Efficient creation method of polyploid poplar

Abstract

The invention discloses an efficient creation method of polyploid poplar, and belongs to the technical field of plant breeding. According to the polyploid poplar creation method, high-temperature induction doubling is conducted 48 h after hybridization on trees and pollination are combined, nutrition supply in the seed development process is guaranteed, and the yield of hybrid filial generations is greatly increased; manual operations such as tedious temperature and humidity control, branch pruning and nutrient solution replacement in branch cutting hybridization are avoided. Experimental results show that the triploid and tetraploid hybrid offspring is successfully obtained by using the method provided by the invention, and the technical bottleneck that the black poplar polyploid is difficult to obtain is broken through. The high-temperature induction chromosome doubling method based on tree hybridization has the advantages of being easy and convenient to operate and low in cost, and an efficient solution is provided for black poplar polyploid breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to a method for efficiently creating polyploid poplar. Background Art

[0002] As an important industrial raw material forest and ecological construction tree species in my country, poplar has an irreplaceable position in the national economy and ecological construction. Research on poplar hybrid breeding in my country began in the 1940s. After nearly a century of development, a complete technical system has been established in the field of polyploid breeding of poplar factions, and rich theoretical foundations and practical experience have been accumulated. However, there are still significant deficiencies in the creation of triploids and tetraploids by hybridization between black poplar and green poplar factions, which is mainly attributed to the technical bottlenecks faced by black poplar faction ploidy breeding: first, the hybridization cycle is long, and cutting hybrids cause early fruit drop due to insufficient nutrient supply; second, the seed harvest is small, and physical or chemical treatments often cause damage to the fruit sequence, resulting in no grain harvest; third, cutting hybridization must inevitably carry out cumbersome temperature and humidity control, branch pruning, and nutrient solution replacement. Therefore, it is urgent to provide a method for creating polyploids by efficient hybridization between black poplar and green poplar. Summary of the invention

[0003] The purpose of the present invention is to provide an efficient method for creating polyploid Populus nigra to solve the problems existing in the above-mentioned prior art. The high-temperature chromosome doubling method based on tree hybridization proposed in the present invention has the advantages of simple operation and low cost, and provides an efficient solution for polyploid breeding of Populus nigra.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a method for creating a polyploid poplar, comprising the following steps:

[0006] Collect pollen from the male poplar tree;

[0007] The female inflorescences of the female poplar trees were bagged;

[0008] After the female inflorescence matures, the pollen is used for on-tree hybridization pollination;

[0009] The female inflorescence after cross pollination on the tree is subjected to high temperature induction, hybrid seeds are collected, sown and seedlings are raised, and the ploidy of the offspring is detected to obtain the polyploid poplar.

[0010] Preferably, the high temperature induction is a treatment at 39-41°C for 2-4 hours 48-96 hours after cross pollination on the tree.

[0011] Preferably, the high temperature induction is 60 hours after cross pollination on the tree, and the treatment is at 41°C for 4 hours.

[0012] Preferably, the high temperature induction is a treatment at 39° C. for 2 hours 96 hours after cross pollination on the tree.

[0013] Preferably, the male poplar is a Populus alba branch; and the female poplar is a Populus nigra branch.

[0014] Preferably, the poplar of the Qingyang School is Populus simonii Carr.; and the poplar of the Black Poplar School is Populus×canadensis.

[0015] Preferably, the female inflorescence matures into a female styla in the middle of the female inflorescence that is shiny and produces mucus.

[0016] The present invention also provides an application of the polyploid poplar obtained according to the above method in hybridization to prepare polyploid poplar.

[0017] The present invention also provides an application of the polyploid poplar obtained according to the above method in poplar improvement and breeding.

[0018] Preferably, the target traits for improved breeding include plant height, ground diameter, leaf size and stoma size.

[0019] The present invention discloses the following technical effects:

[0020] The present invention adopts the on-tree hybridization technology, which effectively guarantees the nutrient supply during the seed development process and greatly improves the yield of hybrid offspring. At the same time, the doubling method provided by the present invention avoids the cumbersome manual operations such as temperature and humidity control, branch pruning and nutrient solution replacement in cutting hybridization. By optimizing the temperature-time coordinated regulation model, a batch of triploid and tetraploid hybrid offspring were successfully obtained, breaking through the technical bottleneck of the difficulty in obtaining black poplar polyploids. The high-temperature induced chromosome doubling method based on on-tree hybridization proposed by the present invention has the advantages of simple operation and low cost, and provides an efficient solution for black poplar polyploid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the result of the ploidy detection instrument identification of the diploid progeny of Populus simonii;

[0023] Figure 2 This is the result of the ploidy detection instrument identification of the triploid progeny of Populus simonii;

[0024] Figure 3 This is the result of the identification of the ploidy detection instrument for the tetraploid progeny of Populus jiajiao×Populus simonii;

[0025] Figure 4 The results of root tip compression of different ploidy progenies of Populus simonii are shown in Figure 1. A is diploid; B is triploid; C is tetraploid;

[0026] Figure 5 This is a statistical comparison of leaf seedling heights of different ploidy progeny of Populus simonii; ** indicates P < 0.01;

[0027] Figure 6 This is a statistical comparison of the leaf diameters at ground level of the progeny with different ploidy of Populus × Populus simonii; ** indicates P < 0.01;

[0028] Figure 7 This is a statistical comparison of leaf length and width of different ploidy progeny of Populus simonii; ** indicates P < 0.01;

[0029] Figure 8 This is a statistical comparison of leaf area of ​​different ploidy progeny of Populus simonii; ** indicates P < 0.01;

[0030] Fig. 9 This is a comparative observation diagram of leaves of different ploidy progeny of Populus xiaoye; A is diploid; B is triploid; C is tetraploid;

[0031] Fig.10 This is a statistical comparison of the stomatal length of the progeny with different ploidy of Populus simonii; ** indicates P < 0.01;

[0032] Fig.11 These are microscopic observations comparing the stomatal density of progenies of Populus simonii with different ploidy. A is a diploid; B is a triploid; C is a tetraploid; the scale bar is 50 μm. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] 1. Experimental methods

[0040] 1. High temperature induces chromosome doubling in Populus

[0041] (1) In mid-March, male flower branches of Populus simonii Carr. with full male flower buds, free of pests and diseases, and strong were collected and stored at low temperature. In late March, the male flower branches were hydroponically cultivated indoors, and pollen was collected using sulfuric acid paper after it matured. After the collected pollen was freed of impurities and dried at room temperature for two days, it was placed in a desiccant and stored at low temperature for a short period of time for subsequent pollination.

[0042] (2) The female plants of Populus × canadensis on the campus of Northeast Forestry University were used as the female parent. When the female flowers just broke through the bud scales, the female inflorescences were isolated by bagging with non-woven hybridization bags.

[0043] (3) When the female stigma in the middle of the female inflorescence becomes shiny and produces mucus, pour an appropriate amount of pollen into the hybridization bag, tie the bag tightly, and shake the hybridization bag to ensure sufficient pollination, thus completing the on-tree hybridization pollination.

[0044] (4) Conducting high temperature tolerance tests on female inflorescences and analyzing the embryo sac development process

[0045] (5) After cross-pollination on the tree, a constant temperature induction treatment was carried out using a constant temperature box developed by Professor Kang Xiangyang of Beijing Forestry University (see patent CN1669408A). 48 hours after cross-pollination on the tree, the megaspore development stage is basically over and enters the embryo sac development stage. Five time points were set at 48 hours, 60 hours, 72 hours, 84 hours and 96 hours after pollination, using two temperature levels of 39°C and 41°C, and two treatment durations of 2 hours and 4 hours (Table 1) to construct an orthogonal experimental design. At least 5-7 female inflorescences were treated for each treatment combination to ensure the reliability of the test results.

[0046] (6) 120 hours after pollination, remove the non-woven bag.

[0047] (7) Before the capsules crack, they are bagged and the seeds are collected. The collected seeds are free of seed fluff and sown for seedling raising.

[0048] Table 1 High temperature induced orthogonal table

[0049]

[0050] 2. Ploidy detection of hybrid offspring

[0051] If the number of seeds in the treatment group was less than 300, all seeds were sown. For the treatment group with more than 300 seeds, 300 seeds were randomly selected and sown uniformly in the seedling tray. After the cotyledons grew, all the progeny were transplanted into 10 cm × 10 cm nutrient pots at the same time. After the seedlings grew true leaves, their ploidy was identified using a ploidy detector (CyFlowPloidyAnalyser produced by Sysmex Partec GmbH).

[0052] Cut fresh leaf samples of about 1 cm in length, rinse them with distilled water, place them in a glass dish, and add a buffered extract solution (Cystain produced by Sysmex Partec GmbH) that has been pre-treated in an ice bath. TM UV Precise P Kit) 750 μL, then quickly mince the leaves with a sharp blade, and then add 750 μL of buffered extraction solution (Cystain produced by SysmexPartec GmbH) TM UV Precise P kit) and 1 μL of staining reagent (Cystain TM UV Precise P kit) was used to filter the obtained leaf mixture through a 30 μm pore size microporous filter membrane into a sampling test tube. Then, after ensuring sufficient mixing, it was allowed to stand for staining for 5 minutes. Finally, the ploidy was accurately detected and identified using a ploidy detector.

[0053] The root apex was pressed to identify its ploidy. The sample was taken at 9:00 in the morning and fixed in Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1) for 24 hours. The fixed root apex was then placed in 70% ethanol and stored at 4°C for later use. The fixed root apex was thoroughly rinsed with distilled water for 5-6 times and then treated with 1 mol·L -1 Treat in HCl (8.33mL concentrated hydrochloric acid is made up to 100mL) for 10-12min, rinse thoroughly 5-6 times, and stain with Carbol fuchsin dye for 9-12h; press slide for microscopic examination, cut the root apex of about 0.5mm with a blade and place it on a slide, place the cover slip on the slide and press slide, use the traditional root apex pressing method to prepare the slide, and use a 10×100x optical microscope to observe and count the number of chromosomes.

[0054] 3. Investigation of polyploid offspring growth and leaf traits

[0055] 16, 28 and 11 diploid, triploid and tetraploid offspring of the hybrid Populus simonii were selected respectively. After the offspring seedlings stopped growing, the height, ground diameter and other indicators of the offspring seedlings were investigated using a tower ruler and a vernier caliper.

[0056] Take the 3rd to 5th mature functional leaves below the morphological top of the progeny seedlings, and evenly apply transparent nail polish on the lower epidermis of the leaves to fix the stomata. After the nail polish is completely dry, gently stick colorless transparent tape on the leaves coated with nail polish and cover them evenly. Press gently with your hands to make them fit tightly, then carefully tear off the tape, cover with a coverslip, place under an ordinary optical microscope to observe and measure the length and width of the stomata in the field of view, and make 30 biological replicates for each sample to investigate the stomatal size.

[0057] The 6th to 8th mature leaves below the morphological top of the progeny seedlings were taken, stored in plastic bags, numbered, and then scanned with a scanner. The leaf size was measured using image J software, with 30 biological replicates for each sample.

[0058] The statistics and analysis of the data were completed using SPAA and Excel software, and the graphics were drawn using Origin.

[0059] (II) Results and analysis

[0060] 1. High temperature induced chromosome doubling and determination of the optimal induction period

[0061] The female inflorescences of the hybrid of Populus xiaoye were treated with high temperature induction at different temperatures (39℃, 41℃) and durations (2h, 4h) 48h, 60h, 72h, 84h and 96h after pollination. The seed yield, seedling survival rate after sowing, and triploid and tetraploid induction rates were statistically analyzed. The results are shown in Tables 2 and 3. The analysis showed that triploids were successfully induced at the five time points from 48h to 96h after pollination. Among them, the seed yield of the combination treated at 41℃ for 4h at 60h after pollination was the lowest (19 seeds). There was no significant difference in the seed germination rate of each treatment combination (all > 70%), indicating that the nutrient supply was sufficient during the experiment and the seeds developed well.

[0062] The surviving seedlings were tested by ploidy detector ( Figure 1-Figure 3 ) and karyotype analysis ( Figure 4 ) found that the combination of 60h after pollination and 41℃ treatment for 4h had the highest triploid induction rate (5.88%). In addition, the combination of 96h after pollination and 39℃ treatment for 2h also showed a high induction rate (3.09%). It is worth noting that tetraploid plants were detected within the time range of 48h to 96h after pollination (Tables 2 and 3), indicating that high temperature treatment can induce the production of triploids and tetraploids at the same time.

[0063] Table 2 Statistics of germination rate and induction rate of hybrid seeds of Populus xiaoye at 39℃

[0064]

[0065] Table 3 Statistics of germination rate and induction rate of 41℃ hybrid poplar × Populus simonii

[0066]

[0067]

[0068] 2. Evaluation of different ploidy progeny of Populus simonii at seedling stage

[0069] Through the ploidy detection of the hybrid progeny of Populus japonici, a total of 28 triploids and 11 tetraploids were detected. The obtained triploids, tetraploids and their diploids were used to perform variance analysis on seedling height, ground diameter, leaf length, leaf width, leaf area, and stoma length and width. The results showed that the seedling height showed extremely significant differences among different ploidies (P<0.01). The triploids and tetraploids were significantly lower than the diploids, which were 21.13% and 29.82% lower than the diploids, respectively ( Figure 5 ); the ground diameter of the diploid offspring was significantly different from that of the triploid and tetraploid offspring (P<0.01), which was 11.58% and 18.15% higher, respectively. There was no significant difference between the triploid and tetraploid offspring ( Figure 6); the leaf lengths of different ploidy species showed extremely significant differences (P<0.01), and the leaf lengths of triploid and tetraploid offspring were 6.38% and 11.99% higher than those of diploid offspring ( Figure 7 ); the leaf width of diploid and tetraploid plants showed a very significant difference (P<0.01), and the leaf width of tetraploid plants was significantly higher than that of diploid plants ( Figure 7 ); The leaf area of ​​diploid was significantly lower than that of triploid and tetraploid, which was 13.82% and 24.80% lower than that of triploid and tetraploid, respectively ( Figure 8 and Fig. 9 ).

[0070] By detecting and analyzing the stomatal characteristics of leaves of different ploidy progeny of Populus simonii ( Figure 10-11 ), the results showed that the stomatal size between different ploidy showed extremely significant differences (P<0.01), the stomatal length and width of triploid and tetraploid were greater than those of diploid, which were 11.15%, 17.02% and 10.49%, 23.49% higher than those of diploid, respectively. By observing the stomatal distribution of different ploidy offspring, it can be seen that the stomatal density of diploid is the largest, and that of tetraploid is the smallest, and the stomatal density from large to small is diploid, triploid and tetraploid.

[0071] 3. Conclusion

[0072] 1. According to the embryo sac development process, the female gamete chromosome doubling technology was studied at five time points: 48h, 60h, 72h, 84h and 96h after hybrid pollination on the tree. It was finally determined that the best induction condition for Populus japonici was a high temperature treatment of 41°C for 4h 60h after pollination, and the induction rate could reach 5.88%.

[0073] 2. A variance analysis was performed on the seedling height, ground diameter, leaf size, stoma size and other traits of the offspring with different ploidy. It was found that the seedling height, leaf size and stoma size among the offspring with different ploidy reached a significant level, and the seedling height and ground diameter of the triploid and tetraploid were smaller than those of the diploid, while the leaf size and stoma size were larger than those of the diploid.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for creating polyploid poplars, characterized in that: The following steps are involved: Collect pollen from the male poplar tree; The female inflorescences of the female poplar trees were bagged; After the female inflorescence matures, the pollen is used for on-tree hybridization pollination; The female inflorescence after cross pollination on the tree is subjected to high temperature induction, hybrid seeds are collected, sown and seedlings are raised, and the ploidy of the offspring is detected to obtain the polyploid poplar.

2. The method according to claim 1, characterized in that The high temperature induction is 48-96 hours after cross pollination on the tree, and the treatment is carried out at 39-41°C for 2-4 hours.

3. The method according to claim 2, characterized in that The high temperature induction is 60 hours after the cross pollination on the tree, and the treatment is carried out at 41°C for 4 hours.

4. The method according to claim 2, characterized in that The high temperature induction is 96 hours after cross pollination on the tree, and the treatment is carried out at 39°C for 2 hours.

5. The method according to claim 1, characterized in that The male parent poplar is a Populus alba branch poplar; the female parent poplar is a Populus nigra branch poplar.

6. The method according to claim 5, characterized in that The poplar tree of the Qingyang School is Populus simonii Carr.; the poplar tree of the Black Poplar School is Populus×canadensis.

7. The method according to claim 1, characterized in that The female inflorescence matures into a female styla in the middle of the female inflorescence that is shiny and produces mucus.

8. Use of a polyploid poplar obtained by the method according to any one of claims 1 to 7 in hybridization to prepare a polyploid poplar.

9. Use of the polyploid poplar obtained by the method according to any one of claims 1 to 7 in poplar improvement and breeding.

10. The use according to claim 9, characterized in that The target traits of the improved breeding include plant height, ground diameter, leaf size and stoma size.

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