Establishment of Efficient System for Isolation and Manipulation of Single Protoplasts Containing Aposporous Embryo Sac Initial Cell in Guinea Grass (Panicum maximum)

Aims: In order to do the molecular analysis of mechanism of aposporous embryo sac initial cell (AIC) appearance, as the first step, we attempted to establish the system of isolating and manipulating single cells containing AIC using different methods in guinea grass (Panicum maximum).

Study Design: At first, single protoplasts were isolated from the ovaries staged in different developmental stages using different pre-treatments and different concentrations of enzyme solutions on ovaries; Based on it, the single protoplasts containing AIC were manipulated with ultra particle electronic syringe picopipet (UPESP) machine set onto microscope.

Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2012 and December 2013.

Methodology: The ovary of facultatively apomictic guinea grass (Panicum maximum) was used as materials in this study for single protoplast isolation. The ovaries were classified and collected from young buds and flowers staged in different developmental stages, according to the colors of stigma and the ovary length. And then, the ovaries were pre-treated with needle into different shapes, and treated with different kinds of enzyme solution with different concentrations of mannitol to increase efficiency of protoplast isolation. In final, the single protoplasts containing AIC isolated from different stages of ovaries were manipulated by handle control.

Results: 1) The ovaries in different stages of before AIC appearance, AIC appearance, and AIC-derived embryo sac formation were collected successfully and respectively, indicating that the stigma colors and length of ovaries are proportionate to stages of ovary mature. And single protoplasts containing AIC were isolated from the ovaries staged in white to yellow color. 2) The ovaries be flooded in enzyme solution, were pre-treated with needle in 4 types, that is, (1) Cut in micropylar end; (2) Cut in chalazal end; (3) Cut in middle part; (4) after 1hr of (3) treatment, cut in micropylar end. As a result, the efficiency of protoplast isolation of (3) and (4) was 1-2 hrs shorter than that of (1) and (2). 12%, 11%, 10% and 9% were proper enzyme concentrations for obtaining perfect shingle protoplasts from the ovaries with white, yellow, peach and purple colors, respectively. 3) The single protoplasts containing AIC were collected and manipulated with UPESP in the performance of controlled aspiration and spit.

Conclusion: In this study, in order to do molecular analysis of the mechanism of AIC appearance, we focus on that, the key points were to isolate AIC single protoplasts from apomictic guinea grass using different methods, and then to establish the method of controlling a single protoplast using UPESP machine. These results obtained in this study will be a useful tool for molecular analysis of AIC, and provide important information for clarification of apomixis reproductive mode.