Production of doubled haploids is potentially an important approach to generate inbreed lines for corn breeding. Culture of immature pollen (microspores) is considered to be one of the most effective means to produce doubled haplids. In this research, tassels of a sweet corn (Zea mays) Seneca 77 were sampled when anthers in the central section of each main tassel contained microspores at the mid- to late- uninucleate stage. Following disinfection in a 20% commercial bleach solution and subsequent rinses with autoclaved distilled water, florets were floated in a liquid medium and incubated at 6-8 C for 7-10 days. Microspores were then released from florets by a micro-blender, and separated from other debris through size-differential filtration and gradient centrifugation using 0.3 M mannitol overlaid on 21% maltose. Microspores were cultured at 26-28 C in a liquid medium containing a mixture of maltose and sucrose, essential mineral nutrients, and appropriate plant growth regulators.

The first cell divisions were observed following 2-3 days of incubation. Embryoids/calli developed in approximately 30-45 days. Upon their transfer to semi-solid media and subsequent cultures for 3-4 weeks, approximately 27.5% of embryoids/calli regenerated into plantlets. Some individual embryoids/calli differentiated into multiple plantlets. These plantlets were then transferred to pots and allowed to grow to maturity in a growth chamber. Over 98% of regenerated plantlets were green, 60-65% of healthy green plants produced seeds upon sib-pollinations. More research needs to be done to test the effectiveness of this system using a wide range of corn germplasm.