In vitro tetraploid induction of Malus × domestica Borkh. using leaf or shoot explants

Abstract

Abstract Triploids and tetraploids of Malus × domestica Borkh. are widely used in breeding programmes because they are characterised by lush growth, larger organs and greater resistance to biotic and abiotic stress. The aim of this study was to develop an in vitro method of apple polyploidisation using leaf and shoot explants of six cultivars. At first, the procedure of efficient in vitro shoot regeneration from leaves was optimised. The leaf regeneration capacity was generally increased by preculture of donor shoots with 4.5 μM thidiazuron (TDZ) compared to a standard preculture in the presence of 4.5 μM benzyladenine (BA). Therefore, for polyploidisation, the leaf explants were collected from four-week shoot cultures that were pretreated with TDZ and the shoot explants derived from standard four-week multiplication subculture. The explants were incubated for six days in darkness on induction medium containing 2.5 μM 1-naphthaleneacetic acid (NAA) and 4.5 μM TDZ or 18 μM BA (leaves) or and 0.5 μM indole-3-butyric acid (IBA) and 4.5 μM BA (shoots) and one antimitotic agent: colchicine, trifluralin, oryzalin or amiprophos methyl (APM). Subsequently, the explants were cultured for four weeks in darkness on the medium without antimitotic agents and then subcultured on multiplication medium containing BA over a 16-h photoperiod. From leaf explants, 58, 38 and 6 tetraploids were obtained for three out of six cultivars, ‘Pinova’, ‘Redchief’ and ‘Sander’, respectively. For leaf explants, the highest polyploidisation efficiency, approximately 20%, was recorded for colchicine at 125 and 250 mg l −1 . For shoot explants, tetraploids were detected for all the cultivars with the higher tetraploid numbers – 13, 26 and 27, for Co-op 32, ‘Free Redstar’ and ‘Redchief’, respectively – and a few tetraploids were obtained for other genotypes (‘Gala Must., ‘Sander’ and ‘Pinova’). For shoot explants, treatment with 10 mg l −1 APM resulted in the highest polyploidisation efficiency of 9.8%. Additionally, mixoploids were detected three times more than tetraploids when shoots were used for polyploidisation, compared to the sporadic occurrence of mixoploids when leaf explants were used.