Chrysanthemum subterraneous stem efficient genetic transformation method independent of tissue culture

Abstract

The invention discloses a chrysanthemum subterranean stem efficient genetic transformation method independent of tissue culture, which comprises the following steps: selecting subterranean stems, cutting the subterranean stems into segments, infecting the segments with agrobacterium tumefaciens, and sequentially performing dark culture and culture under illumination to obtain transgenic positive buds. The method is simple in steps, short in experimental period, high in efficiency and low in cost, a large number of transgenic buds can be obtained in a short time, the transgenic buds can be directly cut into vermiculite to survive, and exogenous genes are stably expressed; the chrysanthemum subterraneous stem transformation system independent of tissue culture is established for the first time, and effective technical support is provided for basic research and scientific research breeding of chrysanthemum.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic transformation, in particular to a method for efficient genetic transformation of chrysanthemum underground stems independent of tissue culture. Background Art

[0002] Chrysanthemum (Chrysanthemum morifolium) is a perennial herbaceous plant of the genus Chrysanthemum in the family Asteraceae, native to my country. It has been recorded in the "Book of Rites·Monthly Orders" of the Western Han Dynasty. Chrysanthemum occupies an important position in the international flower market due to its rich color, varied flower shapes and profound cultural connotations. It is one of the four major cut flowers in the world. Some chrysanthemums have the functions of food, tea and medicine, and have the functions of clearing away heat and detoxifying, calming the liver and improving eyesight. Some companies have built a one-stop service from planting, purchasing, processing to selling, which has greatly increased the profits of enterprises and the income of local residents.

[0003] Chrysanthemum is widely cultivated in China and has a large market, but its genome is highly complex and heterozygous. The traditional chrysanthemum genetic transformation system is often not universal. Even the wild diploid chrysanthemum, Chrysanthemum argentea, has the problem of slow genetic material production and relatively unstable genetic transformation system. This limits the molecular breeding of chrysanthemum. Therefore, a fast, efficient and highly applicable genetic transformation system is particularly important.

[0004] Conventional Agrobacterium-mediated genetic transformation of chrysanthemum leaf discs, that is, using Agrobacterium tumefaciens to infect plant cells, and then using screening substances to screen out transgenic cells for regeneration, thereby obtaining transgenic plants. This method not only requires nearly three months of experimental time, but also involves a series of complex processes such as sterilization, pre-breeding, co-cultivation, screening culture and rooting culture. Some explants even die under the dual stress of antibiotics and bacteriostatins, and the transformation efficiency is low. In addition, this method is only suitable for chrysanthemum genotypes with strong leaf disc regeneration ability. If the chrysanthemum leaf disc has difficulty in regeneration, genetic transformation cannot be achieved through this method.

[0005] Chrysanthemum has a complex genetic background. Exploring the chrysanthemum explant regeneration and genetic transformation system under tissue culture conditions is costly and time-consuming. The most suitable types and ratios of plant growth regulators, Agrobacterium infection time and concentration, and critical values ​​of screening substances for chrysanthemums of different genotypes are also different. In addition, during the genetic transformation process, there are also problems such as browning of chrysanthemum leaf disks and vitrification of regenerated buds. Therefore, a chrysanthemum transformation system that does not rely on tissue culture can not only circumvent the drawbacks of traditional methods, but also reduce technical costs, allowing the genetic improvement of chrysanthemums to better enter the industry.

[0006] Under long-term natural selection, plants have evolved a series of special structures to adapt to adversity. The underground stems of chrysanthemums are one of them. The underground stems have obvious non-green scale leaves. The axillary buds in the leaf axils can form above-ground branches and produce adventitious roots. When the rhizome is cut off by external force, the axillary buds on the stem segment can still regenerate into new plants, providing ideal materials for chrysanthemum transformation that does not rely on tissue culture. Of course, the transformation efficiency of using chrysanthemum underground stems as genetic transformation materials may still be affected by the genotype, but it provides a feasible solution for some genotypes that are still difficult to regenerate using tissue culture technology.

[0007] Therefore, it is necessary to provide a method for efficient genetic transformation of chrysanthemum rhizomes that does not rely on tissue culture. Summary of the invention

[0008] The present invention provides a method for efficient genetic transformation of chrysanthemum underground stems independent of tissue culture.

[0009] In view of this, the scheme of the present invention is:

[0010] A method for efficient genetic transformation of chrysanthemum underground stems independent of tissue culture, comprising the following steps:

[0011] S1. Take the underground stems of the ground-planted chrysanthemum clones that have survived the winter and are in good growth condition, and cut them into stem segments;

[0012] S2. Soak the stem segments in Agrobacterium tumefaciens solution for infection;

[0013] S3. The soaked stem segments were transferred to loose soil for shallow horizontal burial and cultured in the dark for 2 consecutive days;

[0014] S4. The stem segments after dark culture were placed under light for 20 days;

[0015] S5. Select the transgenic positive buds with good growth conditions, cut them off and place them in a vermiculite matrix, keep the air moist, and wait for them to take root, and then you can obtain transgenic chrysanthemum plants.

[0016] Furthermore, the chrysanthemum is Jiangling golden chrysanthemum or wild chrysanthemum.

[0017] Furthermore, in step S1, the length of the stem segment is 2-3 cm.

[0018] Further, in step S2:

[0019] The OD of Agrobacterium tumefaciens culture solution 600 0.6-0.8;

[0020] The soaking and infection time is 20-25 minutes.

[0021] Further, in step S3:

[0022] The shallow burial depth is 1 / 2-2 / 3 of the cross section of the underground stem segment;

[0023] The loose soil is 90% vermiculite and 10% seedling medium soil;

[0024] The temperature of the dark culture process is 25±2°C.

[0025] Furthermore, in step S4, the light cultivation process keeps the soil moist; preferably, the soil moisture is 45%-60%.

[0026] Furthermore, in step S4, the light culture conditions are: temperature of 25±2°C, photoperiod of 16h / 8h, and light intensity of 3000lx.

[0027] Furthermore, in step S5, the transgenic positive buds are obtained by detecting gene tags carried in the buds emerging from the underground stem segments.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The genetic transformation method of the present invention comprises the following steps: selecting underground stems and cutting them into segments, then infecting them with Agrobacterium tumefaciens, and sequentially culturing them in the dark and under light to obtain transgenic positive buds. The method has simple steps, short experimental cycle, high efficiency, low cost, and can obtain a large number of transgenic buds in a short time. The transgenic buds can be directly cut and survived in vermiculite, stably express exogenous genes, and do not require tissue culture technology. The present invention is the first to establish a chrysanthemum underground stem transformation system that does not rely on tissue culture, and provides effective technical support for basic research and scientific research breeding of chrysanthemum. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a schematic diagram of the genetic transformation experiment process of the present invention.

[0031] Figure 2 This is a comparison diagram of the underground stems of 'Jiangling Jinju' before and after genetic transformation in Example 1 of the present invention.

[0032] Figure 3 This is a comparison diagram of the genetic transformation of the underground stems of 'Jiangling Jinju' after GUS staining in Example 1 of the present invention.

[0033] Figure 4 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows:

[0034] Figure 5 This is a comparison diagram of the aboveground stems of 'Jiangling Jinju' before and after genetic transformation in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In one embodiment, Figure 1 As shown, a method for efficient genetic transformation of chrysanthemum underground stems that does not rely on tissue culture is proposed, and the steps include:

[0037] S1. Take the underground stems of the ground-planted chrysanthemum clones that have survived the winter and are in good growth condition, and cut them into stem segments;

[0038] S2. Soak the stem segments in the Agrobacterium tumefaciens solution for 20-25 minutes;

[0039] S3. The soaked stem segments were transferred to loose soil for shallow horizontal burial and cultured in the dark for 2 consecutive days;

[0040] S4. The stem segments after dark culture were placed under light for 20 days;

[0041] S5. Select the transgenic positive buds with good growth conditions, cut them off and place them in a vermiculite matrix, keep the air moist, and wait for them to take root, and then you can obtain transgenic chrysanthemum plants.

[0042] In the above embodiment, the underground stems are cut off and then used to enter the plant cells through the infected wounds by using Agrobacterium tumefaciens, and the T-DNA on the Ti plasmid is inserted into the genome of the plant, thus realizing the transfer and integration of foreign genes into the plant cells. The underground stems of chrysanthemum can be used to regenerate complete plants. By utilizing this germplasm property, the genetic transformation of chrysanthemum can be realized without relying on tissue culture conditions.

[0043] In a preferred embodiment, the chrysanthemum is wild chrysanthemum or Jiangling golden chrysanthemum, more preferably Jiangling golden chrysanthemum variety, which has a higher genetic transformation efficiency.

[0044] Example 1

[0045] The GUS marker gene was transformed into 'Jiangling Jinju', and the steps were as follows:

[0046] S1. Use YEB liquid medium to culture EHA105 Agrobacterium tumefaciens containing the GUS tag vector until OD 600 The OD was 0.6-0.8, and the Agrobacterium was rinsed and resuspended in liquid medium containing 10 mM MgCl2, 10 mM MES and 100 μM acetosyringone. 600 Adjust to 0.6-0.8;

[0047] S2, selecting underground stems from the ground-planted seedlings of the 'Jiangling Jinju' clone that have survived the winter and are in good growth condition, removing them and cutting them into 2-3 cm stem segments with a scalpel;

[0048] S3. Place the underground stem segments from step S2 to OD 600 Soak the stems in a 0.6-0.8% Agrobacterium tumefaciens bacterial solution for 20-25 minutes. During this period, ensure that the bacterial solution covers the stems and shake them gently to increase contact efficiency.

[0049] S4, taking out the underground stem segment described in step S3, placing it in loose soil, the soil should be slightly moist at this time, the humidity is about 45%-60%, the depth of the placement is about 1 / 2-2 / 3 of the cross section of the stem segment, and continuously culturing in the dark for 2 days;

[0050] S4, placing the underground stem segments after continuous dark culture for 2 days in step S4 under light, keeping the soil moist, buds appear after about 3-5 days, and culture for 20 days; Figure 2 As shown, from the comparison of the underground stems of Jiangling golden chrysanthemum before and after genetic transformation, it can be seen that after 20 days of cultivation under light, buds emerged obviously.

[0051] S5. Use X-gluc reagent to perform histochemical staining on the chrysanthemum leaves in S4 to confirm that these seedlings are stably transformed regenerated seedlings; the comparison results after GUS staining of the underground stem genetic transformation are as follows Figure 3 shown.

[0052] S6. Select the transgenic seedlings with good growth condition from S5, cut them off with a scalpel, put them into a vermiculite matrix, keep the air moist, and wait for them to take root, and then the transgenic 'Jiangling Golden Chrysanthemum' seedlings can be obtained. Figure 4 As shown, the survival rate of cuttings of 'Jiangling Jinju' underground stem genetic transformation was good.

[0053] Example 2

[0054] The steps for transforming wild chrysanthemum with GUS marker gene are as follows:

[0055] S1. Use YEB liquid medium to culture EHA105 Agrobacterium tumefaciens containing the GUS tag vector until OD 600 The OD was 0.6-0.8, and the Agrobacterium was rinsed and resuspended in liquid medium containing 10 mM MgCl2, 10 mM MES and 100 μM acetosyringone. 600 Adjust to 0.6-0.8;

[0056] S2, selecting underground stems from the ground-planted seedlings of the wild chrysanthemum clones that have overwintered and are in good growth condition, removing them and cutting them into stem segments of 2-3 cm with a scalpel;

[0057] S3. Place the underground stem segments from step S2 to OD 600 Soak the stems in a 0.6-0.8% Agrobacterium tumefaciens bacterial solution for 20-25 minutes. During this period, ensure that the bacterial solution covers the stems and shake them gently to increase contact efficiency.

[0058] S4, taking out the underground stem segment described in step S3, placing it in loose soil, the soil should be slightly moist at this time, the humidity is about 45%-60%, the depth of the placement is about 1 / 2-2 / 3 of the cross section of the stem segment, and continuously culturing in the dark for 2 days;

[0059] S4, placing the underground stem segments after continuous dark culture for 2 days in step S4 under light, keeping the soil moist, buds appear after about 3-5 days, and culture for 20 days;

[0060] S5, using X-gluc reagent to perform histochemical staining on the leaves of Chrysanthemum indica to confirm that these seedlings are stably transformed regenerated seedlings;

[0061] S6. Select the transgenic seedlings in good growth condition from S5, cut them off with a scalpel, put them into a vermiculite matrix, keep the air moist, and wait for them to take root, thereby obtaining transgenic wild chrysanthemum seedlings.

[0062] Comparative Example 1

[0063] The steps of transforming 'Jiangling Jinju' with the GUS marker gene are the same as those in the embodiment, except that in step S2, the aboveground stems are selected from the ground-planted seedlings of the 'Jiangling Jinju' clones that have survived the winter and are in good growth condition. Figure 5 As shown, the comparison of the aboveground stems of 'Jiangling Jinju' before and after genetic transformation showed that few buds emerged after 20 days of cultivation under light.

[0064] The above Examples 1-2 and Comparative Example 1 respectively carried out genetic transformation on the same number of explants, and compared the number of positive buds, the number of chimeras and the positive efficiency. The results are shown in Table 1.

[0065] Table 1: Genetic transformation efficiency

[0066]

[0067] It is not difficult to see from Table 1 that when underground stems were selected as the object of genetic transformation, the positive efficiency of 'Jiangling Golden Chrysanthemum' was the highest, followed by wild chrysanthemum; when the aerial stems were used, no positive buds were obtained; in addition, in comparison, the positive efficiency of genetic transformation of the underground stems of 'Jiangling Golden Chrysanthemum' was higher than that of the underground stems of wild chrysanthemum, indicating that the difference in genotype may be the reason for the different genetic transformation effects.

[0068] It can be seen from the above examples that the experimental materials of this scheme are easy to obtain, no tissue culture technology is required, the steps are simple, the experimental cycle is short, the efficiency is high, the cost is low, and a large number of transgenic buds can be obtained in a short time. The transgenic buds can be directly cut and survive in vermiculite, and stably express exogenous genes. In addition, most chrysanthemums have underground stems, and it is speculated that this method may be applicable to chrysanthemums of other genotypes. The transformation efficiency may be different, but for some genotypes that are still difficult to regenerate using tissue culture technology, a feasible solution is provided; the present invention is the first to establish a chrysanthemum underground stem transformation system that does not rely on tissue culture conditions, providing effective technical support for basic research and scientific research breeding of chrysanthemums.

[0069] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for efficient genetic transformation of chrysanthemum underground stems independent of tissue culture, characterized in that the steps include: S1. Take the underground stems of the ground-planted chrysanthemum clones that have survived the winter and are in good growth condition, and cut them into stem segments; S2. Soak the stem segments in Agrobacterium tumefaciens solution for infection; S3. The soaked stem segments were transferred to loose soil for shallow horizontal burial and cultured in the dark for 2 consecutive days; S4. The stem segments after dark culture were placed under light for 20 days; S5. Select the transgenic positive buds with good growth conditions, cut them off and place them in a vermiculite matrix, keep the air moist, and wait for them to take root, and then you can obtain transgenic chrysanthemum plants.

2. The genetic transformation method according to claim 1, characterized in that The chrysanthemum is Jiangling golden chrysanthemum or wild chrysanthemum.

3. The genetic transformation method according to claim 1, characterized in that In step S1, the length of the stem segment is 2-3 cm.

4. The genetic transformation method according to claim 1, characterized in that In step S2, the OD of the Agrobacterium tumefaciens solution is 600 0.6-0.8; And / or, the soaking and infection time is 20-25 minutes.

5. The genetic transformation method according to claim 1, characterized in that In step S3, the shallow burial depth is 1 / 2-2 / 3 of the cross section of the underground stem segment; And / or, the loose soil is 90% vermiculite and 10% seedling medium soil; And / or, the temperature of the dark culture process is 25±2°C.

6. The genetic transformation method according to claim 1, characterized in that In step S4, the light cultivation process keeps the soil moist.

7. The genetic transformation method according to claim 6, characterized in that The soil moisture is 45%-60%.

8. The genetic transformation method according to claim 1, characterized in that In step S4, the light culture conditions are: temperature of 25±2°C, photoperiod of 16h / 8h, and light intensity of 3000lx.

9. The genetic transformation method according to claim 1, characterized in that In step S5, the transgenic positive buds are obtained by detecting the gene tags carried in the buds emerging from the underground stem segments.

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