Journal of Water and Land Development

Abstract

Using doubled haploid technologies inbreeding can significantly reduce the time to obtain homozygous parental lines required for the production of F1-hybrid of vegetable crops. This study aims to investigate the influence of factors on the efficiency of carrot embryogenesis in isolated microspore culture to optimise the elements of protocol for producing doubled haploids. Microspores were isolated from inflorescences of 21 genotypes and incubated in NLN13 medium supplemented with 0.1 mg•dm -3 2,4-dichlorophenoxyacetic acids, 0.1 mg•dm -3 1-naphthyl acetic acids, 130 g•dm -3 sucrose, and 400 mg•dm -3 casein hydrolysate and its modifications. Embryoids and their groups were formed after 2-6 months, in some cases after 12 months of cultivation. Depending on the variant, the embryogenesis efficiency averaged from 0 to 4.9 embryoids or groups of embryoids per Petri dish (10 cm 3 ). Embryoids within the group were formed from different microspores. No significant effects of inflorescence position on the plant (branching order), sucrose, and casein hydrolysate concentration in the medium were observed. Significant advantages (p ≥ 0.05) for some genotypes were shown: 1) microspore suspension density 4•10 4 cells•cm -3 (5.0 embryoids per Petri dish were formed at a microspore suspension density of 4•10 4 cells•cm -3 , 0.0 embryoids per Petri dish at a density of 8•10 4 cells•cm -3 ); 2) cultivating microspores of tetrad and early mononuclear stage (4.9 ±3.1 embryoids per Petri dish were obtained by culturing tetrads and early mononuclear microspores, while 0.6 ±0.7 embryoids per Petri dish were obtained by culturing of later developmental stages); 3) high-temperature treatment duration of five days (4.9 ±2.1 embryoids per Petri dish were obtained after five days of high-temperature treatment, 2.7 ±2.6 embryoids per Petri dish formed after two days of high-temperature treatment; 9.8 ±4.7, 10.1 ±6.1, 0.0 ±0.0 embryoids per Petri dish formed after two, five and eight days of high-temperature treatment respectively); 4) adding colchicine 0.5 mg•dm -3 to the nutrient medium for two days of high-temperature treatment, followed by medium replacement (3.3 ±2.6 embryoids per Petri dish were obtained by using a nutrient medium with colchicine, while 1.7 ±1.5 embryoids per Petri dish were obtained by culturing in the reference variant).