In order to produce interspecific somatic hybrids between Brassica campestris (2n = 20, AA) and Brassica oleracea (2n = 18, CC), we isolated protoplasts from cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with 0.2 mg/L 2,4-dichorophenoxyacetic acid (2,4-D) +0.5 mg/L 6-benzylaminopurine (6-BA)+0.1 mg/L naphthaleneacetic acid (NAA)+ 0.1 mg/L Kinetin (Kin), 0.3 mol/L sucrose and 0.3 mol/L glucose were used as osmoticum. At the eight-to ten-cell stage, divided cells were transferred to Kao's basal medium supplemented with 0.3 mol/L sucrose as carbon source and 0.1% agarose, 2 mg/L 6-BA+ 2 mg/L Zeatin (ZEA)+1 mg/L NAA+ 0.5 mg/L Kin for callus induction. After 35 days, when small calli reached 2-3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/L Zeatin (ZEA) and 2 mg/L indole-3-acetic acid (IAA). After the length of the shoots reached 1-2 cm, the shoots were transferred to 1/2 MS+0.2 mg/L NAA for root induction. Morphological, cytological and molecular biological analysis methods were used for identification of somatic hybrids. The results showed that, the first cell division occurred during 2-7 days of culture. Five weeks after culture initiation, the plating efficiency attained 0.66%. Finally, the shoot regeneration frequency was 3.7%. A total of eleven regenerated plants were obtained and verified as somatic hybrids by morphological observation and flow cytometry. Cytological studies showed that all tested plants had a chromosome number of 38, the sum of both parents. Hybridity was also confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis, indicating that these regenerated plants were all true hybrids of B. campestris and B. oleracea. All amphidiploid somatic hybrids showed low pollen fertility. Pollen fertility was gradually recovered in the first and second progenies.
Brassica ; genetics ; growth & development ; physiology ; Breeding ; methods ; Genes, Plant ; Hybridization, Genetic ; genetics ; Protoplasts ; physiology ; Random Amplified Polymorphic DNA Technique