Method for improving tobacco interspecific hybridization maturing rate through plant hormone treatment

Abstract

The invention discloses a method for improving the tobacco interspecific hybridization maturing rate through plant hormone treatment, and belongs to the technical field of crop breeding. The method comprises the following steps: selecting tobacco parents with consistent flowering phases for interspecific hybridization, and applying GA3 to stigmas within 20 minutes before pollination; 1 day after pollination, IAA is used for spraying pistils; 3 days after pollination, 6-BA is sprayed to pistils; cultivated tobacco is used as a female parent in the tobacco parents. According to the method, the pistil of the tobacco female parent is treated by different hormones in three stages before and after pollination, and the three different hormones are combined to take effect and play a role together, so that the aim of improving the tobacco interspecific hybridization setting rate is fulfilled, and the method has important value in the field of tobacco interspecific hybridization breeding.

Description

Technical Field

[0001] The invention relates to the technical field of crop breeding, and in particular to a method for improving the fruit setting rate of interspecific hybridization of tobacco by treating with plant hormones. Background Art

[0002] Excellent varieties are the basis of tobacco production and directly affect the quality and yield of tobacco. Therefore, when improving the production efficiency of tobacco, its inherent genetic factors must be fully considered. At present, the varieties of tobacco production in my country are relatively concentrated, facing many problems such as variety degeneration, reduced disease resistance and stress resistance, and convergence of tobacco quality. It is urgent to solve the difficulties such as the lack of excellent varieties and the single variety structure. In addition, the quality characteristics of the main flue-cured tobacco varieties are mostly derived from varieties such as G28, K326, Honghua Dajinyuan and NC82. The genetic basis of these varieties is derived from the original species of common tobacco, Orinoco, resulting in insufficient diversity of genetic resources within the species. Wild tobacco species have accumulated abundant resistance genes in the process of evolution, forming unique aroma and special quality, such as the resistance of Nicotiana aplumbaginifolia and Nicotiana alongiflora to black shank, the resistance of Nicotiana glutinosa to mosaic disease, the resistance of Nicotiana rustica to bacterial wilt, and the aromatic smell of Nicotiana alata, which are important gene banks for breeding new tobacco varieties and genetic improvement. However, due to the long-term temporal and spatial isolation between different species, there is reproductive isolation between many wild species and cultivated varieties, and there is often hybrid incompatibility when interspecific hybridization is carried out. Conventional hybridization methods have problems such as failure to set fruit and low fruit setting rate, which seriously limits the effective utilization of wild tobacco germplasm resources.

[0003] Therefore, it is urgent to develop a hybridization method that can improve the fruit set rate of interspecific hybridization in tobacco and promote the effective utilization of wild tobacco germplasm resources. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the fruit setting rate of tobacco interspecific hybridization by using plant hormone treatment to solve the problems existing in the above-mentioned prior art. The present invention selects three stages before and after pollination to treat the pistil of the tobacco mother plant with different hormones, and the three different hormone treatments are combined to take effect and work together, thereby achieving the purpose of improving the fruit setting rate of tobacco interspecific hybridization, which has important value in the field of tobacco interspecific hybridization breeding.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for improving the fruit setting rate of interspecific hybridization of tobacco by using plant hormone treatment, comprising the following steps:

[0007] Select tobacco parents with the same flowering period for interspecific hybridization, apply GA3 to the stigma within 20 minutes before pollination; spray the pistil with IAA one day after pollination; and spray the pistil with 6-BA three days after pollination;

[0008] Among the tobacco parents, cultivated tobacco is used as the female parent.

[0009] Optionally, the concentration of GA3 is 200-300 mg / L; the concentration of IAA is 100-200 mg / L; and the concentration of 6-BA is 15-25 mg / L.

[0010] Optionally, the concentration of GA3 is 250 mg / L; the concentration of IAA is 200 mg / L; and the concentration of 6-BA is 20 mg / L.

[0011] Optionally, before pollination, the immature flower buds and flower buds as well as the opened flowers and fruits of the female parent are removed, and only the flowers in bud are retained, and the stamens are removed.

[0012] Optionally, the tobacco parents include Nicotiana rustica var. brasilia, Nicotiana glutinosa, Nicotiana longiflora and Nicotiana tabacum cv. K326.

[0013] Optionally, the female parent is the common tobacco cultivar K326.

[0014] Optionally, the tobacco parents include the Brasilia variety of Nicotiana rustica, Nicotiana glutinosa and Nicotiana longiflora as male parents.

[0015] Optionally, the tobacco parent is the Brasilia variety of Nicotiana rustica as the male parent.

[0016] The present invention discloses the following technical effects:

[0017] The present invention uses gibberellin (GA3) to treat the female parent stigma within 20 minutes before interspecific hybridization pollination of tobacco, which can effectively promote pollen germination on the stigma; uses auxin (IAA) to treat the pistil 1 day after pollination, which can effectively promote the top growth of the pollen tube in the pistil; uses cytokinin (6-BA) to treat the pistil 3 days after pollination, which can effectively promote the elongation of the pollen tube in the pistil and the formation of the embryo. The different hormone treatments in the three stages before and after pollination of the present invention are combined and work together to achieve the purpose of improving the fruit setting rate of interspecific hybridization of tobacco, and have important value in the field of interspecific hybridization breeding of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The fruit setting rates of three interspecific hybrid combinations (A: N. tabacum cv. K326×N. rustica var. brasilia; B: N. tabacum cv. K326×N. glutinosa; C: N. tabacum cv. K326×N. longiflora) under different pollination modes, "*" indicates significant difference (P<0.05), "**" indicates extremely significant difference (P<0.01);

[0020] Figure 2 The results show the germination of pollen at different time points of the hybrid combination of N. tabacum cv. K326×N. rustica var. brasilia under conventional pollination. The results show that pollen activity is inhibited at 4-12 hours, recovers at 24 hours, grows pollen tubes at 48 hours, and enters the style tissue at 72 hours.

[0021] Figure 3 The results show that pollen germination of N. tabacum cv. K326×N. rustica var. brasilia hybrid combination at different time points under plant hormone treatment. The results show that pollen gradually recovers vitality after 4-8 hours, germinates after 12 hours, grows a large number of pollen tubes after 24 hours, and enters the style tissue after 48 hours.

[0022] Figure 4 The growth of pollen tubes of the hybrid combination N. tabacum cv. K326×N. rustica var. brasilia at 120h under conventional pollination. The pollen tubes stopped growing when they grew to 3-5mm of the style tissue. The pollen tubes were curled and bifurcated. The tips of the pollen tubes were swollen, bent, and had callose plugs.

[0023] Figure 5 This is the growth of pollen tubes of the hybrid combination of N. tabacum cv. K326×N. rustica var. brasilia under the treatment of plant hormones for 120 hours. The pollen tubes stopped growing when they grew to the 1 / 2 of the style tissue.

[0024] Figure 6The fruit setting rate of each hybrid combination (A: N. tabacum cv. K326×N. rustica var. brasilia; B: N. tabacum cv. K326×N. glutinosa; C: N. tabacum cv. K326×N. longiflora) under different treatments. Different lowercase letters indicate significant differences among treatments (P<0.05). DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The present invention utilizes plant hormones to treat pistils to improve the fruit setting rate of interspecific hybridization of tobacco. The specific method comprises taking cultivated tobacco as the female parent, conducting interspecific hybridization, and sequentially using gibberellin (GA3), auxin (IAA), and cytokinin (6-BA) to treat the pistils before and after hybridization to improve the fruit setting rate of interspecific hybridization.

[0031] Optionally, the parents of the interspecific hybridization of the tobacco include Nicotiana rustica var. brasilia, Nicotiana glutinosa, Nicotiana longiflora and Nicotiana tabacum cv. K326.

[0032] In a specific embodiment of the present invention, the parental combinations for interspecific hybridization of tobacco include N. tabacum cv.K326×N.rustica var.brasilia, N.tabacum cv.K326×N.glutinosa, and N.tabacum cv.K326×N.longiflora.

[0033] In the above hybrid combinations, N. tabacum cv. K326 was used as the female parent.

[0034] The plant hormone treatment of the pistil of the present invention specifically comprises applying gibberellin (GA3) to the stigma within 20 minutes before pollination; spraying the pistil with auxin (IAA) one day after pollination; and spraying the pistil with cytokinin (6-BA) three days after pollination.

[0035] Optionally, the concentration of gibberellin (GA3) is 200-300 mg / L, the concentration of auxin (IAA) is 100-200 mg / L, and the concentration of cytokinin (6-BA) is 15-25 mg / L.

[0036] In a specific embodiment of the present invention, the concentration of gibberellin (GA3) is 250 mg / L, the concentration of auxin (IAA) is 200 mg / L, and the concentration of cytokinin (6-BA) is 20 mg / L.

[0037] In the method described in the present invention, the hybridization method can adopt conventional hybridization methods in the art.

[0038] As an exemplary illustration, the hybridization method is specifically as follows:

[0039] When wild tobacco is in its early flowering stage, pick the buds (mature but not open flowers) between 7 and 9 in the morning, take out the anthers with clean tweezers, place them in a sterile culture dish, and place it in a 4°C refrigerator to allow the anthers to naturally split, thereby obtaining the male parent's pollen.

[0040] When the mother plant is in full bloom, between 9 a.m. and 12 p.m., remove the immature flower buds and buds of the mother plant, and also pick the opened flowers and fruits, leaving only the flowers that are about to bloom. Then tear off the petals from the middle of the flower to expose the stigma, remove the stamens, then use a cotton swab to dip a small amount of pollen, gently smear it on the stigma, and finally put it on the net bag. In order to ensure the authenticity of the hybrid, the flowers and fruits should be combed every 7 days until the fruit is ripe.

[0041] Those skilled in the art should know that in order to make the flowering period of wild tobacco and cultivated tobacco overlap, it is necessary to choose a suitable time to sow wild tobacco and cultivated tobacco according to the different flowering periods of wild tobacco and cultivated tobacco.

[0042] Unless otherwise specified, the test methods used in the examples of the present invention are conventional methods.

[0043] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0044] The technical solution of the present invention is demonstrated in detail below in conjunction with specific embodiments.

[0045] Example 1

[0046] 1. Test materials

[0047] Male parents: Nicotiana glutinosa (2N=2n=24), Nicotiana longiflora (2N=2n=20) and Nicotiana rustica var. brasilia (2N=4n=48);

[0048] Female parent: common tobacco cultivar K326 (Nicotiana tabacum cv. K326, 2N=4n=48).

[0049] 2. Test methods

[0050] Parent cultivation method: In order to make the flowering periods of the father and mother parents coincide, the father parent is sown in three batches, one month before the sowing of the mother parent, when the mother parent is sown, and one month after the sowing of the mother parent; the floating seedling method is used to raise seedlings, and after the seedlings are grown, the mother parent is transplanted to the experimental field and normal water and fertilizer management is carried out; the father parent is transplanted to the greenhouse using the potting method and fine management is carried out.

[0051] Hybridization method: When the female and male parents are in the initial flowering period, pick the budding flowers between 7 and 9 in the morning, take out the anthers with clean tweezers, place them in a sterile culture dish, and place them in a 4°C refrigerator to allow the anthers to naturally crack, thereby obtaining the male parent's pollen; when the cultivated tobacco is in full flowering period, remove the immature flower buds and buds of the female parent between 9 and 12 in the morning, and also remove the opened flowers and fruits, leaving only the budding flowers, then tear off the petals from the middle of the flower to expose the stigma, remove the stamens, then dip a small amount of pollen with a cotton swab, gently smear it on the stigma, and finally put it on a net bag; in order to ensure the authenticity of the hybrid, thin the flowers and fruits every 7 days until the fruit is ripe, and count the fruit set rate of the hybrid.

[0052] Technology of treating pistils with plant hormones: within 20 minutes before pollination, apply 250mg / L GA3 to the stigma and put it on the net bag immediately after pollination; one day after pollination, gently untie the net bag, spray the pistil with 200mg / L IAA, and then put the net bag on; three days after pollination, gently untie the net bag, spray the pistil with 20mg / L 6-BA, and then put the net bag on; similarly, thin out the flowers and fruits every 7 days until the fruits are ripe, and count the fruit set rate of hybridization.

[0053] Pollen tube observation: Taking N. tabacum cv. K326 × N. rustica var. brasilia as an example, the pollinated flowers were picked at 4h, 8h, 12h, 24h, 48h, 72h, and 120h after pollination. Ten flowers were sampled each time, and the whole pistil was peeled off and placed in FAA fixative (50% ethanol 90mL, glacial acetic acid 5mL, 37% formaldehyde 5mL) for at least 48h. The stigma of the fixed pistil was cut off along the top of the ovary, washed twice in distilled water, and then transferred to 2mol / LNaOH in a 60℃ water bath to soften for 4h, and then washed twice with distilled water, and then stained with 0.1% water-soluble aniline blue solution for 2h, and the stained stigma was placed on a slide, and appropriate glycerol was added, and the cover glass was gently pressed to observe the growth of pollen tubes under an Axio Imager.M2 fluorescence microscope.

[0054] 3. Test results

[0055] The statistical results of hybrid fruiting rate showed that the fruiting rate of interspecific hybridization of tobacco was significantly increased after treatment with plant hormones ( Figure 1 and Table 1), in particular, the hybrid combination of N. tabacum cv. K326×N. rustica var. brasilia could not set fruit normally under conventional cross-pollination, but after treatment with plant hormones, its fruit setting rate was as high as 8.33%.

[0056] Pollen tube observation results showed that: under normal pollination, pollen vitality was inhibited 4 to 12 hours after pollination, pollen vitality recovered after 1 day, pollen tubes grew after 2 days, and pollen tubes entered the style tissue after 3 days ( Figure 2 ), and at 5 days, the pollen tubes grew 3 to 5 mm in the style tissue and then were inhibited ( Figure 4 ), the pollen tubes could not continue to grow; however, under the treatment of plant hormones, the pollen gradually recovered its vitality after 4-8 hours, a small amount of pollen germinated after 12 hours, a large number of pollen tubes grew after 1 day, and the pollen tubes entered the style tissue after 2 days ( Figure 3 ), and at 5 days, the pollen tube grows to the 1 / 2 of the style tissue ( Figure 5 ). It can be seen that the use of plant hormones can promote the germination of heterologous pollen, induce the growth of the apex of the pollen tube, and extend the growth distance of the pollen tube in the pistil.

[0057] Table 1 Effects of plant hormone treatment on fruit setting rate

[0058]

[0059] Example 2

[0060] In order to study the effects of different hormones at different stages on the intermediate hybridization fruit setting rate of tobacco, the specific operation of hormone treatment was adjusted based on Example 1 "Technique of treating pistil with plant hormones".

[0061] 1. Test methods

[0062] The test materials, parents, parent breeding method and hybridization method are the same as those in Example 1.

[0063] When the female parent is in full bloom, remove the opened flowers and fruits, and remove the immature buds and flower buds. Select the flowers that are about to bloom and emasculate them. Perform distant hybridization according to the treatment method in Table 2. Hybridize 60 flowers in each treatment and perform 3 biological replicates. Bag the plants immediately after pollination, and comb the flowers and fruits every 7 days until the fruits are ripe. After the fruits are ripe, count the number of seed-bearing fruits in each treatment, and use Excel 2019 to calculate the fruit setting rate of each treatment and its mean and standard deviation (fruit setting rate = number of seed-bearing fruits / number of hybrid flowers).

[0064] Table 2 Grouping by different hormone treatments

[0065]

[0066] 2. Test results

[0067] The fruit set rates of the three interspecific hybrid combinations under different pollination methods are shown in Table 3 and Figure 6As shown. In the hybrid combination of N.tabacumcv.K326×N.rustica var.brasilia, the fruit setting rates of T1 and T2 treatments (1.07%, 1.08%) were higher than those of CK, but the difference was not significant. The fruit setting rates of T2 treatments of the two hybrid combinations of N.tabacum cv.K326×N.glutinosa and N.tabacum cv.K326×N.longiflora (2.34%, 68.37%) were higher than those of CK (1.64%, 62.21%), but the difference was not significant. The fruit setting rates of T3 treatments of the three hybrid combinations were all higher than those of the control group, and the differences were significant. Therefore, the effects of T1 and T2 treatments on the fruit setting rates of three distant hybrid combinations of N. tabacum cv. K326×N. rustica var. brasilia, N. tabacum cv. K326×N. glutinosa and N. tabacum cv. K326×N. longiflora were not significant, while T3 treatment could significantly increase their fruit setting rates.

[0068] Table 3 Effects of different treatments on fruit setting rate

[0069]

[0070] 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 improving the fruit set rate of interspecific hybridization of tobacco by using plant hormone treatment, characterized in that: The following steps are involved: Select tobacco parents with the same flowering period for interspecific hybridization, apply GA3 to the stigma within 20 minutes before pollination; spray the pistil with IAA one day after pollination; and spray the pistil with 6-BA three days after pollination; Among the tobacco parents, cultivated tobacco is used as the female parent.

2. The method according to claim 1, characterized in that The concentration of the GA3 is 200-300 mg / L; the concentration of the IAA is 100-200 mg / L; and the concentration of the 6-BA is 15-25 mg / L.

3. The method according to claim 2, characterized in that The concentration of the GA3 is 250 mg / L; the concentration of the IAA is 200 mg / L; and the concentration of the 6-BA is 20 mg / L.

4. The method according to claim 1, characterized in that: Before pollination, the immature flower buds and flower buds as well as the opened flowers and fruits of the female parent are removed, only the flowers in bud are retained, and the stamens are removed.

5. The method according to claim 1, characterized in that The tobacco parents include Nicotiana rustica var. brasilia, Nicotiana glutinosa, Nicotiana longiflora and Nicotiana tabacum cv. K326.

6. The method according to claim 1, characterized in that The female parent is the common tobacco cultivar K326.

7. The method according to claim 6, characterized in that The tobacco parents include the Brasilia variety of Nicotiana rustica, Nicotiana glutinosa and Nicotiana longiflora as male parents.

8. The method according to claim 6, characterized in that The tobacco parent is the Brasilia variety of Nicotiana rustica as the male parent.

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