CRIS -Creative Research Institution- Hokkaido University

【Division of Innovative Research】Yoichiro HOSHINO

Yoichiro HOSHINO Non-Traditional Science
Identification of the molecular program of fertilization / embryogenesis control in higher plants by fertilization in vitro
Yoichiro HOSHINOAssistant Professor

Introduction

 Since fertilization event and following embryogenesis succeed in deeply inside of the ovule in higher plants, analyzing that how the process is controlled and regulated is difficult. Recently, procedure for isolating living egg cells and sperm cells has been developed and in vitro fertilization, which is fusion of isolated single egg and sperm cell, successes. By using this procedure, it became possible to observe the process of embryogenesis directly. As for fertilization of the higher plants, there are similarities to the animals, such as inhibition of polyspermy, but pollen germination, pollen tube elongation, pollen tube invasion into synergid, double fertilization, formation of apical cell and basal cell from zygote are specified in higher plants. The molecular program controlling these processes from fertilization to embryogenesis plays important roles in development of the plants. The clarification of these processes will contribute further improvements of useful plants by clearing the problem of distantly cross hybridization and embryo abortion. Thus, in vitro fertilization in plants will contribute to develop novel breeding procedure in the future.

At present, we try to isolate gametophytic cells and to analyze fertilization as well as following embryogenesis. In addition, analysis of the mechanism which can develop gthe flower h as the reproductive organ, and research for the function of the endosperm which is the product of double fertilization.

 

Formation of male gamete: Pollen germination and sperm cell isolation from pollen tubes

 Pollen grains of Alstroemeria are binucleate. For isolating sperm cells, it is necessary to form sperm cells in pollen tube after pollen germination. We developed novel in vitro system for pollen germination in liquid medium and could observe processes of sperm cell formation from generative cell. After sperm cell formation, we succeed to dissect pollen tubes with glass needles. Finally, sperm cells were successfully isolated and recovered by microcapillary. Isolated sperm cells are provided for experiments of in vitro fertilization.

 

Egg cell isolation for in vitro fertilization

 Recently, techniques have been developed for the isolation and culture of female gametes, and for plant regeneration from in vitro-fertilized egg cells of maize (Kranz and Lorz, 1993) and zygote protoplasts of barley (Holm et al., 1994). The isolated gametes are expected to be used for various new prospects, such as direct observations of fertilization processes in vitro, studies of the mechanism of cell to cell recognition, adhesion and fusion of gametes, and in vitro fertilization studies for breeding by crossing between distantly related species. However, it is still difficult to manipulate female gametophytes in most angiosperms, because their development generally takes place deep inside of the sporophytic ovule tissues. We tried to isolate egg cells and zygotes from Alstroemeria ovules to develop in vitro fertilization technique. Ovules of A. aurea were histologically observed by clearing procedure, which showed localization and sizes of embryo sacs and egg apparatus. For isolating egg cells, ovules were cut into pieces with a surgical blade and treated with enzyme solutions. Subsequently, these ovule pieces were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillary. At present, we make an attempt to establish in vitro fertilization technique between isolated egg cells and sperm cells and keep to study for further research of fertilization and embryogenesis in higher plants.

 

Analysis of the zygomorphy in Alstroemria flower

 We focus on the mechanism controlling development of the flower as a reproductive organ. One of the dicotyledonous plant, snapdragon has bilateral symmetry flower (zygomorphic flower). Recently, CYCLOIDEA (CYC) and DICHOTOMA (DICH) genes concerning with formation of zygomorphic flower were isolated by analyzing snapdragon mutant. In our study, research for zygomorphy in monocotyledonous plant, Alstroemeria which is one of important floricultural crop is now in progress. We try to isolate CYC-homolog genes and to reveal the function of these genes during flower development. This study will contribute further improvement of Alstroemeria flower as well as analysis of relationship between zygomorphic and regular flower (radially symmetric flower) from viewpoint of evolution and diversification in higher plants.

 

Functional analysis of endosperm development: Production of endosperm-derived triploid plants

 As a result of double fertilization, triploid endosperm and diploid embryo are formed from central cell and egg cell, respectively. Although both central cell and egg cell are originated from same female gamete, prospective fate of each cells is largely different. Endosperm can store nutrient for supporting embryo growth and never regenerate plants. As first attempt, we investigated the regeneration ability of develop developing endosperm and could success to produce endosperm-derived triploid plants in Haskap (Lonicera caerulea L. var. emphyllocalyx Nakai). These regenerated plants will be also utilized for breeding materials of this plant. By comparing with the developing processes of embryo, we analyze the mechanism of double fertilization and following embryo and endosperm development.