Induction of haploid callus through anther cultured on citrus sp.

Abstract

Anther culture is one method to produce haploid callus and plants. By the doubling of their chromosome, double haploid plants can be generated and used to produce F1 hybrids. Haploid callus can also be used as tissues for somatic hybriditation to produce triploid Citrus and other non-conventional breeding program. The objective of this research were to study microspore development of four Citrus spesies (Tangerine Garut, Tangerine Batu 55, Siam and Pamelo), effect of cold pretreatment, media type, and plant growth regulator on of callus induction from citrus anther. The development of microspores can be seen by measuring the ratio of the size of the sepals and petals flower. Microspores that have high percentage of uninukleat given different levels cold pretreatment to improve the ability to form callus. After treated with cold pretreatment, callus was induced with various formulations of media, resulting haploid callus then carried out chromosome counting. The results shown that the microspore uninucleat was highest (78,2% to 91,4%) in medium-size flowers with a ratio of the sepal: petal (1:4 - 2:6) mm in flower Tangerine Garut, Tangerine Batu 55, Siam Citrus, and (6:14 - 6:17) mm in flower Pamelo. Microspores uninucleat in Garut citrus flowers ranged from 78.2 to 91.4%, at tangerine Batu 55 flowers ranged from 78.2 to 85.6%, the citrus flower Siam from 79.4 to 90.5%, and citrus flower Pamelo ranged from 78.2 to 85.4% of the take total microspores. Cold pretreatment of anther for 5 days was able to inducted callus citrus keprok Garut up to 2%. MT medium + 3 mg / l BAP + 500 mg / l extract malt on solid media were able to induce callus 14.5% in tangerine Batu 55, while the treatment of liquid media is only able to induce callus up to 4.5%, and solid + liquid media treatments able to induce callus 3.6%. The data is observed when the culture aged 6 weeks after planting. The best medium to induce callus Siam Citrus are MT medium + 3 mg / l 2,4-D + 500 mg / l extract malt, is able to induce callus up to 1,6%. MT medium with 3 mg/l BAP and with 1 mg/l NAA was able to induce callus Pamelo up to 2,6%. Chromosome counting is performed to determine ploidy level of callus produced. The result showed that all chromosome of the anther derived callus was 9 as half of diploid chromosome.

Kultur antera merupakan salah satu metode kultur jaringan untuk menghasilkan kalus atau tanaman haploid. Penggandaan kromosom akan menghasilkan tanaman double haploid yang dapat digunakan sebagai tetua dalam pemuliaan konvensional untuk menghasilkan hibrida F1 atau untuk bahan dalam program pemuliaan tanaman. Penelitian ini bertujuan untuk mendapatkan kalus haploid pada beberapa spesies jeruk. Penelitian ini diawali dengan studi perkembangan inti mikrospora pada keempat spesies jeruk (Keprok Garut, Keprok Batu 55, Siam, dan Pamelo). Perkembangan mikrospora dapat dilihat dengan mengukur perbandingan ukuran sepal dan petal masing - masing bunga untuk mendapatkan ukuran bunga yang mempunyai mikrospora inti tunggal yang banyak. Mikrospora yang mempunyai banyak inti tunggal diberi berbagai tingkat praperlakuan suhu dingin untuk meningkatkan kemampuan antera jeruk membentuk kalus. Setelah mendapatkan praperlakuan dingin terbaik kemudian dilakukan induksi kalus pada antera dengan berbagai formulasi media, dan untuk mengetahui kalus yang dihasilkan antera merupakan kalus haploid maka dilakukan analisis kromosom.