| Bougainvillea is a general name for the plants of Bougainvillea,belong to Nyctaginaceae.It is native to South America and is an important woody flower in many southern provinces of China,and is also an important variety for Yunnan to build world-class “green food brand” hundred-billion industry.Bougainvillea have beautiful bracts,prosperous flower sense and extremely long flowering period.It has high ornamental and economic value.However,due to low temperature,it is susceptible to cold damage.Ploidy breeding is an important breeding strategy that can improve the adaptability of plants by creating genetic variations.In this study,Bougainvillea glabra ‘Elizabeth Angus’,a main Bougainvillea cultivar with excellent cold resistance,was used as the experimental material to investigate meiosis of pollen mother cells,correlation between external characteristics of flower organs and meiosis process,2n pollen induction technology,and mutagenesis of terminal buds by dropping liquid method through studying its flower characteristics,meiosis process,2n pollen induction,terminal bud mutation,and ploidy identification,so as to provide a scientific basis for sexual and asexual polyploid breeding of Bougainvillea.The main results and conclusions are as follows:(1)The pollen mother cell undergoes a total of 16 periods of development into mature pollen from the interphase Ⅰ.The type of cytokinesis is simultaneous.There were many Abnormal behaviors of chromosomes from the anaphase I to the tetrad stage.Among them,abnormal meiosis such as lagging chromosome,chromosome bridge and micronucleus in tetrad will lead to the reduction of pollen vitality.(2)The development process of pollen mother cells and the longitudinal diameter of flower buds have a sequential rule and the longitudinal diameter of flower buds can be used as a morphological indicator to determine the development process of pollen mother cells.In the same flower bud,the ratio of the main division phase of the upper anther development to the main division phase of the lower anther development is 86.67%,so the development of the upper anther in the same flower bud slightly precedes the development of the lower anther.In addition,the development of pollen mother cells within the same anther is also inconsistent.The asynchronous development of anthers in the same flower bud and the existence of different division phases in the same anther are beneficial to the prolongation of the anther dispersal period and the effective pollen action time.(3)Colchicine induced B.glabra ‘Elizabeth Angus’ 2n pollen,and the highest induction rate was 10.62% when the flower buds were treated with 1% colchicine at leptotene.The average diameter of natural 2n pollen was(40.30±0.21)μm,1.30 times that of 1n pollen,and the average diameter of colchicine induced 2n pollen was(48.24±0.19)μm,1.55 times that of 1n pollen.The two types of 2n pollen were of different sizes and different formation mechanisms existed.Natural 2n pollen is produced by abnormal spindle localization in metaphase II,and the mechanism of colchicine induced 2n pollen formation is most likely the formation of recombinant nuclei due to the failure of the first meiotic division.The estimated frequency of 2n pollen occurrence was 7.57% based on the proportion of each sporads,and the actual frequency of 2n pollen occurrence was 10.56% in the same year.The estimated value is about 3 percentage points lower than the actual value,which has reference value and can prove the occurrence of 2n pollen.(4)The main toxic effects of colchicine injection on flower buds of B.glabra‘Elizabeth Angus’ are flower dropping and stamen development inhibition,and the toxicity rate will increase with the increase of colchicine concentration.Both distilled water and colchicine injection can affect the number of anthers.The determination of pollen germination rate and germination speed induced by colchicine showed that the germination rate of 2n pollen was still 0 after 72 h.The germination speed of 1n pollen treated with colchicine was slower than that of untreated pollen.The control pollen could germinate at 4 h,while 1n pollen treated with colchicine germinated at 8h.The germination rate of 1n pollen treated with colchicine was significantly lower than that of untreated pollen.The germination rate of control pollen was(9.60 ±1.00)% at 4 h,while the germination rate of 1n pollen treated with colchicine was(1.24 ± 0.40)% at 8 h.(5)The maximum morphological variation of 30% was induced by the dropping liquid method for B.glabra ‘Elizabeth Angus’ terminal buds,with a combination of0.5% colchicine and a treatment time of 48 h.With the increase of treatment time,the variation rate basically showed an upward trend;As the concentration of colchicine increases,the variation rate first presents an upward trend,and then presents a downward trend.In combination with the mortality rate,when the treatment time was chosen to be 10 h,the colchicine concentration should be chosen to be 0.5%,at which time the terminal bud mortality rate was 0 and the variation rate was 16.67%;When the treatment time was chosen for 24 h,the colchicine concentration should be selected as 0.3%,at which time the terminal bud mortality rate was 0 and the variability was 16.67%;when the treatment time was chosen for 48 h,the colchicine concentration should be selected as 0.3%,at which time the terminal bud mortality rate was 6.67% and the variability was as high as 26.67%.(6)The dropping liquid method induced the terminal buds of B.glabra‘Elizabeth Angus’,and the morphological variations were mainly manifested as leaf shape variation,leaf margin variation,leaf surface variation,and leaf hair variation.Leaf surface variation and leaf hair variation are the main variation indicators of mutant plants.(7)The number of chloroplasts in a single stoma of the mutant plant was 28.17 ±0.39,with a variation range of 15 to 40,while the number of chloroplasts in a single stoma of the control plant was 18.27 ± 0.49,with a variation range of 14 to 25.The number of chloroplasts in a single stoma guard cell of the mutant plant was much larger than that of the control plant,but the variation range had intersections.The length and width of stoma and the length and width of guard cell of the mutant increased significantly compared with the control,and the difference was extremely significant;However,there was no significant difference in the ratio of stomatal length to width and the ratio of guard cell length to width between the mutant and the control,indicating that the shape of stomatal guard cell did not change.The stomatal density in a single field of the mutant was extremely significantly lower than that of the control plant.(8)Flow cytometry identified three chimeras,and DNA content measurement showed that the three mutant were all composed of diploid and tetraploid,with an induced colchicine concentration of 0.3%.Therefore,the optimal colchicine concentration for mutagenesis of B.glabra ‘Elizabeth Angus’ terminal buds by dropping liquid method is 0.3%.It is possible to induce polyploidy after treatment for10,24,and 48 h.One of the chimeras was produced by adding 1% DMSO,so although DMSO can lead to an increase in mortality,it is beneficial for inducing polyploidy. |
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