Triploid plant formation of tangerine (Citrus Nobilis Lour) var simadu through endosperm culture

Abstract

Seedless fruit is one of the criteria necessary to improve the quality of citrus. Traditionally, formation of seedless citrus plant is difficult due to the barriers of sterility gametes which are owned by gene sources of seedless citrus to be transfer to Simadu tangerine. The formation of a triploid plant from a interploidy crossing could not be done because tetraploid citrus plants is not available. Triploid citrus is unable to form the fertile seeds, so it can be used as an alternative to the production of seedless fruits. In vitro, formation of triploid plant can be aimed by regenerate endosperm tissue that is naturally triploid. Shoots obtained from endosperm tissue expected also to be a triploid plant. The aim of this research was to obtain triploid Simadu tangerine derived from culture of endosperm tissue. There were four stages of research conducted to accomplish the research objective. Study of Developmental stage of endosperm tissue of Simadu tangerine. Research conducted to determine the developmental of endosperm tissue that could be cultured in vitro. Plant materials observed was a young fruit from 2 to 14 weeks after anthesis (WAA). The observation was made against fruit morphology, fresh and preserved slice of seeds. The observation was performed with a stereo and inverted microscope. The results showed that endosperm tissues could be isolated from the fruit of 10-14 weeks after anthesis. This tissue had formed cell walls and gradually form compactendosperm tissues and could be isolated and separated from zygotic and nucellar embryos. Induction of somatic embryogenesis and formation of triploid plants derived from varied developmental stages of endosperm tissue of Simadu tangerine (Citrus Nobilis Lour). Research conducted to obtained in vitro technique that could be regenerated endosperm tissue to forming triploid plants. A plant material was an endosperm tissue that was isolated from 10-14 WAA fruit. Endosperm tissues isolated with a binocular microscope with 40x magnification. The results showed that the endosperm tissues from 11-13 WAA could be induced the formation of embryogenic callus with best response shown by endosperm tissue that was 13 WAA and cutured on MS medium modified by MW vitamine. Mixoploid Calli was resulted from the endosperm tissues so the regeneration should be done by somatic embryogenesis. Somatic embryogenesis could be avoiding formation of mixoploid plants. Maturation of somatic embryos cultured on solid medium and 3 g L-1 phytagel without addition of plants growth regulators. Plantlets sub cultured on the same media with the addition of 2.5 mg L-1 GA for shoots elongation. Clonal Multiplication of in vitro shoots that regenerated from endosperm tissue of Simadu tangerine. The research was done to obtain the medium formulation and population of in vitro shoots that regenerated from endosperm tissue. Plant material was shoots that obtained from the somatic embryogenesis derived from endosperm of tissue. Shoots cultured on multiplication medium and observed formation of normal shoots and directly of adventives shoots. The results showed that the MT medium (medium for in vitro culture of citrus), was growth of abnormal shoots higher than a MS medium by vitamin MW modified. Addition of Kinetin, BA and NAA on both basic media only slightly induces the formation of adventitious shoots and its node, consequently shoot multiplication was cultured on MS medium and MW modified vitamins with the addition of thidizuron. On this medium, there 5 was increased of adventitious shoots formation and nodes, successively 2.15±1.34 and 2.90±1.51. Cytology and morphology variation of in vitro shoots derived from Simadu tangerine endosperm tissues. Research conducted to evaluated variation of in vitro shoots derived from endosperm tissue and amount of triploid shoots. Plants material were 52 in vitro normal shoots derived from endosperm tissues.The cytology variation measured were the ploidy level and chromosome number of in vitro shoots. Determination of ploidy level was determined by measuring of nuclear DNA content of in vitro leaves by flow cytometry (Partec Cyflow). Chromosome counting was done by squash method on the tip of the in vitro root. Morphology variables observed was in vitro leaves and stomata. Control plant used for the observation was diploid plant of Simadu tangerine and in vitro shoots derived from nucellar embryo sprouts. The observation showed thatfrom 52 regenerated shoot from endosperm tissues there were: 21 haploid (40.38%), 11 diploid (21.15%) and 20 triploid shoots (38.46%). Determination of ploidy level were done by counting of chromosome, nuclear DNA and chloroplast number of stomata guard cells. Till the end of this dissertation writing, the embryonic cells of endosperm tissues keep on regenerate shoots. Continously sub cultured of embryos and abnormal shoots at eight weeks interval on medium free plant growth regulators will produce normal shoots. It showed that endosperm tissue culture could be produced more than 52 normal shoots.