High Frequency Sporophytes Regeneration from the Spore Culture of the Endangered Aquatic Fern Isoetes coreana

Abstract

Using a mixed culture of megaspores and microspores from I. coreana, we established high frequency sporophyte re- generation system. After 20 days of culturing in MS basal medium, microscopic examination showed significant mor- phological changes and the microspore released numerous small vesicles into the culture medium. Megaspores also showed dramatic morphological changes during its incubation time in culture. The spore wall was cracked by the ex- pansion of the megaspore (about 2 times increase in diameter). Simultaneously, brown spots were observed on the sur- face of the megaspores. The frequency of female gametophytes developing from immature megaspores cultured in MS basal liquid medium (pH 7) supplemented with 1 mgl−1 GA3 was 46%. However, these female gametophytes derived from megaspore only culture could not differentiate into sporophytes. The mixed culture of microspores and mega- spores resulted in successful sporophyte regeneration. The highest frequency (12.3%) of green sporophyte regeneration from mixed spore culture occurred when the cultures were maintained at 25˚C under cool-white fluorescent light (40 μmol·m−2·s−1) with a 16 h photoperiod. Regenerated sporophytes were transferred to a test tube containing vermiculite and a sand mixture and left there until they had three leaves. After root growth and the fifth leaf had emerged, more than 95% of the regenerated sporophytes were successfully transferred to the soil and grown to mature plants. The sporophyte regeneration system established in this study could be successfully used for the restoration of the endan- gered aquatic species, I. coreana.