Philippine Journal of Science 149 (S1): 159-173, Special Issue on Nuclear S&T ISSN 0031 - 7683 Date Received: 19 Jul 2019 Gamma Radiation and In Vitro Induced Banana Bunchy Top Virus (BBTV) Resistant Mutant Lines of Banana cv ‘Lakatan’ (Musa sp., AA) Olivia P. Damasco1*, Fe M Dela Cueva1, Jonathan C. Descalsota1, and Ryan Rodrigo P. Tayobong2 1Institute of Plant Breeding 2Institute of Crop Science College of Agriculture and Food Science University of the Philippines Los Banos College, Laguna 4031 Philippines Gamma irradiation coupled with in vitro technology was used to develop BBTV resistance in banana cv ‘Lakatan.’ Ten (10) resistant lines were selected after several generations of evaluation and selection. The selected lines (M1V3–M1V4) showed low disease incidence in the field under high disease pressure and, likewise, low disease incidence with aphid inoculation of the virus. Further disease evaluation (M1V4–M1V5) on these lines consistently showed low BBTV disease incidence (11.62–28.57%) 30 mo from planting (MAP). The genetic variability in morpho-agronomic traits derived from SHAN cluster analysis grouped the selected lines into four major clusters for qualitative traits at 0.636 coefficient of similarity and three clusters for quantitative traits at 0.11 Euclidian distance coefficient. Selections based on agronomic traits showed significantly earlier flowering in three mutant lines and shorter stature in two mutant lines. Short strand repeat (SSR) analysis using 11 primers detected a high level of polymorphism in mutant lines. Mutant lines were differentiated from the ‘Lakatan’ control by the absence of one or few alleles in mutant lines with four primers and/or addition of one or few alleles in mutant lines with two primers. SSR analysis revealed genetic differences among mutant lines and between mutant lines and ‘Lakatan' control. The results of the study further affirmed stable BBTV resistance in advanced generation evaluation (M1V4M1V5). Five out of ten resistant lines were selected for further evaluation in multi-location field trials as a requirement for registration and release of new BBTV resistant mutant variety of ‘Lakatan.’ Keywords: banana, BBTV, gamma radiation, ‘Lakatan’, resistant lines, somaclonal variation INTRODUCTION primarily by small-scale banana farmers for the local market. ‘Lakatan’ contributes about 12.6% in the total Banana (Musa spp) is one of the most important fruit banana production with a total area of about 55,000 ha and crops of the Philippines, both as a source of local and a production volume of 0.92 M metric tons (PSA 2018). international revenues for farmers and an important The biggest commercial production areas are located in component of the daily diet of all Filipinos. The banana cv Davao Region in Mindanao and Cagayan Valley in Luzon. ‘Lakatan’ (AA) is a popular dessert type banana produced The banana bunchy top disease (BBTD) caused by the *Corresponding Author: [email protected] banana bunchy top virus (BBTV) (Babuvirus, Nanoviridae) 159 Special Issue on Nuclear Science and Technology Damasco et al.: BBTV Resistant Lakatan Mutant Lines is one of the most economically important diseases of several Musa accessions (Jain 2010, Lamo et al. 2017). bananas in many production regions of the world including Banana mutant lines with improved agronomic traits the Philippines (Dale 1987). The virus is transmitted in such as height reduction, larger fruit size, and earliness a persistent and circulative but non-propagative manner to flowering have been identified (Jain 2010, Lamo et al. by the banana aphid Pentalonia nigronervosa (Hafner et 2017). Moreover, banana mutant lines with resistance to al. 1995, Hu et al. 1996). Initial symptoms in infected Fusarium oxysporum f. sp cubense (Hwang and Ko 1987, plants include marginal chlorosis and dark green dots and Bhagwat and Duncan 1998, Jain 2010), Mycosphaerella streaks along the veins that often extend down the midrib fijiensis (Roux 2004), and BBTV (Damasco et al. and petiole. Infected plants become progressively stunted 2007) have been identified and further evaluated for with malformed and more upright leaves, eventually confirmation of resistance trait. resulting in a ‘bunchy’ appearance. Plants infected early with BBTV do not bear fruit, and fruits of later infected According to the Mutant Variety Database (IAEA-FAO plants are typically deformed and unmarketable. Hence, 2017), only three officially released banana cultivars yield losses in severely infected plants can be as high as generated from gamma irradiation treatment showed 100%. Additionally, the virus spreads to suckers through significant improvements in agronomic traits. These the rhizome and the entire banana mat eventually becomes include the cv ‘Al-Beely’ with 30% yield improvement, infected (Dale and Harding 1998). The cv ‘Lakatan’ is cv ‘Klue Hom Thong KU1’ with large cylindrical shape very susceptible to BBTV; the disease has wiped out bunch, and cv ‘Novaria’ – an early flowering mutant with the ‘Lakatan’ industry in some areas of the country improved fruit quality. and, at present, is continuously decimating the industry. The first reported BBTV resistant mutant lines in Micropropagation, coupled with disease indexing for mass ‘Lakatan’ generated by gamma irradiation coupled with production of disease-free planting materials, has now in vitro techniques (Damasco et al. 2007, 2008) have become one of the most adopted technologies in managing undergone further generations of evaluation and selection BBTV spread (Dela Cueva et al. 2010). While the use for the stability of BBTV resistance trait and identification of micro-propagated disease-free planting materials is a of useful agronomic traits. viable BBTV disease management option, its effectiveness is limited where residual or alternate inoculum sources This paper presents the results on the advanced generation are present. Regular replanting has to be done once the evaluation and selection (M1V3-M1V5) of stable initially disease-free plants got infected within one or BBTV resistant ‘Lakatan’ mutant lines/variants and the after a few growing seasons. It has been reported that the morphological and molecular characterization done in rate of BBTV re-infection in the field ranges from 5% in these selected resistant lines. well-managed farms to 20% and 75% in medium- and traditionally managed farms, respectively (Magnaye 1998). In addition, only big commercial growers and corporate farms benefit from this micropropagation MATERIALS AND METHODS technology, and small-scale banana farmers and growers cannot afford the high cost of tissue cultured planting Plant Material materials. Thus, built-in BBTV resistance remains to be The selected BBTV resistant lines of banana cv ‘Lakatan’ the most effective disease control measure. were originally generated from gamma irradiation at 5–45 Gy (Damasco et al. 2007) and in vitro induced variation Conventional breeding is difficult in edible Musa spp. (somaclonal variation) generated from higher subculture due to polyploidy and male and female sterility. Almost treatments (Damasco et al. 2008). Disease-free suckers all edible bananas and plantains could have originated from selected lines after each generation of evaluation through spontaneous mutation (Buddenhagen 1987). (M1V3-M1V4) were collected from the field, indexed Because of this limitation, BBTV resistance could not for viruses, and micro-propagated following the standard be introgressed into banana by conventional breeding banana tissue culture procedure (Damasco and Barba methods. In other crops, resistance to pests and diseases 1984). Shoots were multiplied, rooted, potted out, and were generated using irradiation, chemical mutagens, established in the nursery. somaclonal variation, or by a more direct method of in vitro selection in the presence of the stress factor (Jain The selection of promising lines at each generation of 2010, Lamo et al. 2017). evaluation was done following the selection procedure adopted for mutant lines and somaclonal variants (Figure Gamma irradiation has been reported to be the main 1). Selected lines from each generation/cycle of evaluation physical mutagen for mutation induction in Musa, were advanced into the next generation of evaluation. although chemical mutagenesis has also been used in 160 Special Issue on Nuclear Science and Technology Damasco et al.: BBTV Resistant Lakatan Mutant Lines Figure 1. Procedure for evaluation and selection of BBTV resistant lines from radiation and in vitro induced mutation in banana cv ‘Lakatan’ (modified from Donini and Sonnino 1998). Field Evaluation and Selection of BBTV Resistant to serve as a source of inoculum. Artificially infected Lines (M1V3–M1V4) ‘Lakatan’ plants were planted after three test plants within Three-month-old micro-propagated plantlets from 33 every block. Using this layout, the disease pressure in the selected lines (30 mutant lines and three somaclonal experimental area was maintained at a very high level. variants) were established in three experimental plots in The BBTV incidence in the field was monitored regularly a BBTV -infected field located at the Institute of Plant every month until the fruiting stage. Plants were examined Breeding Experimental Station in Tranca, Bay, Laguna. individually for BBTV symptoms and the presence of the Selected mutant lines/variants were planted together with aphid vector. The percent relative advantage over control the ‘Lakatan’ control (original parent clone) in each plot. in terms