Breeding for Seedless Kinnow: A Biotechnology Approach

Abstract

Kinnow mandarin became popular due to its vigorous growth habit, profuse bearing, heavy yields, color and size of the fruit and excellent eating quality. It has emerged as an excellent source of foreign exchange. Though Kinnow has many merits, there are some drawbacks as well. A significant complaint from the consumer is the high number of seeds in the Kinnow fruit. The goal is this research venture has been to produce genetically improved seedless Kinnow by creating its triploid strains. A combination of conventional breeding and biotechnological manipulation has been employed. 1. Interploid crosses were made to produce triploid by exploiting tetraploid and diploid Kinnow, Succari and Sweetlime parents. Diploid Kinnow was found to be the best seed parent and tetraploid Kinnow as a better pollen parent for fruit set but seed set was better when tetraploid was used as seed parent. The problem of embryo abortion was successfully solved by invitro embryo rescue. Embryogenic callus from zygotic embryos was also induced and regeneration was achieved. Morphological comparison was also made between diploid, tetraploid, and hybrid which indicated many variations in angle of leaf petiole, length of leaf blade (cm), petiole and stem girth. All the hybrids were placed under similar growth conditions in a greenhouse. In visual observation the differences were found in surface, texture and shape of leaf, upper and lower surface and color of leaf. Number of chloroplasts was directly proportional to the ploidy level of the plant. The chloroplast number of tetraploid plants was almost twice than that of diploid plants. On the basis of number, width, length of stomata and number of chloroplasts, several hybrids were found authenticated triploids on the basis of four stomatal parameters. One can not fully rely on stomatal parameters for characterization of hybrid because difference in these parameters is dependent on cultivar type. Also, climatic conditions alter the growth of plants and in adverse conditions, wide differences can be observed in morphological parameters. For an accurate characterization of hybrid cytological studies/chromosomes counts were required. Cytological studies revealed high proportion of triploids in crosses of 4x crossed with 2x Kinnow. Low proportions were found in 4x Kinnow used as pollen parent with 2x Kinnow and Succari seed parent. Tetraploid Kinnow seed parent crossed with diploid Succari also yielded low number of triploids. 2. Endosperm of diploid citrus cultivars has been the promise to produce triploids cultivars. Callus was induced from endosperm of Kinnow mandarin and Succari sweet orange. Succari initiated early and maximum callus than Kinnow mandarin. Callus multiplication was also tested by subculturing callus on different media. Murashige and Tucker (1996) medium supplemented with 500mg/l malt extract yielded the maximum callus proliferation in Succari. No embryogenesis was observed on any media. Both Kinnow and Succari callus remained alive after 6 months of subculturing in liquid media. Further standardization of media formulations is desired. 3. Treatment of citrus buds with colchicine is a useful approach for creating polyploid breeding parents to enhance the germplasm base for the interploidy hybridization. The colchicine treatment induced significant morphological differences for all parameters studied. Statistical analysis showed significant differences for shoot length, leaf length and width, chloroplast density and non-significant differences for sprouting percentage and days to bud sprout. Differences were observed in surface, texture and shape of leaf, upper and lower surface and color of leaf. 4. Induction of callus and embryogenesis from juice vesicles, albedo tissues and immature embryos of diploid and tetraploid Kinnow was performed. Callus initiation occurred from the juice vesicles and albedo tissues on all the nutrient medium within 15 and 30-40 days, respectively. Callus initiation from embryos was much delayed i.e., two months. Maximum callus growth was observed on cultures from albedo tissues of tetraploid origin and in juice vesicles of diploid origin. This research venture have been helped in the development of different protocols for the in vitro regeneration of seedlessness/triploidy in Kinnow leading towards the elimination of the complaints regarding juice bitterness during processing.