Creation method of populus nigra triploid

Abstract

The invention discloses a method for creating poplar triploid, and belongs to the technical field of poplar polyploid induction. The creation method comprises the following steps: performing cross pollination on female flower buds of populus japonica by using pollen of a male parent of a poplar to obtain a female inflorescence, treating the female inflorescence at 39-41 DEG C for 2-4 hours, managing the inflorescence, harvesting seeds, sowing and raising seedlings, and identifying seedlings to obtain the populus japonica triploid plant. According to the method, on the basis of systematically analyzing the development process of the female gametes of the populus japonica, a high-temperature induced female gamete chromosome doubling technology is innovatively adopted, a populus japonica female gamete chromosome doubling induction system is successfully constructed, the triploid induction rate is increased by 63.95%, and a solid foundation is laid for populus japonica triploid germplasm creation.

Description

Technical Field

[0001] The invention relates to the technical field of poplar polyploid induction, in particular to a method for creating a triploid Populus ussuriensis. Background Art

[0002] Populus ussuriensis is a native tree species unique to Northeast my country. It has strong cold resistance (can tolerate low temperatures of -40°C) and wide ecological adaptability (can grow from humid mountains to semi-arid plains), and plays an irreplaceable role in the construction of regional ecological barriers and short-cycle industrial timber forests. However, the increased frequency of droughts and extreme low temperature fluctuations caused by global climate change pose a severe challenge to the growth stability of traditional Populus ussuriensis germplasm.

[0003] Chromosome doubling technology significantly improves the stress resistance and rapid growth of trees through the genome dosage effect, and achieves a synergistic improvement in drought resistance and rapid growth through cell structure remodeling (such as thickened cuticle and developed xylem vessels). This synergistic enhancement of "stress resistance-rapid growth" traits makes triploid Populus cathayana a key germplasm resource for meeting the needs of ecological restoration and low-carbon economy in semi-arid areas.

[0004] The triploid induction schemes for different poplar varieties need to take into account their respective reproductive development characteristics, hybrid combinations and environmental conditions, so the induction schemes between different poplar varieties cannot be directly applied. At present, the triploid induction of Populus ussuriensis mainly relies on the male gamete doubling technology, but due to the weak competitiveness of 2n pollen in the pollination process, the triploid induction rate is only 0.22%. Therefore, how to overcome this technical bottleneck, successfully construct the female gamete chromosome doubling induction system of Populus ussuriensis, and then improve the triploid induction rate, and obtain triploid Populus ussuriensis is of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a method for creating a triploid Populus ussuriensis to solve the problems existing in the above-mentioned prior art. The present invention adopts high temperature induced female gamete chromosome doubling technology to successfully construct a female gamete chromosome doubling induction system of Populus ussuriensis, with an induction rate of 14.29%, which is 63.95% higher than the induction rate of 0.22% achieved by the triploid Populus ussuriensis in the prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A method for creating a triploid Populus ussuriensis, comprising the step of placing the pollinated female inflorescence at 39-41°C for 2-4 hours.

[0008] Furthermore, the steps include: cross-pollinating female flower buds of Populus ussuriensis with pollen from a male poplar parent to obtain female inflorescences, treating the female inflorescences at 39-41°C for 2-4 hours, managing the infructescences, harvesting seeds, sowing and raising seedlings, and identifying the seedlings to obtain the triploid Populus ussuriensis plants.

[0009] The pollen of the male poplar parent needs to be collected in advance. The collection includes first hydroponically cultivating the male flower branches indoors, collecting the pollen when it matures, removing impurities from the collected pollen, drying it in a dryer for one day, and then storing it at low temperature for a short period of time for subsequent pollination.

[0010] Furthermore, the poplar male parent is European black poplar.

[0011] Furthermore, the female inflorescence is a female inflorescence 48-96 hours after hybrid pollination.

[0012] The hybrid pollination is as follows: each female flower branch preserves 1-2 full female flower buds, and is hydroponically cultivated in a plastic greenhouse, where the temperature is maintained at 18-25° C.; after the female flowers have just broken through the bud scales, they are bagged to prevent premature pollination; after the female stigma is shiny and produces mucus, a proper amount of male flower pollen is dipped in a brush for pollination;

[0013] Furthermore, the female inflorescence is the female inflorescence 48 hours after hybrid pollination.

[0014] Furthermore, the treatment temperature of the female inflorescence is 41°C.

[0015] Furthermore, the treatment time of the female inflorescence is 2 hours.

[0016] Furthermore, the identification is to identify the chromosome ploidy of the seedlings.

[0017] The identification is to use a ploidy detector to detect chromosome ploidy after the seedlings grow true leaves.

[0018] The present invention discloses the following technical effects:

[0019] The present invention adopts high temperature induced female gamete chromosome doubling technology, and successfully constructs a female gamete chromosome doubling induction system of Populus ussuriensis, with an induction rate of 14.29%, laying a solid foundation for the creation of Populus ussuriensis triploid germplasm. The high temperature induced female gamete chromosome doubling technology system of the present invention achieves a double breakthrough in triploid induction efficiency and stability through object innovation, parameter optimization and species adaptability research, and provides a new paradigm of efficient, safe and replicable polyploid breeding of poplars, which is of milestone significance for forestry germplasm resource innovation and ecological engineering construction. It has important theoretical and practical significance for the construction of a new and efficient poplar triploid breeding technology system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 These are pictures of different embryo sac development stages of Populus ussuriensis (20 μm); A is a mononuclear embryo sac; B is a dinuclear embryo sac; C is a tetranuclear embryo sac; D is an octanuclear embryo sac;

[0022] Figure 2 This is the result of flow cytometry identification of diploids;

[0023] Figure 3 This is the result of flow cytometry identification of triploids;

[0024] Figure 4 The results of root apex compression examination, where A is diploid; B is triploid;

[0025] Figure 5 The height of the offspring of Populus alba × Populus nigra with different ploidy;

[0026] Figure 6 The ground diameter of the offspring with different ploidy of Populus alba × Populus nigra;

[0027] Figure 7 The leaf area of ​​the offspring of Populus ussuriensis×Populus nigra with different ploidy;

[0028] Figure 8 The leaf length of the offspring of Populus ussuriensis×Populus nigra with different ploidy;

[0029] Fig. 9 This is a comparison of leaf sizes of different ploidy offspring of Populus alba × Populus nigra, where A is diploid and B is triploid;

[0030] Fig.10 Comparison of stomatal length and width in different ploidy progenies of Populus ussuriensis×Populus nigra;

[0031] Fig.11 Comparison of stomatal density of progenies with different ploidy of Populus ussuriensis×Populus nigra (50 μm), among which A is diploid and B is triploid. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The female flowering branches of Populus alba were collected from Mohe, Daxinganling, Heilongjiang Province; the male flowering branches of Populus nigra were collected from Manas Plain Forest Farm in Xinjiang.

[0038] Example 1

[0039] 1. Experimental Methods

[0040] 1. Cutting hybridization and chromosome doubling

[0041] (1) In mid-March, female branches of Populus dasyphylla and male branches of Populus nigra with full flower buds and no pests and diseases, and 2-3 cm in diameter, were collected and stored at low temperature. In late March, male branches were hydroponically cultivated indoors and pollen was collected when it was mature. The collected pollen was free of impurities, placed in a desiccator for one day, and then stored at low temperature for a short period of time for subsequent pollination;

[0042] (2) In late April, prune the female flower branches, keep 1-2 full female flower buds on each branch, and hydroponically cultivate them in a plastic greenhouse, with the temperature in the greenhouse maintained at 18-25°C;

[0043] (3) Bag the female flowers of Populus ussuriensis just after they break through the bud scales to prevent premature pollination;

[0044] (4) After the female stigma becomes shiny and produces mucus, use a brush to pick up an appropriate amount of pollen for pollination;

[0045] (5) Conduct a test on the high temperature tolerance of the female inflorescence of Populus ussuriensis and analyze the embryo sac development process;

[0046] (6) After pollination, the embryo sac was induced by high temperature using a thermostat invented by Professor Kang Xiangyang of Beijing Forestry University (patent number ZL200510011598.5). At 48 h after pollination, the megaspore development stage was basically completed and the embryo sac development stage entered. The orthogonal experimental factors and levels for doubling the embryo sac after pollination were set at 48 h, 60 h, 72 h, 84 h, and 96 h after pollination, and were treated at 39 ° C and 41 ° C for 2 h and 4 h, respectively (Table 1). At least 5 female inflorescences were treated for each treatment combination;

[0047] Table 1 High temperature induced orthogonal table

[0048]

[0049] (7) After treatment, continue to hydroponically culture the cut flower branches, change the water every 3 days, and properly clean and trim the lower part of the branches. Add 1 / 2MS culture medium and rooting powder to the water to ensure that the hydroponics branches receive sufficient nutrient supply until the capsule matures and cracks to release seeds.

[0050] 2. Ploidy detection of hybrid offspring

[0051] (1) Before the capsule cracks, bag the seeds and collect them. Remove the seed fluff from the collected seeds, place them in a desiccator, and store them at 4°C. Sow all the seeds in a seedling tray. After the cotyledons grow, all the progeny are transplanted into 10 cm × 10 cm nutrient pots at the same time. After the seedlings grow true leaves, use a ploidy detector (CyFlow PloidyAnalyser produced by Sysmex Partec GmbH) to identify their ploidy.

[0052] (2) Cut a fresh leaf sample of about 1 cm in length, rinse it with distilled water, place it 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 μm, the leaves were quickly minced with a sharp blade, and then 750 μm of buffered extraction solution and 1 μm of staining reagent (Cystain produced by Sysmex Partec GmbH) were added. TMUV Precise P kit), filter the obtained leaf mixture through a 30μm pore size microporous filter membrane into a sampling test tube. Then, ensure that it is fully mixed and let it stand for 5 minutes. Finally, use a ploidy detector to accurately detect and identify its ploidy;

[0053] (3) Use root apex compression to identify its ploidy. Samples were collected 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 placed in 70% mass concentration ethanol at 4°C for later use. The fixed root apex was rinsed 6 times with distilled water, treated with 1mol·L-1HCL (8.33mL concentrated hydrochloric acid to 100mL) for 12 minutes, rinsed 6 times, and stained with Carbofuxin dye for 12 hours; the root apex was cut with a blade and placed on a slide. The cover glass was placed on the slide and the slide was pressed. The traditional root apex compression method was used to prepare the slide. The number of chromosomes was observed and counted under a 10×100 optical microscope.

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

[0055] (1) Select diploid and triploid plants of the hybrid offspring of Populus ussuriensis × Populus nigra, and after the offspring seedlings stop growing, use a pyramid ruler and vernier caliper to investigate the offspring seedling height, ground diameter and other indicators;

[0056] (2) 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, and place under an ordinary optical microscope to observe and measure the length and width of the stomata in the field of view. Make 30 biological replicates for each sample to investigate the stomatal size;

[0057] (3) Take the 6th to 8th mature leaf below the morphological top of the progeny seedling, store it in a plastic bag, number it, and then scan the leaf with a scanner. Use image J software to measure the leaf size. Each sample has 30 biological replicates.

[0058] (4) Data statistics and analysis were completed using SPAA and Excel software, and graphics were drawn using Origin.

[0059] 2. Experimental Results

[0060] 1. Cytological observation of embryo sac development process and statistics of development period of Populus ussuriensis

[0061] The three mitosis during embryo sac development provides a critical window period for artificially induced chromosome doubling. Through systematic statistics of the embryo sac development process (Table 2), it was found that 48 hours after pollination, the megaspore period was nearly completed (accounting for 14.29%), most embryo sacs were in the mononuclear (29.85%) and dinuclear (32.29%) stages, and octanuclear embryo sacs began to appear at the same time (6.90%). 60 hours after pollination, mature embryo sacs began to form, and the inducible period (mononuclear to octanuclear embryo sacs) accounted for more than 80.00%, among which the proportion of octanuclear embryo sacs increased significantly (10.23%). Cytological observations of different embryo sac development periods of Populus ussuriensis can be seen in Figure 1 .

[0062] Table 2 Statistics of embryo sac development period of Populus ussuriensis

[0063]

[0064]

[0065] Note: The inducible period is the sum of the mononuclear period, the dinuclear period and the tetranuclear period.

[0066] 72h after pollination, megaspore meiosis was completely completed, the proportion of inducible period reached a peak (84.15%), and the proportion of two-nuclear embryo sac rose to the highest (35.36%). At 84h after pollination, embryo sac development was further advanced, the proportion of eight-nuclear embryo sac continued to increase (15.39%), the proportion of mononuclear (16.67%) and two-nuclear embryo sac (25%) decreased, and the four-nuclear embryo sac reached a peak (32.25%). 96h after pollination, the proportion of mononuclear embryo sac dropped to below 10.00%, and the proportion of eight-nuclear embryo sac (28.57%) and mature embryo sac (21.84%) increased significantly.

[0067] 120h after pollination, the mononuclear and dinuclear embryo sac stages were completely completed, and the proportion of tetranuclear embryo sacs dropped to 21.43%. At 144h after pollination, the proportion of tetranuclear embryo sacs continued to decline (18.18%), and the octanuclear embryo sac reached a peak (44.44%). Finally, at 168h after pollination, all embryo sac mitosis was completed, and all embryo sacs entered the octanuclear embryo sac stage and subsequent developmental stages.

[0068] In summary, 48 hours after pollination, most embryo sacs were in the mononuclear and binuclear embryo sac stages; 96 hours after pollination, the proportion of the mononuclear embryo sac period dropped to below 10.00%, and the proportion of the eight-nuclear embryo sac and mature embryo sac periods increased to 28.57% and 21.84%, respectively, indicating that some embryo sacs had completed development.

[0069] 2. Triploid induction system

[0070] The female inflorescences of Populus ussuriensis × Populus nigra hybrids were subjected to high temperature induction treatments at different temperatures (39°C, 41°C) and durations (2h, 4h) 48h, 60h, 72h, 84h and 96h after pollination. The seed yield, seedling survival rate and triploid induction rate were statistically analyzed. The results are shown in Tables 3 and 4.

[0071] Table 3 Statistics of germination rate and induction rate of inflorescences of hybrids of Populus dasyphylla × Populus nigra after pollination at 39℃

[0072]

[0073] Table 4 Statistics of germination rate and induction rate of inflorescences of hybrids of Populus dasyphylla × Populus nigra after pollination at 41℃

[0074]

[0075]

[0076] The results showed that triploids were successfully induced at three time points: 60h, 72h and 84h after pollination. The number of progeny seedlings in different treatment combinations was significantly different. Among them, the seed yield of the combination treated at 39℃ for 4h after 96h pollination was the highest (125 seeds), while no seeds were obtained in the combination treated at 48h, 60h, 72h and 41℃ for 4h after pollination. It is speculated that this is due to the long ripening period of P. ussuriensis capsules, insufficient nutrient supply of hydroponic cuttings, and damage to the female inflorescence caused by high temperature treatment.

[0077] Germination rate analysis showed that the germination rates of the combinations of 72h, 41℃ treatment for 2h, 96h, 39℃ treatment for 2h, 84h, 39℃ treatment for 4h, 96h, 41℃ treatment for 2h and 4h after pollination all reached 100%. Figure 2-Figure 4 ) found that the combination of 72h after pollination and 41℃ treatment for 2h (72h / 41℃ / 2h) had the highest triploid induction rate, reaching 14.29%, which was significantly better than other treatment combinations.

[0078] 3. Evaluation of triploid progeny of Populus dasyphylla × Populus nigra at seedling stage

[0079] Variance analysis was performed on 9 5-month-old triploid and diploid plants for seedling height, ground diameter, leaf length, leaf width, leaf area, leaf size, stomatal length, and stomatal width. The results showed that the seedling height of triploid and diploid plants showed extremely significant differences (P<0.01). The seedling height of triploid was significantly lower than that of diploid, which was 22.83% lower than that of diploid. The seedling height of progeny with different ploidy was as follows: Figure 5 As shown; there is no significant difference in ground diameter, and the ground diameter of the triploid offspring is slightly lower than that of the diploid offspring ( Figure 6 ); blade length( Figure 8 )、Blade Width( Fig. 9) and leaf area ( Figure 7 ) were significantly higher than those of the diploid offspring, which were 8.73%, 27.57% and 26.89% higher than those of the diploid offspring, respectively.

[0080] The analysis of stomatal size of triploid and diploid hybrid offspring of Populus ussuriensis showed that the stomatal length and stomatal width of triploid offspring showed extremely significant differences (P<0.01). The stomatal length and stomatal width of triploid offspring were significantly higher than those of diploid offspring, which were 12.32% and 13.43% higher than those of diploid offspring, respectively (P<0.01). Fig.10 ), and found that the density of triploids was more than 1 times higher than that of diploids ( Fig.11 ).

[0081] In summary, the present invention clarifies the cytological characteristics of the embryo sac development period of Populus ussuriensis, and statistically analyzes the proportion of each development period, and finds that the cytological development of the embryo sac is asynchronous, with up to 5 development periods existing simultaneously. Megaspore development ends 48 hours after pollination, and the megaspore meiosis period ends completely 72 hours after pollination, which is the period with the highest induction rate of Populus ussuriensis triploid, and the proportion of the inducible period reaches a maximum of 84.15%. At 96 hours after pollination, part of the embryo sac is developed.

[0082] According to the embryo sac development process, the invention studies the female gamete chromosome doubling technology at five time points, namely 48h, 60h, 72h, 84h and 96h after pollination, and finally determines that the optimal induction condition for Populus ussuriensis is: applying a high temperature treatment of 41°C for 2h 72h after pollination, and the induction rate can reach 14.29%.

[0083] The present invention also conducts variance analysis on the seedling height, ground diameter, leaf size, stoma size and other traits of the triploid offspring induced by Populus ussuriensis × Populus nigra, and finds that the seedling height, leaf size and stoma size among the offspring with different ploidy all reach a significant difference level, and the seedling height and ground diameter of the triploid are both smaller than those of the diploid, while the leaf size and stoma size are both larger than those of the diploid.

[0084] 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 producing triploid Populus ussuriensis, characterized in that: The method comprises the step of placing the pollinated female inflorescence at 39-41° C. for 2-4 hours.

2. The method according to claim 1, characterized in that: The steps include: cross-pollinating female flower buds of Populus ussuriensis with pollen of a male poplar parent to obtain female inflorescences, treating the female inflorescences at 39-41°C for 2-4 hours, managing the infructescences, harvesting seeds, sowing and raising seedlings, and obtaining the triploid Populus ussuriensis plants after identifying the seedlings.

3. The method according to claim 2, characterized in that: The poplar male parent is European black poplar.

4. The method according to claim 2, characterized in that: The female inflorescence is the female inflorescence 48-96 hours after cross pollination.

5. The method according to claim 2, characterized in that: The female inflorescence is the female inflorescence 48 hours after cross pollination.

6. The method according to claim 2, characterized in that: The treatment temperature of the female inflorescence was 41°C.

7. The method according to claim 2, characterized in that: The treatment time of the female inflorescence is 2 hours.

8. The method according to claim 2, characterized in that: The identification is to identify the chromosome ploidy of the seedlings.

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