Recovery of haploid plants from asparagus microspore culture

Abstract

Asparagus (Asparagus officinalis L.) microspore culture was performed in an array of experiments that assessed the roles of plant growth and culture conditions. The following protocol provided the best results. Flowers with microspores at the late uninucleate stage of development were collected from greenhouse plants grown at 22:18 °C (light:dark) and stored at 5 °C for 3 days. One millilitre of MS medium plus 0.2 g/L yeast extract, 500 mg/L casein hydrolysate, 800 mg/L glutamine, 2.0 mg/L naphthaleneacetic acid, 1.0 mg/L benzyladenine, and 6% sucrose (MSFY) was conditioned with 10 anthers/mL for 1 week, after which it was filtered. One hundred anthers were added to shed their microspores (1.6 × 105 per mL) and were removed after 3 weeks when 0.5 mL of fresh medium was added. Cultures were incubated at 35 °C for 1 week, then 30 °C for 5 weeks. Microcalli were collected subsequently on a 100-μm screen and placed on induction medium (MSFY minus yeast extract, plus 3 g/L gelrite) in darkness at 35 °C for 4 weeks and then in light at 25 °C for 4 weeks. Shoots, roots, and bipolar embryos were produced. The latter were transferred to maturation medium (MS plus 0.1 mg/L naphthaleneacetic acid, 0.5 mg/L kinetin, 3% sucrose, 3 g/L gelrite, and 0.65 mg/L ancymidol) for 4 weeks, then to germination medium (MS plus 1.0 mg/L gibberellic acid, 3% sucrose, 3 mg/L gelrite). Plantlets were grown and maintained on maturation medium. Approximately 0.3% of the cultured microspores produced calli, and 85% of calli produced plantlets. Of 10 plants analyzed, 2 were haploid, 7 were diploid and, 1 was tetraploid. Key words: asparagus, haploid, microspore.