Abstract
Using a mixed culture of megaspores and microspores from I. coreana, we established high frequency sporophyte regeneration system. After 20 days of culturing in MS basal medium, microscopic examination showed significant morphological 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 expansion of the megaspore (about 2 times increase in diameter). Simultaneously, brown spots were observed on the surface 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 megaspores 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℃ 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 endangered aquatic species, I. coreana.
Highlights
Isoëtes L., known as the quillworts, is a cosmopolitan genus of lycopsids that consists of approximately 150 or more species found in lake, wetland and terrestrial habitats. [1,2]
Brown spots were observed on the surface of the megaspores
Spore cultures of ferns require surface sterilization before germination procedures [17] sterilization affected the viability of the spore to defend itself from fungal infections [13]
Summary
Isoëtes L., known as the quillworts, is a cosmopolitan genus of lycopsids that consists of approximately 150 or more species found in lake, wetland (swamp and marsh) and terrestrial habitats. [1,2]. The development of an in vitro culture system for Isoëtes is required for the study of its life cycles. There are no successful reports about in vitro culture systems for the mass proliferation of Isoëtes species. Based on our recent field surveys, the habitats of I. coreana have degraded and the plant has disappeared from several locations [15]. This species is considered to be rare and endangered by the Korean government. This study reports on spore germination and early gametophyte and sporophyte formation of I. coreana in response to changes in pH, temperature, GA3, and light intensity
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