Induction of haploids in Populus maximowiczii via embryogenic callus

Abstract

Anthers of Populus maximowiczii with microspores at the mononucleate stage were cultured at 20°C in the dark on agar-solidified Murashige and Skoog medium after 4 days of cold treatment (4°C). After 4 to 8 weeks anthers on medium supplemented with 0.5, 1.0 or 2.0 mg l-1 2,4-D in combination with 0.1 mg l-1 kinetin developed calli that were characterized by smooth surface and gel-like consistency. These calli were comprised of expanding microspores surrounded by a mucilaginous matrix. After transfer of anthers with embryogenic calli to MS medium with low hormone levels (NAA at 0, 0.1 and 0.1 mg l-1 and BA at 0, 0.1 and 1.0 mg l-1) microspores started to divide and initiated independent meristematic nests, which developed into embryoidal structures, resembling globular to bi-polar heart-shaped embryoids. The embryoids germinated precociously without developing cotyledons. After transfer to medium with a range of levels of BA (1.0, 2.5 and 5.0 mg l-1), adventitious shoots developed mainly from the roots. Shoots were rooted in half strength MS medium supplemented with 0.025 mg l-1 NAA. Via this pathway anther response in the best treatment combination was 10%.