CN115362934B

CN115362934B - Method for inducing tetraploid hybrid tulip tree strain south Lin Jinsen E1 by colchicine

Info

Publication number: CN115362934B
Application number: CN202210583055.4A
Authority: CN
Inventors: 陈金慧; 杨顶杰; 殷健超; 陆叶; 黄世清; 杨立明; 成铁龙; 施季森
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-05-26
Filing date: 2022-05-26
Publication date: 2023-05-02
Anticipated expiration: 2042-05-26
Also published as: CN115362934A

Abstract

The invention discloses a method for inducing hybrid liriodendron tulipifera fine variety No. Lin Jinsen E1 tetraploid by colchicine, which comprises the following steps: 1) Homogenization treatment of embryo callus of hybrid tulip tree of 'nan Lin Jinsen E1'; 2) Colchicine is dripped to treat the callus in the induction process of the somatic embryo; wherein, the mg/mL concentration of colchicine water solution is 0.01% -0.1%; 3) Transferring the dark culture material in the step 2) to illumination culture to induce plant regeneration; 4) Preliminary screening of tetraploid plants and transplanting of seedlings; 5) Ploidy identification of regenerated plants. The invention takes embryogenic callus as an explant, obtains the pure isogenic tetraploid hybrid tulip tree No. Lin Jinsen E1 through colchicine treatment, overcomes the defect that chimerism appears when the conventional method takes seeds, stem tips and the like as the explant to induce chromosome doubling, is easy to operate and stable in induction, and is a method suitable for inducing the polyploid of the tulip tree plants.

Description

Method for inducing tetraploid hybrid tulip tree strain south Lin Jinsen E1 by colchicine

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing tetraploid hybrid tulip tree improved variety 'nan Lin Jinsen E1' by colchicine.

Background

The improved hybrid tulip tree seed No. Lin Jinsen E1 is an interspecific improved hybrid seed of the tulip tree plant of the magnoliaceae. The genus tulip tree contains two species, one species is tulip tree and the other species is tulip tree. Since the 60 s of the 20 th century, the university of Nanjing forestry She Peizhong teaches that the two were artificially hybridized to obtain hybridized tulip trees with obvious hybrid vigor. In 2003, a hybrid tulip tree efficient somatic embryo (hereinafter collectively referred to as "somatic embryo") generation system and a plant regeneration system were created by professor Chen Jinhui of the university of Nanjing forestry and the like, and large-scale application was realized. From this point, the subject group tries to artificially hybridize between different parents each year, and combines with the embryogenesis technology system to successfully obtain a plurality of hybridized tulip tree embryogenic calli with high embryogenesis efficiency, thereby realizing the beautiful scenery of 'giving I a cell and you a forest'. The subject group obtains immature embryo by artificial hybridization seed production, successfully induces embryogenic callus, and finds that the subject group has good performance on the induction speed and efficiency of the embryo through embryo induction experiments, and the regenerated plant of the embryo is widely popularized in the southern province of China, shows excellent characters such as fast growth, high quality and the like, and is distinguished as an improved variety 2019, and is named as improved variety "south Lin Jinsen E1" of hybrid tulip tree.

Chen Tingting, through phenotypic observation, physiological experiments, transcriptome and proteome sequencing analysis and other researches, the new tetraploid hybrid tulip tree variety's sejinxiang' has the excellent characteristics of organ enlargement, biomass increase, photosynthesis enhancement and the like compared with the diploid. However, chromosome doubling of tulip species has been lacking in stable and efficient induction methods, which have limited intensive research in this direction and the yield and scale application of tetraploid varieties of this genus. Colchicine, as a conventional cell division inhibitor and ploidy mutagen, can inhibit mitosis, destroy the spindle, and arrest the chromosome in metaphase, in which case the chromosome is slit but the cell is not divided and two daughter cells cannot be formed, thus doubling the chromosome. In recent years, colchicine has been widely used in the induction work of woody plants such as tetraploid poplar, loquat, locust, etc. However, it has been recently reported in the study of the induction of the ploidy variation of tulip tree species.

Disclosure of Invention

Aiming at the defects existing in the prior art, the technical problem to be solved by the invention is to provide a method for inducing the tetraploid of the improved hybrid tulip tree variety 'nan Lin Jinsen E1' by using colchicine, and inducing the pure contract source tetraploid of the improved hybrid tulip tree variety 'nan Lin Jinsen E1' by using embryogenic callus as an explant.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for inducing hybrid tulip tree improved variety 'nan Lin Jinsen E1' tetraploid by colchicine comprises the following steps:

1) Homogenizing embryo callus of improved hybrid tulip tree strain No. Lin Jinsen E1;

2) Colchicine is dripped to treat the callus in the induction process of the somatic embryo; wherein, the mg/mL concentration of colchicine water solution is 0.01% -0.1%;

3) Transferring the dark culture material in the step 2) to illumination culture to induce plant regeneration;

4) Preliminary screening of tetraploid plants and transplanting of seedlings;

5) Ploidy identification of regenerated plants.

In the step 1), the embryo callus homogenizing treatment process of the hybrid tulip tree improved variety 'nan Lin Jinsen E1' is as follows: transferring embryo callus of improved hybrid tulip tree variety 'nan Lin Jinsen E1' into MS+5mg/L VC+2mg/L2,4-D+0.2mg/L BA+30g/L sucrose liquid culture medium, shaking thoroughly, sieving with 500 mesh cell sieve to remove liquid, re-sieving callus, inoculating to newly prepared MS+5mg/L VC+2mg/L2,4-D+0.2mg/L BA+30g/L sucrose+6.5 g/L agar solid culture medium, and recovering for 2 weeks.

In the step 2), the callus process in the embryo induction process of colchicine dropwise addition treatment is as follows: inoculating the callus in the step 1) on a somatic embryo induction culture medium of MS+5mg/L VC+1g/L active carbon+30 g/L sucrose+6.5 g/L agar, and culturing induced somatic embryos in dark at 23 ℃; dripping colchicine water solution with the concentration of 0.05% -0.1% in the 1d and 7d of the induced somatic embryo twice respectively to enable the colchicine solution to soak the callus blocks;

in the step 2), the colchicine water solution with the concentration of mg/mL of 0.05% and filtered by a 0.22 mu m filter head is respectively dripped twice at 1d and 7d after the induction of the somatic embryo, so that the colchicine solution permeates the callus blocks.

In the step 3), the regeneration process of the dark culture material in the step 2) through transferring the light culture induced plant is as follows: transferring the material of the post-embryo development stage of the dark culture in the step 2) to light for culturing for 1-2 weeks, wherein the light intensity is 15 mu mol.m ^-2 ·s ^-1 The illumination time is 16h/d, and the culture temperature is 24 ℃; picking seedling after the cotyledon turns green, inoculatingAnd (3) continuously carrying out illumination culture for two months in a seedling picking culture medium of freshly prepared MS+5mg/L VC+30g/L sucrose+7.5 g/L agar, so as to obtain regenerated plants.

In the step 4), the preliminary screening and seedling transplanting process of the tetraploid plants comprises the following steps: seedlings with the characteristics of thickened leaves, fleshy quality, reduced leaf area, dark green leaf color, thickened roots and stems, reduced lateral roots and the like are initially screened from the obtained regenerated plants, and transplanted into humus nutrient soil and perlite=7:3 soil to adapt to soil environment.

In the step 5), the ploidy identification process of the regenerated plant is as follows: after the number of new-born lateral roots of seedlings growing in soil is vigorous for one month, taking root tips of the seedlings to be placed in paradichlorobenzene saturated solution for pretreatment for 6-8 hours, washing the seedlings with clear water, placing the seedlings in carnot fixing solution for fixing for 12-24 hours, washing materials with 95% ethanol, transferring the materials into 1mol/L HCl preheated to 60 ℃ in advance, carrying out water bath dissociation for 7-8 minutes at 60 ℃, washing the materials with tap water, adding alkaline carbomer fuchsin dye liquor capable of overflowing the materials for overall dyeing for more than 2 hours, slightly scraping off root tip meristematic regions with deeper dyeing by using an dissecting needle, and then tabletting, and determining the ploidy of regenerated plants through chromosome observation and counting.

The beneficial effects are that: compared with the prior art, the method takes embryogenic callus as an explant, obtains the pure isogenic tetraploid hybrid tulip tree improved variety 'nan Lin Jinsen E1' through colchicine treatment, overcomes the defect that chimerism appears when the traditional method takes seeds, stem tips and the like as the explant to induce chromosome doubling, is easy to operate and stable to induce, and is a method suitable for polyploid induction of the hybrid tulip tree improved variety 'nan Lin Jinsen E1'.

Drawings

FIG. 1 is a view of embryogenic callus under a stereoscopic microscope;

FIG. 2 is a drawing showing the state of callus development induced for 30 days in embryos treated with different concentrations of colchicine, light gray arrows indicating the enlarged body shape obtained after treatment with appropriate concentrations of colchicine, white arrows indicating malformed embryos resulting from treatment with high concentrations of colchicine;

FIG. 3 shows the phenotype and chromosome number of diploid and tetraploid cotyledon embryos, scale of a is 2mm, scale of b and scale of c is 10 μm;

FIG. 4 shows the phenotype and chromosome number of diploid and tetraploid regenerated plants, scale of a to d is 1cm, scale of e and f is 10. Mu.m.

FIG. 5 shows the number of diploid and tetraploid embryo regenerated plants and chromosomes without light cultivation.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Materials: the materials used in the following examples were embryogenic callus derived from immature embryo of the improved variety "nan Lin Jinsen E1" of hybrid tulip tree [ Chen Jinhui, shi Jisen, ge Jiang, and Huang Minren (2003), "hybrid tulip tree somatic embryogenesis research," forestry science, 39 (4), 49-53.], as shown in FIG. 1, which was fine granular, pale yellow, loose in texture, fast in cell division, and high in embryo induction rate.

Example 1

1) And (3) carrying out embryo callus homogenization treatment on the improved strain 'nan Lin Jinsen E1' of the hybrid tulip tree.

The embryo callus of the improved hybrid tulip tree variety 'nan Lin Jinsen E1' is transferred into MS+5mg/L VC+2mg/L2,4-D+0.2mg/L BA+30g/L sucrose liquid medium, fully shaken up, filtered out by 500 meshes of cell sieve to remove liquid, and inoculated onto newly prepared MS+5mg/L VC+2mg/L2,4-D+0.2mg/L BA+30g/L sucrose+6.5 g/L agar solid medium for 2 weeks for the next colchicine treatment.

2) Colchicine is added dropwise to treat callus in the induction process of somatic embryos.

The callus (about 0.5cm in diameter) from step 1) was inoculated onto a somatic embryo induction medium of MS+5mg/L VC+1g/L activated carbon+30 g/L sucrose+6.5 g/L agar, and the induced somatic embryos were dark-cultured at 23 ℃. Dripping 0.01%,0.05%,0.1%,0.5% (w/v) colchicine water solution sterilized by suction through 0.22 μm filter head (20 μl for the first time and 30 μl for the second time) twice (1 d and 7d respectively after somatic embryo induction to soak the callus blocks, taking untreated callus as CK, and dripping equal amount of ddH ₂ O was callus MOCK.

3) Ploidy identification and tetraploid induction rate statistics of colchicine-treated somatic embryos.

Taking cotyledon embryo of which the somatic embryo is induced for 25-30d, placing the cotyledon embryo in p-dichlorobenzene saturated solution for pretreatment for 2-4h, placing the cotyledon embryo in Carnot fixing solution (acetic acid: 95% ethanol=1:3) for fixing for 12-24h after cleaning with clear water, then using 95% ethanol to clean the material, transferring the material into 1mol/L HCl which is preheated to 60 ℃ in advance, dissociating the material in a water bath at 60 ℃ for 6-7 min, cleaning the material with tap water, adding alkaline Kabao fuchsin dye solution which can be used for immersing the material for integral dyeing for more than 2h, lightly grinding the material with forceps, and placing the material under an optical microscope for observation after light scattering of a dye solution and a cover glass. The ploidy of regenerated plants was determined by chromosome counting. Tetraploid induction rate under each treatment was counted. The statistical results showed (FIG. 2 and Table 1) that tetraploid embryos could not be obtained without treatment or treatment of the calli with water and high concentration colchicine (0.5%) by this dripping method, and that the 0.01%,0.05%,0.1% colchicine treatments could respectively obtain 3.3%,5.9%,4.5% tetraploid embryos, with 0.05% -0.1% being considered to be the suitable treatment concentration for this method.

TABLE 1 Induction efficiency of tetraploid embryos after colchicine treatment

4) And (3) transferring the dark culture material in the step (2) to illumination culture to induce plant regeneration.

Transferring the material (hypocotyl elongation, cotyledon yellowing) of the post-embryo development stage of dark culture of about 40d in step 2) to light for culturing for 1-2 weeks with light intensity of 15 μmol.m ^-2 ·s ^-1 The illumination time is 16h/d, and the culture temperature is 24 ℃. And after the cotyledons turn green, picking seedlings, inoculating the cotyledons into a seedling picking culture medium of freshly prepared MS+5mg/L VC+30g/L sucrose+7.5 g/L agar, and continuing to carry out illumination culture for two months to obtain regenerated plants.

5) Preliminary screening of tetraploid plants and transplanting of seedlings.

And 4) preliminarily screening seedlings with the characteristics of thickened leaves, fleshy quality, reduced leaf area, dark green leaf color, thickened roots and stems, reduced lateral roots and the like from the regenerated plants obtained in the step 4), and transplanting the seedlings into soil (humus nutrient soil: perlite=7:3) to adapt the seedlings to soil environment.

6) Ploidy identification of regenerated plants.

After the number of new-born lateral roots of seedlings growing in soil is vigorous, taking root tips of the seedlings to be placed in a paradichlorobenzene saturated solution for pretreatment for 6-8 hours at 9:00-10:00 am, placing the seedlings in a carnot fixing solution (acetic acid: 95% ethanol=1:3) for fixing for 12-24 hours after cleaning by clear water, then cleaning materials by using 95% ethanol, transferring the materials into 1mol/L HCl which is preheated to 60 ℃ in advance, dissociating the materials in a water bath at 60 ℃ for 7-8 minutes, cleaning the materials by using tap water, adding an alkaline Baobao fuchsin dye solution which can be used for dyeing the materials for more than 2 hours in the whole, gently scraping off a root tip meristematic region with a dissecting needle, and then tabletting, and determining the ploidy of regenerated plants through chromosome observation and counting.

The results are shown in fig. 3 and 4: part of cotyledon embryo bodies obtained by colchicine treatment are enlarged, cotyledons are more stretched and thick (figure 3 a), the number of chromosomes is found to be 76 by identification of the number of chromosomes, the number of the chromosomes is twice that of diploid controls (38) of the cotyledon embryo bodies (figures 3b and c), the cotyledon embryo bodies are confirmed to be tetraploid cotyledon embryo bodies, and dipoles (root ends and stem ends) of the cotyledon embryo bodies are complete and are not deformed, which implies that the tetraploid cotyledon embryo bodies induced by colchicine have the integrity of bipolar functions and can continue to develop into complete regeneration plants; through screening of regenerated plants, part of the plants are found to show the characteristics of thickening leaves, fleshy quality, reduced leaf area, miraculous leaf shape, green-increasing leaf color and the like (figures 4 a-d), which shows that part of the plants in the regenerated plants have variant phenotypes, and the variant plants are confirmed to be tetraploids through chromosome number identification of root tip meristematic regions (figures 4e and f). The result shows that the complete tetraploid regenerated plant can be obtained by the method of dropping colchicine to treat embryogenic callus in the induction process of somatic embryo.

Example 2

Materials: and subjected to homogenization treatment as in example 1.

1) Colchicine is added dropwise to treat callus during cell proliferation.

Callus blocks with the diameter of about 0.3cm are inoculated on a callus proliferation solid culture medium of MS+5mg/L VC+1 mg/L2,4-D+0.2 mg/LBA+30g/L sucrose+6.5 g/L agar. In one subculture period (22-25 d), the colchicine aqueous solution with concentration gradient of 0.05%,0.1% and 0.2% is added dropwise 4 times (15-30 uL each time) respectively at 1d,6d,11d and 16d after callus inoculation, preferably, the colchicine solution is used for soaking the callus blocks.

2) Somatic embryo induction after colchicine treatment of the callus.

The callus treated by colchicine in the step 1) is inoculated on a solid culture medium of MS+5mg/L VC+1g/L active carbon+30 g/L sucrose+6.5 g/L agar, somatic embryos (spherical embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo) of each development stage can be obtained after induction for 20-25d, and somatic embryo regeneration plants can be obtained after 35-40 d.

3) And (5) identifying ploidy of the somatic embryo regenerated plants.

The root tip of the regenerated plant of the somatic embryo is taken for chromosome flaking, the operation procedure is the same as that of the step 6) in the example 1, and the ploidy of the regenerated plant of the somatic embryo is determined by chromosome counting. Statistical results show that chromosome doubling can be induced by the method, and 0.1% and 0.2% are effective colchicine concentrations, which respectively correspond to tetraploid induction rates of 3.1% and 4.2%, tetraploid yellowing plants are shown in figure 5 (the whole plant is dyed and used for ploidy identification because the root tips of somatic embryo regeneration plants are tiny and difficult to obtain). The above results show that the method can induce the embryogenic callus to double, and the doubled embryogenic cells can induce the regeneration of somatic embryos and plants, so that the tetraploid induction efficiency of the method can be further improved if the optimal concentration of colchicine and the time window for vigorous callus proliferation are determined.

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Claims

1. A method for inducing tetraploid hybrid tulip tree improved variety 'nan Lin Jinsen E1' by using colchicine is characterized by comprising the following steps:

1) Homogenizing embryo callus of improved hybrid tulip tree strain No. Lin Jinsen E1; the process is as follows: transferring embryo callus of improved hybrid tulip tree variety 'nan Lin Jinsen E1' into MS+5mg/L VC+2mg/L2,4-D+0.2mg/L BA+30g/L sucrose liquid medium, shaking thoroughly, sieving with 500 mesh cell sieve to remove liquid, re-sieving callus, inoculating to newly prepared MS+5mg/L VC+2mg/L2,4-D+0.2mg/L BA+30g/L sucrose+6.5 g/L agar solid medium, and recovering for 2 weeks;

2) Colchicine is dripped to treat the callus in the induction process of the somatic embryo; the process is as follows: inoculating the callus in the step 1) on a somatic embryo induction culture medium of MS+5mg/L VC+1g/L active carbon+30 g/L sucrose+6.5 g/L agar, and culturing induced somatic embryos in dark at 23 ℃; dripping colchicine water solution with w/v concentration of 0.05% -0.1% twice after somatic embryo induction for 1d and 7d respectively, so that the colchicine solution permeates the callus blocks;

3) Transferring the dark culture material in the step 2) to illumination culture to induce plant regeneration; the process is as follows: transferring the material of the post-embryo development stage of the dark culture in the step 2) to light for culturing for 1-2 weeks, wherein the light intensity is 15 mu mol.m ^-2 ·s ^-1 The illumination time is 16h/d, and the culture temperature is 24 ℃; after the cotyledons turn green, picking seedlings, inoculating the cotyledons into a seedling picking culture medium of freshly prepared MS+5mg/L VC+30g/L sucrose+7.5 g/L agar, and continuing to carry out illumination culture for two months to obtain regenerated plants;

4) Preliminary screening of tetraploid plants and transplanting of seedlings;

5) Ploidy identification of regenerated plants.

2. The method for inducing tetraploid hybrid liriodendron tulipifera fine variety "nan Lin Jinsen E1" by colchicine according to claim 1, wherein in step 2), colchicine aqueous solution with w/v concentration of 0.05% and sterilized by suction filtration through a 0.22 μm filter head is added dropwise to 1d and 7d after somatic embryo induction, respectively, so that the colchicine solution is soaked in the callus pieces.

3. The method for inducing tetraploid hybrid tulip tree improved variety "nan Lin Jinsen E1" by colchicine according to claim 1, wherein in step 4), the preliminary screening and seedling transplanting process of tetraploid plants is: preliminarily screening seedlings with characteristics of thickened leaves, fleshy leaves, reduced leaf areas, dark green leaves, thickened roots and stems and reduced lateral roots from the obtained regenerated plants, and transplanting the seedlings to humus nutrient soil: perlite=7: 3, adapting the soil to the soil environment.

4. The method for inducing tetraploid hybrid tulip tree elite "nan Lin Jinsen E1" using colchicine according to claim 1, wherein in step 5), the ploidy identification process of the regenerated plant is: after the number of new-born lateral roots of seedlings growing in soil is vigorous for one month, taking root tips of the seedlings to be placed in a paradichlorobenzene saturated solution for pretreatment for 6-8 hours, washing the seedlings with clear water, placing the seedlings in a Carnot fixing solution for fixing for 12-24 hours, washing materials with 95% ethanol, transferring the materials into 1mol/L HCl preheated to 60 ℃ in advance, carrying out water bath dissociation for 7-8 minutes at 60 ℃, washing the materials with tap water, adding alkaline Kabao fuchsin dye liquor which is used for material to carry out overall dyeing for more than 2 hours, gently scraping off the root tip meristematic regions with deeper dyeing by using an anatomical needle, and then tabletting, and determining the ploidy of regenerated plants through chromosome observation and counting.