50%) despite the high aphid colony count indicate resistance of the host plant to the virus. Vector-virus-host relationship studies showed that the BBTV resistance mechanism in selected mutant line, and somaclonal variant was either due to less preference of the vector to host plant or resistance of the host to the virus. The relationships among mutant lines based on disease reaction parameters analyzed by SAHN cluster analysis using NTSys program clustered the mutant lines into five groups. The lines 28-30-2; LK control and line 23-30-2 were in separate groups while lines 23-28-7 and 9-28-1 grouped together. The biggest group was composed of lines 22-28-2, 13-30-2, 9-28-3, 9-28-2, 7-29-1, and somaclonal variant 6-30-2. The line 28-30-2 was an outlier and entirely separated from the rest of the mutant lines which is an indication of a different disease reaction. The top five lines based on disease resistance reaction parameters (13-30-2, 22-28-2, 9-28-2, 9-28-3, and 28-30-2) were evaluated in multi-location sites in Cavite (2 sites in Indang) and in Laguna (Bay and Siniloan) and the other five lines (ranked 6 to 10 namely 7-29-1, 23-28-7, 6-30-2, 9-29-1, 23-30-2) were evaluated in farmers' field in San Juan, Batangas. Low incidences of the BBTV were observed in all multi-location trial sites, despite the presence of infected plants and vectors in the experimental area and within the vicinity of trial site. The highest percentage incidence of the BBTV was recorded at 11.6% in the case of the Bay, Laguna trial site. BBTV resistant mutant lines were comparable with LK control in terms of plant height, girth, total number of fruits per bunch, number of hands per bunch and number of fingers per hand. Number of days to flowering was significantly earlier for five mutant lines compared with LK control (lines 13-30-2(26 days), 22-28-2 (21 days), 28-30-2 (17 days), 23-28-7 (35 days) and 6-30-2 (17 days)). Molecular characterization of the mutant lines was conducted using SSR analysis. Of the 36 primes, 6 (16.7%) amplified products resulting in distinct repeatable polymorphic bands; 17 (47.2%) produced polymorphic bands in some mutant lines but with no amplification products in 1 or 2 mutant lines; 9 (25%) produced monomorphic products, and 4 (11.1%) did not amplify any products. The genetic relationships among mutant lines based on SSR markers, derived from Jaccard coefficient of dissimilarity, clustered the mutant lines into 7 groups. The semaclonal variant 6-30-2 was a distinctly separate group from the major groups. The mutant line 13-30-2 and LK control exhibited very high similarity and could not be differentiated by the primers used. Eleven SSR markers were able to discriminate most of the mutant lines studied; likewise the somaclonal variant 6-30-2 was discriminated fro the other mutant lines and LK control by several primers. Generation 4 suckers from selected resistant mutant lines were collected indexed micropropagated. To date, 400 cultures (~3,000 plantlets) from selected mutant lines are maintained in vitro for future utilization and dissemination.