Environmental Stress And Mechanisms Regulating Production Of Diploid Male Gametes In Dianthus Caryophyllus L

2n gametes without reduction of the somatic chromosomes during sexual polyploidization, also called unreduced gamete.2n gametes have played a great role in producing new varieties and breeding of polyploid ornamental and horticultural crops. Unreduced gametes are useful in enabling crosses between plants of different ploidy levels and could overcome unbalanced parental contributions in the developing seed. Meanwhile 2n gametes from a distantly related hybrid can be a source of intergenomic recombinant chromosomes and lead to higher fitness and heterozygosity in the offspring. It was not clear that environmental stress, cytological and genetic mechanism was involving in 2n gamete formation in carnation which was disadvantageous for polypoid breeding using 2n gamete.Environmental stress regulating production of diploid male gametes in Dianthus Caryophyllus was studied. We used microscopy to study cytological mechanisms controlled the formation of 2n gamete and reproductive barriers in 4x-2x crosses in D. caryophyllus. PS1 and OSDl-Like genes connected 2n formation in carnation was cloned and correlation between PS1 expression and the frequency of 2n gamete formation was researched. We are going to use new genome editing derived from CRISPR/Cas to obtain the mutants by targeted mutagenesis of PS1 and OSD1-like genes. Research achievements are as follows:The viability of pollen grains and frequency of 2n pollen of D. caryophyllus cultivars were observed and estimated under microscope. Viability of pollen in the same cultivars differed as affected by the seasons, March and December were lower and June was higher, and various cultivars in the same season differed viability of pollen. It indicated viability of pollen was affected by the genotype and also the environment such as temperature. The frequency of 2n pollen was lower (<5%) which also was influenced by varieties and environment. To see if there was a meiotic defect leading to unreduced gamete formation we used microscopy to follow meiotic progression and found that the two cytological mechanisms controlled the formation of 2n gametes. One is the omission of the second meiotic cell divisions, the other is abnormal spindle geometry in the second meiosis, both of which produce dyads and triads leading to form 2n gametes.Study on reproductive barriers in 4x-2x crosses in D. caryophyllus. Pollen germination, pollen tube growth, fertilization and embryo development were inspected using fluorescence microscopy and paraffin section. Pre-fertilization and postfertilization barriers were detected in these combinations. The embryo rescue might be contributed to obtain hybrid F1 plants. The ploidy of the hybrids and the biological character of parents and hybrids in 4x-2x was analyzed. The experiment results showed that triploid and tetraploid progeny which had different colors and shapes can be produced, and excellent characters of parents can be delivered in 4x-2x. Flower diameters of hybrid progeny were bigger than one or both parents which suggested the size of the flower would increase with the ploidy level. Tetraploid hybrids appeared, it is possible that male parent produce 2n gametes. Polyploid varieties owning excellent traits can be cultivated in the hybrid offsprings by 4x-2x.In order to defect genetic mechanism of 2n formation, DcPS1 gene connected 2n formation in carnation was cloned. The DcPS1 cDNA is 3534-bp long and contains an open reading frame of 3234 bp, plus a 153-bp untranslated 5’region and a 147-bp untranslated 3’region. The DcPS1 encode a predicted protein of 1077 amino acid residues. An Forkhead Associated domain (FHA) was predicted at the N-terminus (CD-search:52-129 aa), while the C-terminal conserved region shows a PilT N terminus domain (PINc) (CD-search:846-3002 aa). We monitored the expression of DcPS1 and found higher expression levels in microspore mother cell phases or meiotic stages in carnation, which suggest that DcPS 1 may be related to meiotic. Transcripts of DcPS1 were also more abundant in the ovary tissue in different cultivars of carnation, these results suggest that DcPS1 may be also related to ovary development. There is clear correlation between the decrease of DcPS1 expression and the frequency of 2n gamete formation. We have demonstrated that unreduced pollen formation in various cultivation was sensitive to adverse climatic environments, which was probably regulated by DcPS1 gene expression. The frequency of 2n formation can be controlled by adjusting temperature in carnation breeding. We researched carnation genetic transformation system and found that the frequencies of transgenic plant regeneration in carnation were different in different varieties and expression vectors.’Master’was lower,’Promesa’and’Nogalte’were higher in frequencies of regenerated plant. We also successfully established CR1SPR/Cas system in carnation, and obtained four positive transgenic plants which target loci occurred a base mutation, and two plants occurred amino acid variation, other two plants were not generated. These findings can have potential applications in gene function research and plant breeding programs by CRISPR/Cas system.OSD1-Like genes connected 2n formation in carnation was cloned. The results showed that OSD1-Like gene in carnation has three homologous genes, named OSDL1a, OSDL1b and OSDL1c genes. The OSDL1a, OSDL1b and OSDL1c cDNA is 1180-bp,1288-bp and 971-bp long respectively and encode a predicted protein of 223,237 and 177 amino acid residues respectively. OSD1-Like has two putative cell-cycle related domain:a D-box and C-terminal MR-tail, and not a GxEN/KEN-box. We monitored the expression of OSD1-Like and found higher expression levels of OSDL1a in microspore mother cell phases and meiotic stages. OSDL1b and OSDL1c were higher expression levels in microspore mother cell phases and ovary which suggest that three OSD1-Like genes in carnation may be related to meiosis, and OSDL1b and OSDL1c in carnation may be related to ovary development. We successfully established Cas 9 vector of OSD1-Like in carnation, and obtained transgenic plants. These findings can lay the foundation for further clarify the function OSDL1a, OSDL1b and OSDL1c gene and obtain high frequency 2n gametes germplasm.