Physical Induction Method of Poplar 2n Pollen

Abstract

The invention discloses a physical induction method of poplar 2n pollen. The invention uses low temperature as an inducer to induce and treat poplar pollen mother cells to obtain poplar 2n pollen; further, by optimizing the technical conditions for inducing chromosome doubling of poplar pollen by low temperature treatment, the optimal technical parameters such as effective treatment period, treatment temperature and continuous treatment time are screened, and finally high-frequency poplar 2n pollen is obtained, and a technical system for low-temperature induction of poplar pollen chromosome doubling and breeding triploids is established, which greatly improves the ratio of poplar 2n pollen, and the male inflorescence of poplar treated by low temperature induction can shed pollen normally and has high pollen vitality, which is suitable for hybrid pollination of triploids; compared with the current colchicine induction and high temperature induction methods, the low temperature induction of 2n pollen has the advantages of simple operation, low cost, high relative safety, etc., and has theoretical and practical significance in constructing a new and efficient poplar triploid breeding technology system.

Description

Technical Field

[0001] The invention relates to a method for inducing plant 2n pollen, in particular to a method for physically inducing poplar (Populus) 2n pollen, and belongs to the field of inducing poplar 2n pollen. Background Art

[0002] Triploid breeding combines ploidy effect and hybrid vigor. It can achieve comprehensive genetic improvement of multiple target traits such as growth, wood properties and adaptability through a single round of breeding process. It is an important way and method for selecting new poplar varieties and is increasingly attracting the attention of tree breeders. Hybridization of natural 2n gametes with haploid gametes is an important way to obtain plant triploids. However, due to the generally low frequency of natural 2n pollen or natural 2n female gametes, the triploid yield is generally low, which severely limits the application of this approach in breeding practice. In the practice of polyploidy of forest trees, in order to increase the yield of triploids, it can be achieved by artificially inducing high-frequency 2n gametes to hybridize with haploid gametes.

[0003] Plant 2n gamete mutagens can be roughly divided into two categories: one is chemical mutagens, such as colchicine, fenpropimorph, oryzalin, trifluralin (Huang Quanjun, Zhang Zhiyi, Kang Xiangyang. Study on four anti-microtubule substances inducing 2n pollen grains of Populus tomentosa. Journal of Beijing Forestry University, 2002, 24(1): 12-15) and other anti-microtubule inhibitors. The other is physical mutagens, such as high temperature, ultraviolet rays, gamma rays, etc. (Zhou Q, Cheng XT, Kong B, et al. Heat shock-induced failure of meiosis I to meiosis II transition leads to 2n pollen formation in a woody plant. Plant Physiology. 2022, 189(4): 2110-2127).

[0004] Colchicine is a common and highly effective 2n gamete mutagen that has been successfully used to induce poplar (Kang Xiangyang, Zhang Pingdong, Gao Peng, et al. Discovery of a new pathway for colchicine-induced triploid in poplar. Journal of Beijing Forestry University, 2004, 26(1): 1-4; Zhao CG, Tian MD, Li Y, et al. Slow-growing pollen-tube of colchicine-induced 2n pollen responsible for low triploid production rate in Populus. Euphytica, 2017, 213(4): 94. https: / / doi.org / 10.1007 / s10681-017-1881-9; Tian MD, Zhang Y, Liu Y, et al. High temperature exposure did not affect induced 2n pollen viability in Populus. Plant Cell & Environment, 2018, 41(6): 1383-1393), Eucommia ulmoides (Gao Peng. Study on the doubling technology of gamete and zygotic chromosomes in Eucommia ulmoides. Beijing: Beijing Forestry University, 2006; Gao Peng, Kang Xiangyang, Wang Shangde, et al. Comparative study on the induction of 2n pollen in Eucommia ulmoides by bottle immersion and cotton immersion. Hebei Forestry Science and Technology, 2008, 6: 28-29), Eucalyptus urophylla (Yang J, Yao PQ, Li Y, et al. Induction of 2n pollen with colchicine during microsporogenesis in Eucalyptus. Euphytica, 2016, 210(1): 69-78; Yang J, Wang J, Liu Z, et al. Megaspore chromosome doubling in Eucalyptus urophylla STBlake induced by colchicine treatment to produce The artificial induction of 2n gametes of trees such as triploids. Forests, 2018, 9(11): 728.) has yielded 2n female gametes or 2n pollen up to 20.0%-88.0%. However, colchicine is highly toxic and relatively expensive, so its purchase is strictly controlled. In addition, the use of colchicine for doubling treatment can easily lead to poisoning of operators.

[0005] High temperature is an efficient 2n gamete physical mutagen, which has been widely used to induce poplar (Populus spp.) (Geng Xining, Ren Yongyu, Han Zhiqiang, Du Kang, Kang Xiangyang. High temperature induced megaspore chromosome doubling to breed hybrid triploids of Populus tomentosa. Journal of Beijing Forestry University, 2018, 40(11): 12-18; Kang Ning, Bai Fengying, Zhang Pingdong, Kang Xiangyang. High temperature induced embryo sac chromosome doubling to obtain hybrid triploids of Populus tomentosa. Journal of Beijing Forestry University, 2015, 37(02): 79-86; Tian Mengdi, Li Yanjie, Zhang Pingdong et al. High temperature induced pollen chromosome doubling to create hybrid triploids of Populus tomentosa. Forestry Science, 2018, 54(03): 39-47), Eucommia ulmoides (Li Y. et al. Induction of unreduced megaspores in Eucommia ulmoides by high temperature treatment The breeding practice of triploid selection by doubling megaspore, embryo sac and pollen chromosomes in woody plants such as Eucalyptus (Yang Jun. Research on the basis of reproductive biology and chromosome doubling technology of eucalyptus. Beijing: Beijing Forestry University, 2015), and Rosa sp. (Pécrix Y. et al. Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp. [J]. Journal of Experimental Botany, 2011, 62 (10): 3587-3597) has obtained a large number of triploids in Populus tomentosa, Populus pseudo-simon×P.nigra'Zheyin3#', Populus tomentosa×P.bolleana, Populus canescens, Populus nigra ... alba×P. glandulosa) Eucommia ulmoides and eucalyptus triploid.In recent years, Zhou et al. (Zhou Q, Cheng XT, Kong B, Zhao YF, Li ZQ, Sang YR, Wu J, Zhang PD*. Heat shock-induced failure of meiosis I to meiosis II transition leads to 2n pollenformation in a woody plant. Plant Physiology. 2022, 189 (4): 2110-2127) analyzed the genetic composition of the triploid silver-gray poplar obtained by high temperature induction and found that the 2n gametes obtained by high temperature induction were all SDR type, and the heterozygosity of their genetic parents was only half of that of the FDR type 2n gametes, which seriously limited the application of high temperature in polyploid breeding practice. Therefore, it is very necessary to develop new plant 2n gamete mutagens.

[0006] Low temperature is also an important physical mutagen, which has been used to induce Arabidopsis (Arabidopsis) (DeStorme N, Copenhaver GP, Geelen D. Production of diploid male gametes in Arabidopsis by cold-induced destabilization of postmeiotic radial microtubule arrays [J]. Plant Physiology, 2012, 160 (4): 1808-1826.), pepper (Capsicum annuum L.) (Zhang Xinzhong, Liu Guojian, Yan Liying, Zhao Yongbo, Chang Ruifeng, Wu Luping. Cytogenetic mechanism of heat shock or low temperature induction of pepper 2n pollen production [J]. Hebei Agricultural Sciences, 2003, 7 (3): 7-13; Zhang Xinzhong, Liu Guojian, Zhang Deming. Effects of heat shock and low temperature treatment on pepper 2n pollen production [J]. Acta Agriculturae Boreali-Sinica, 1999 (01), 144.), Japanese persimmon (Diospyros kaki Thunb.) (Yamada A, Tao R, Sugiura A. Influence of low temperature before flowering on the occurrence of unreduced pollen in Japanese persimmon (Diospyros kaki Thunb.) [J]. Hort Science, 2005, 40 (1): 24-28), Dioscorea parviflora (Dong Zhiyuan, Guo Huachun. Research on artificial induction of unreduced pollen in Dioscorea parviflora [J]. Western Forestry Science, 2008, (03): 6-12), Brassica L. hybrids (Mason AS, Nelson MN, Yan G, Cowling W A. Production of viable male unreduced gametes in Brassica interspecific hybrids is genotype specific and stimulated by coldtemperatures [J]. BMC Plant Biology, 2011, 11 (1): 103) and other species have their cell chromosomes doubled and obtained a certain frequency of 2n gametes.However, since key technical parameters such as the effective treatment period, treatment temperature and continuous treatment time for inducing 2n gametes in plants by low temperature treatment are not clear, the frequency of occurrence of low temperature-induced 2n gametes is low, and there are few reports on obtaining plant triploids induced by low temperature treatment. Summary of the invention

[0007] One of the main purposes of the present invention is to provide a method for physical induction of poplar 2n pollen, which can obtain high-frequency 2n pollen, and a certain number of triploid poplar plants can be obtained by hybridization of the induced 2n pollen.

[0008] In order to achieve the above object, the technical solution adopted by the present invention includes:

[0009] A physical induction method for poplar 2n pollen comprises: using low temperature as a mutagen to induce and treat poplar pollen mother cells in a developmental stage.

[0010] In a preferred embodiment of the present invention, the low temperature may range from 8°C to -20°C, preferably from 4°C to -20°C, and most preferably is -20°C.

[0011] A preferred specific embodiment of the present invention, the poplar pollen mother cells in the development stage are poplar pollen mother cells in the meiotic period; the present invention finds that when the pollen mother cells in the male flower buds of poplars are in the meiotic period and are subjected to low-temperature induction treatment, they can induce the production of poplar 2n pollen, and the meiotic period is the period from the pachytene stage to the telophase II, which sequentially includes diakinesis, metaphase I, anaphase I, telophase I, prophase II, metaphase II, anaphase II and telophase II; wherein, compared with other treatment periods, when most of the pollen mother cells are in any of the developmental periods of diakinesis, anaphase I or metaphase II, low-temperature treatment of the male flower buds can obtain a higher frequency of 2n pollen.

[0012] In a preferred embodiment of the present invention, the induction treatment time may be 0.5h-120h, preferably 0.5h-72h, most preferably 0.5h-48h.

[0013] The present invention further combines the treatment periods of low temperature treatment and induction treatment and the induction treatment time to investigate the induction effects of different combinations on poplar 2n pollen and conducts a significant difference analysis. The results of the significant difference analysis show that when the poplar pollen mother cells are subjected to low temperature induction treatment when they are in different meiotic stages, different treatment periods have a significant effect on the 2n pollen induction rate of Populus tomentosa, and different low temperature induction treatment times also have a very significant effect on the 2n pollen induction rate of Populus tomentosa: when the poplar pollen mother cells are induced at a low temperature of 4°C, the average 2n pollen induction rate of the poplar pollen mother cells when the pollen mother cells are induced at metaphase II is significantly higher than the induction rates of other meiotic stages except diakinesis and anaphase I; the average 2n pollen induction rate of the continuous treatment for 48 hours is significantly higher than the 2n pollen induction rate of the induction treatment for 24 hours and 72 hours.

[0014] Therefore, the present invention provides an optimal induction parameter treatment combination for inducing Populus tomentosa 2n pollen at 4°C, namely: continuous induction treatment at 4°C for 48h when most of the pollen mother cells of the poplar are in metaphase II of meiosis.

[0015] The present invention found that even if the above-mentioned optimal optimization treatment combination (using 4°C low temperature continuous induction treatment for 48h when most poplar pollen mother cells are in metaphase II of meiosis) is used for induction, the 2n pollen of poplar obtained is still low, which is difficult to meet the requirements of poplar triploid breeding practice. To this end, the present invention uses a low temperature of -20°C to treat the pollen mother cells of Populus tomentosa in metaphase II for different induction times. The results show that there are significant differences in the 2n pollen induction rate of male flowering branches of Populus after different lengths of continuous treatment at -20°C. Among them, the 2n pollen induction rate of continuous treatment at -20°C for 4h is the highest, with an average of 39.17%; the 2n pollen induction rate of continuous treatment at -20°C for 0.5h is the lowest, only 10.45%. The difference significance analysis results of the 2n pollen induction rate of different time periods of continuous treatment at -20°C show that the effect of different continuous treatment times on the 2n pollen induction rate has reached extremely significant differences (F=218.69, P<0.001). The 2n pollen induction rate when treated at -20℃ for 4h or 5h was significantly higher than that when treated for 0.5h, 1h, 2h and 3h.

[0016] Therefore, the present invention provides an optimal induction parameter treatment combination for inducing 2n pollen of Populus tomentosa by low temperature of -20°C, comprising: continuous induction treatment at -20°C for 4h-5h when most of the pollen mother cells of the poplars are in metaphase II of meiosis; wherein, the 2n pollen induction rate of continuous induction treatment at -20°C for 4h when most of the pollen mother cells of the poplars are in metaphase II of meiosis is significantly higher than that of other treatments, and the average 2n pollen induction rate reaches 39.17%.

[0017] As a preferred specific embodiment of the present invention, the male flower buds of poplar described in the present invention can be male flower buds on male flower branches of poplar collected in winter and hydroponically cultivated in a greenhouse; for example, male flower branches of poplar can be collected from December of the current year to January of the next year, wherein the length of the male flower branches of poplar collected is preferably 70-90 cm, and the collected male flower branches are hydroponically cultivated at room temperature (10-25°C), and the height of the male flower branches of poplar immersed in water is preferably 30-40 cm; during the hydroponics, the water is changed every two days to keep the water used for cultivating the lower part of the flower branches clean; after the male flower buds of poplar are induced by low temperature, the flower branches are continued to be hydroponically cultivated until pollen is released, and pollen of different treatment combinations is collected.

[0018] The poplar described in the present invention includes but is not limited to any one of Populus bolleana, Populus canescens, Populus tomentosa×Populus bolleana, Populus tomentosa×Populus bolleana, Populus adenopoda, Populus alba or Populus tomentosa, and Populus tomentosa is most preferred.

[0019] The present invention utilizes low temperature treatment to induce poplar 2n pollen, which greatly improves the ratio of 2n pollen of Populus tomentosa. The male inflorescence of the poplar treated with low temperature induction can shed pollen normally and has high pollen vitality, and is suitable for hybrid pollination in cultivating triploids. Compared with the commonly used colchicine induction and high temperature induction methods, the low temperature induction of 2n pollen is simple to operate, low in cost, and relatively safe.

[0020] The present invention further optimizes the technical conditions for inducing chromosome doubling of poplar pollen by low-temperature treatment, clarifies key technical parameters such as effective treatment period, treatment temperature and continuous treatment time, obtains high-frequency poplar 2n pollen, and establishes a technical system for low-temperature-induced chromosome doubling of poplar pollen and breeding of triploids, which has important theoretical and practical significance for developing new mutagens for plant 2n gametes and constructing a new and efficient poplar triploid breeding technology system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of observation and photography of low temperature (4°C) induced 2n pollen (a) and natural 2n pollen (b) of Populus tomentosa under an optical microscope; a. low temperature induced 2n pollen (arrow); b. natural 2n pollen in the control group (arrow); scale bar = 20 μm.

[0022] Figure 2This is the pollen diameter distribution of the mid-term II treatment group and the control group of Populus tomentosa.

[0023] Figure 3 These are the photographic results of low-temperature (-20℃)-induced 2n pollen of Populus tomentosa observed under an optical microscope; A. 2n pollen treated at -20℃ for 0.5h (arrow); B. 2n pollen treated at -20℃ for 1h (arrow); C. 2n pollen treated at -20℃ for 2h (arrow); D. 2n pollen treated at -20℃ for 3h (arrow); E. 2n pollen treated at -20℃ for 4h (arrow); F. 2n pollen treated at -20℃ for 5h (arrow); Scale bar = 20μm. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the scope of protection of the present invention. Experimental Example 1 Experiment on the optimization and screening of low temperature treatment to induce 2n pollen in Populus tomentosa and induction treatment parameters

[0025] 1. Materials and Methods

[0026] 1.1 Biomaterials

[0027] After the leaves fall in winter, healthy male and female flowering branches of Populus tomentosa with full flower buds and no pests or diseases are collected from Jinsha Botanical Garden in Shuozhou, Shanxi Province. They are wrapped in plastic sheeting and transported to the greenhouse of Beijing Forestry University, where they are stored in a cool and windproof place until use.

[0028] 1.2 Optimization of the treatment period for low temperature-induced chromosome doubling of Populus tomentosa pollen

[0029] The male flowering branches of Populus tomentosa were pruned to retain flower buds of similar size on the branches and hydroponically cultured in a greenhouse at 10-20℃. Every 2h, 2-3 flower buds were randomly collected and fixed with Carnoy's fixative. The meiotic process of the flower buds was observed using the acetic acid magenta pressing method. When most of the pollen mother cells were observed to enter the pachytene, diakinesis, metaphase I, anaphase I, telophase I, prophase ⅠⅠ, metaphase ⅠⅠ, anaphase ⅠⅠ, and telophase ⅠⅠ, 6-9 male flowering branches were hydroponically cultured in a 4℃ refrigerator, and the treatment lasted for 1d, 2d, and 3d, for a total of 27 treatment combinations, and each treatment combination was numbered and recorded.

[0030] 1.3 Optimization of treatment temperature and treatment duration for low temperature treatment to induce chromosome doubling in pollen of Populus tomentosa

[0031] On the basis of determining the effective treatment period, this experiment tried to optimize it by lower treatment temperature and shorter treatment time. The specific approach is as follows:

[0032] The male flower branches of Populus tomentosa were pruned, and flower buds of similar size were retained on the branches and hydroponically cultivated in a greenhouse at 10-20℃. Every 2 hours, 2-3 flower buds were randomly collected and fixed with Carnoy's fixative, and the meiotic process of the flower buds was observed using the acetic acid magenta pressing method. When most of the pollen mother cells were observed to enter metaphase II, 6-9 male flower branches were taken and placed in a -20℃ refrigerator, and treated for 0.5h, 1h, 2h, 3h, 4h, 5h and 6h, respectively, for a total of 7 treatment combinations, and each treatment combination was numbered and recorded.

[0033] 1.4 Pollen collection

[0034] The treated male flower branches were hydroponically cultivated in the greenhouse, and the water was changed every 2-3 days. After the anthers matured and cracked, the pollens of different treatments were collected and stored in 10 mL centrifuge tubes, with appropriate amount of silica gel added, marked, sealed with plastic wrap, and stored in a -20°C refrigerator for later use.

[0035] 1.5 Statistics of 2n pollen occurrence frequency

[0036] A small amount of pollen from different treatment combinations was picked up with tweezers and evenly sprinkled on a glass slide dripped with acetic acid magenta to make a temporary smear. Five fields of view were randomly selected for observation and photography under an Olympus BX-51 optical microscope, and the pollen grain diameter was further measured using Image J software. The 1.28 times average diameter of the pollen grains in the control group was used as the standard for distinguishing 2n pollen (Zhang PD, and Kang X Y. Occurrence and cytological mechanism of numerically unreduced pollen in diploid Populus euphratica. Silvae Genet. 2013, 62(6): 285-291). The total number of pollen grains and the number of 2n pollen grains in each field of view were counted, and the percentage of 2n pollen to the total number of pollen grains was used as the frequency of 2n pollen occurrence, and the frequency of 2n pollen occurrence in different treatment combinations was calculated. The experiment was repeated 3 times, and 200-300 pollen grains were measured each time.

[0037] 1.6 Data Analysis

[0038] The percentage data of 2n pollen induction rate were first subjected to arcsine transformation, and then SPSS 20.0 statistical software was used for significant difference analysis and multiple comparisons.

[0039] 2 Experimental results

[0040] 2.1 Optimization of treatment period for low temperature-induced chromosome doubling of Populus tomentosa pollen

[0041] The pollen from the 4℃ low temperature treatment group and the control group were stained with acetic acid magenta and observed and photographed under an optical microscope. Most of the pollen grains showed a uniform red color, which indicated that the pollen grains had good vitality ( Figure 1 ). When treated at 4℃, male inflorescences of Populus tomentosa at different developmental stages could obtain a certain amount of 2n pollen ( Figure 1 a), while a small amount of naturally formed 2n pollen was also found in the control group pollen ( Figure 1 b), the occurrence frequency is (0.33±0.24)%.

[0042] Comparison of the pollen diameter distribution of the three treatment combinations and the control group in mid-term II showed that ( Figure 2 ) showed that after low temperature treatment, the distribution range of pollen diameter became wider and the number of large pollen grains increased, further confirming the effectiveness of 4℃ low temperature treatment in inducing pollen chromosome doubling to obtain 2n pollen.

[0043] The 2n pollen induction rates of each treatment combination are shown in Table 1.

[0044] Table 1 2n pollen induction rate of different low temperature (4℃) treatment combinations

[0045]

[0046]

[0047] As can be seen from Table 1, the 2n pollen induction rate was significantly different among different treatment combinations. The highest 2n pollen induction rate was obtained when the pollen mother cells were treated with 4℃ low temperature for 2 days during the development of metaphase II, and the average 2n pollen induction rate reached (9.51±1.33)%. The worst induction effect was obtained when the pachytene stage was treated with 4℃ low temperature for 1 day, and the average 2n pollen induction rate was only (1.36±1.18)%. On this basis, this experiment further analyzed the significance of the 2n pollen induction rate of different treatment combinations of low temperature treatment. The results showed that the meiotic stage had a significant effect on the 2n pollen induction rate of P. tomentosa, and the treatment days had a very significant effect on the 2n pollen induction rate of P. tomentosa, while the interaction between the treatment stage and the treatment days had no significant effect on the 2n pollen induction rate of P. tomentosa (Table 2).

[0048] Table 2 Variance analysis of 2n pollen induction rate of Populus tomentosa under different low temperature treatment combinations

[0049]

[0050] Note: ** indicates extremely significant difference (P<0.01).

[0051] Multiple comparisons were further performed on the meiotic period and treatment days, and the results of the multiple comparisons are shown in Table 3.

[0052] Table 3 Multiple comparisons of the average 2n pollen induction rate of Populus tomentosa at different meiotic stages and different treatment days

[0053]

[0054] Note: Different lowercase letters represent significant differences (P<0.05)

[0055] The results in Table 3 show that the average 2n pollen induction rate in metaphase II is significantly higher than that in other meiotic stages except diakinesis and anaphase I; the average 2n pollen induction rate in continuous treatment for 2 days is significantly higher than that in treatment for 1 day and 3 days. It can be seen that the optimal treatment combination for 4℃ low temperature inducing pollen chromosome doubling and obtaining 2n pollen in Populus tomentosa is: continuous low temperature treatment for 2 days in metaphase II of meiosis of pollen mother cells.

[0056] 2.2 Optimization of treatment temperature and treatment duration for low temperature treatment to induce chromosome doubling in pollen of Populus tomentosa

[0057] Although the above experiments determined that the optimal treatment period for low temperature treatment to induce pollen chromosome doubling of Populus tomentosa to obtain 2n pollen is metaphase II of meiosis, the proportion of 2n pollen of Populus tomentosa obtained by low temperature induction at 4°C is still low, which is difficult to meet the requirements of triploid breeding practice. The reason may be closely related to the fact that the treatment temperature is not low enough. Therefore, this experiment once again used a low temperature of -20°C to treat the pollen mother cells of Populus tomentosa in metaphase II for 0.5, 1, 2, 3, 4, and 5 hours to explore the effects of treatment temperature and duration of continuous treatment on the induction rate of 2n pollen. After the low temperature stress treatment, the flower branches continued to be hydroponically cultured in the greenhouse. It can be observed that a small number of male flower buds were killed by low temperature damage, and pollen can be collected from the surviving flower buds. After the pollen was stained with acetic acid magenta stain, most of the pollen grains appeared bright red ( Figure 3 ), indicating that the pollen grains induced by -20℃ low temperature treatment have better vitality.

[0058] The statistical results of 2n pollen induction rate of male flowering branches of Populus tomentosa at different treatment times after low temperature treatment at -20℃ are shown in Table 4.

[0059] Table 4-2n pollen induction rate at different time periods of low temperature treatment at 20℃

[0060]

[0061] Note: Different capital letters represent significant differences (P<0.01)

[0062] As can be seen from Table 4, except for the death of male flower buds of Populus tomentosa when treated with -20℃ for 7h and 8h, pollen was not collected in all other treatments. In the treatments where pollen was collected, there were significant differences in the 2n pollen induction rate of male flower branches of Populus tomentosa after different lengths of -20℃ low temperature treatment. Among them, the 2n pollen induction rate of 4h treated with -20℃ was the highest, with an average of 39.17%; the 2n pollen induction rate of 0.5h treated with -20℃ was the lowest, only 10.45%. The results of the significant difference analysis of the 2n pollen induction rate of different time treated with -20℃ showed that the effect of different treatment time on the 2n pollen induction rate reached an extremely significant difference (F=218.69, P<0.001). The 2n pollen induction rate of 4h or 5h treated with -20℃ was significantly higher than that of 0.5h, 1h, 2h and 3h treated with -20℃. Considering that the 2n pollen induction rate of -20°C low temperature treatment for 4 hours is higher than that of 5 hours, the present invention determines that the continuous treatment of pollen mother cells of Populus tomentosa in metaphase II at -20°C for 4 hours is the optimal treatment parameter combination for low temperature induction of 2n pollen of Populus tomentosa.

Claims

1. A method for inducing poplar 2n pollen, characterized in that: include: When the developmental stage of poplar pollen mother cells was in metaphase II of meiosis, -20℃ low temperature was used as an inducer to continuously induce the poplar pollen mother cells for 4h or 5h.

2. The induction method according to claim 1, characterized in that: The poplar is any one of Xinjiang poplar, silver-gray poplar, hairy new poplar, silver gland poplar, clacking leaf poplar, silver white poplar or hairy white poplar.

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