Studies On In Vitro Gynogenesis In Strawberry(Fragaria L.)

By means of the culture in vitro of unfertilized ovary of strawberry (Fragaria L), the gynogenesis was induced and the gynogenic plants with half number chromosomes were obtained. Factors influencing induction were compared and analysed. The gynogenic plant was described. The chromosomal ploidy was identified. The embryology and the procedure of the induction of gynogenesis were studied. The main results were as follows:1. It was a creative research method that by means of the culture in vitro of unfertilized ovary of strawberry to induce the gynogenesis and obtain the gynogenic plants with half number chromosomes.2. The main factors influencing the gynogenesis of the unfertilized ovary of strawberry were genotypes, kinds and concentrations of hormones, sucrose concentrations, the time of cold pretreatment, growth environment and development state and culture conditions. Genotypes of varieties such as Mingxu, Fengxlang, Shisheng, Tianjin No5, UC10), J10, EMC could be induced while Hani, Fuzhou, Xinjing No3. not. 2,4-D was the necessary hormone during the induction and its optimum concentration was 2.0mg/L and induction effect increased with increase of the concentration of 2,4-D when its concentration was smaller than 20mg/L. The cytokinin substance of KT and BA inhibited gynogenesis, but promoted the development of somatic callus. The induction effect increased with increase of the sucrose concentration when it lowed 6%. The seed rate decreased as the sucrose concentration exceeded 6%. The induction effect increased with increase of the time of pretreatment at 4℃ when it was not exceeded 48h, and the 48h of pretreatment at 4℃ was the optimum time. The induction frequency influenced by the growth environment and development state of the experiment materials could be sequenced from high to low as follows: the first grade buds of outdoor plants in spring, the second grade buds of outdoor plants in spring, the firstgrade buds of greenhouse plants, the second grade buds of greenhouse plants. The optimum conditions of culture in vitro in early stage were: alternative temperature at 25℃/16℃, darkness>alternative temperature at 25℃, light /16℃, darkness> constant temperature at 25℃ , darkness > alternative temperature at 25℃/16℃, light > constant temperature at 25℃, light.3. The high frequent and steady and repeatable experiment system of induction of the gynogenic plants with half number of the chromosomes was established. The first grade buds of the octoploid variety Mingxu, which was planted outdoor in spring, were studied 2 days before their blossoming in spring. After 48h pretreatment, the buds whose perianths and stamina were removed were planted on the medium of MS+2mg/L 2,4-D+6% sucrose+0.8% agar. After 7d dark alternative induction at 25℃ and 16℃ per 12h, the buds were cultivated 23d in light (25℃) and dark (16℃) alternatively per I2h. Finally some seeds matured and the seed rate was 29.7%. Under the germfree condition, the seeds were planted on the medium of 1/2 MS+2% sucrose+0.8% agar. About lOd later the seeds sprouted and the highest sprout rate was 20%.4. 12 gynogenic plants of various genotypes with half number of chromosomes had been obtained in this experiment. The multiple of the genogenic plants of strawberry was identified by means of flow cytometry techniques measuring the content of DNA as well as the traditional method counting the number of the chromosomes. The results showed that 9 gynogynic plants possessed half number chromosomes and the rate was 75%. 2 gynogenic plants possessed the same number of chromosomes as their maternal plants and the rate was 16.7%. 1 mixoploid plant was obtained and the rate was 8.3%. The results indicated that the rate of the gynogenic plants with half number chromosomes was highest.5. The embryological studies of unfertilized ovaries of strawberry in vitro showed that the gynogenesis had three pathways. ① egg cell started to divide. ② individual polar nucleus divided into few endosperm like free nuclei or the structure of free nuclei group, but at last stopped dividing and gradually disappeared,.③occasionally some antipodal cells started to divide into cell group,but the forward developing was unclear. The apogamic of synergid was not observed.6. The embryoid could be classified into three types. ①the typical embryoid. ②the chemiric embryoid. ③the callusized embryoid. The embryoid came from the parthenogenesis of egg cells. The development of typical embryoid was similar to the normal embryoid of the plant. The gynogenic plants primarily developed from the typical embryoid.7. The embryological studies of strawberry showed that the ovule of strawberry were hemitropous, the ovule had the obvious hypostace structure, the type of its embryo sac was Polygonum, the embryo sac was long and narrow, the type of development of embryo was the Geum type of the Asterad type, the endosperm was Nuclear type and the somatic embryo sacs happened. The integral transparency technique combined with the technique of microdissection was used in this experiment. The combination was an innovation in the techniques of section.8. The culture in vitro of unfertilized ovaries of strawberry promoted the development of somatic embryo sacs and raised the frequency of the matured double embryo sacs.9. The cultured strawberry had the possibility of somatic apospory apomixes.