Construction method of direct organogenesis type regeneration path of blackberry leaves

Abstract

The invention discloses a method for inducing a direct organ generation type regeneration path of blackberries by using leaves as explants, which belongs to the technical field of plant biologication.The method comprises the steps of obtaining of sterile materials, regeneration culture of the leaves, subculture of regenerated buds, rooting culture of regenerated seedlings and acclimatization and transplantation. According to the method, after the tender stem explants are collected from a field and induced to obtain the sterile blackberry seedlings, the adventitious buds are subjected to direct organ generation type regeneration induction by using the leaves of the sterile seedlings, and finally the complete plants are obtained, so that a new explant source is provided for blackberry tissue culture, and a construction method of a direct organ generation type regeneration way is established; and a foundation is laid for somatic cell mutagenesis polyploid breeding and exogenous gene transformation of improved varieties.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a method for constructing a direct organogenesis regeneration pathway of blackberry leaves. Background Art

[0002] Blackberry is a perennial plant belonging to the genus Rubus, Rosaceae. It has antioxidant, anti-aging, and immunity-enhancing effects, and has special effects in lowering cholesterol, preventing heart disease, and fighting cancer. It is praised as the "fruit of life" and "black diamond".

[0003] Traditional blackberry genetic breeding and improvement has a long cycle and low efficiency, but the application of genetic transformation technology based on plant tissue culture and in vitro tissue regeneration in blackberries can greatly promote the germplasm innovation and new variety selection process of blackberries. The premise of plant genetic transformation is to establish a genetically stable and short-cycle regeneration system. The organogenesis pathway in the process of plant tissue culture can be divided into indirect organogenesis and direct organogenesis. Among them, the indirect organogenesis pathway needs to go through the callus stage. This pathway is unstable and is not suitable for genetic engineering breeding with the purpose of trait-oriented improvement. The direct organogenesis pathway can skip the callus stage and directly differentiate organs. Compared with the indirect organogenesis pathway, the direct organogenesis pathway has a short cycle, low mutation rate, and genetic stability, which is more suitable for genetic transformation.

[0004] At present, most blackberry explants are stem segments with axillary buds, but the genetic transformation technology using stem segments with axillary buds as explants is unstable, and the probability of chimeras in transgenic plants is high. At the same time, using leaves as explants is mostly an indirect organogenesis pathway, and there are low induction rates and low differentiation efficiency. Therefore, the adventitious bud induction medium of blackberry leaves was screened, and a direct organogenesis regeneration pathway of blackberry leaves was established, in order to lay a foundation for the genetic transformation and germplasm improvement of blackberry. Summary of the invention

[0005] 1. Technical problems solved

[0006] In view of the above problems, the present invention provides a direct organogenesis regeneration pathway of blackberry leaves. The purpose of the present invention is to explore the factors affecting the regeneration of blackberry leaves using blackberry leaves as materials, establish a direct organogenesis regeneration pathway system of blackberry leaves, provide a method and culture medium for regenerating adventitious buds from blackberry leaves efficiently and quickly, establish an efficient and stable regeneration system for blackberry, and solve the problems of difficulty in regenerating blackberry varieties and difficulty in applying genetic transformation systems in blackberry.

[0007] 2. Technical solution

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] (1) Acquisition of sterile materials: Cut young shoots with buds of the current year in the field, with a length of 5 to 8 cm. Remove the leaves, wash and trim them into stem segments with axillary buds, disinfect them, and inoculate them into MS medium to obtain sterile in vitro plants.

[0010] (2) Leaf regeneration culture: Select the sterile in vitro plant leaves, scratch them, and place them in MS medium to induce regeneration buds;

[0011] (3) Subculture of regenerated buds: The regenerated buds were cultured in WPM medium and subcultured multiple times to obtain regenerated plants.

[0012] (4) Rooting culture of regenerated plants: The regenerated plants were inoculated into 1 / 2 MS medium for rooting culture.

[0013] (5) Hardening and transplanting: Remove the bottle caps of the rooted tissue culture seedlings about 3 to 5 cm in height and place them under natural light for 5 to 7 days. Then, take the tissue culture seedlings out of the culture bottles, wash off the root culture medium, and plant them in a matrix mixed with peat and vermiculite (3:1).

[0014] As one of the preferred embodiments of the present invention, when collecting explants in (1), sunny days for more than 3 consecutive days should be selected, and the new buds should be the stem segments with buds at the top of branches that are healthy and free of diseases and insect pests.

[0015] As one of the preferred embodiments of the present invention, the cleaning method in (1) is: soaking with a dilute detergent solution and rinsing with tap water.

[0016] As one of the preferred embodiments of the present invention, the method for pruning into stem segments with axillary buds in (1) is: cutting the new shoots into single axillary bud stem segments of about 1 to 1.5 cm.

[0017] As one of the preferred embodiments of the present invention, the disinfection treatment (1) is as follows: sterilize on a clean bench, sterilize the surface with 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak and shake in 2% NaClO solution for 7 minutes, and rinse with sterile water for 5 times.

[0018] As one of the preferred embodiments of the present invention, the culture medium in (1) is:

[0019] MS+6-BA2.0mg / L+NAA0.1mg / L+sucrose30g / L+agar6g / L, pH 5.8. Then light culture, light intensity is 2000lx, light time is 16h / d; the temperature of the culture room is 25±2℃.

[0020] As one of the preferred embodiments of the present invention, the culture medium for leaf regeneration culture in (2) is: MS+TDZ 4.0 mg / L+6-BA 1.5 mg / L+NAA 0.3 mg / L+sucrose 30 g / L+agar 6 g / L, with a pH of 5.8.

[0021] As one of the preferred embodiments of the present invention, the scratching method in (2) is to make 2 to 4 cuts perpendicular to the main veins of the leaf, and then place the leaf with the back side facing downward.

[0022] As one of the preferred embodiments of the present invention, the leaf regeneration culture environment conditions in (2) are first dark culture for 10 days, with the culture room temperature at 25±2°C, and then light culture, with a light intensity of 2000 lx, a light time of 16 h / d, and a culture room temperature of 25±2°C.

[0023] As one of the preferred embodiments of the present invention, the (3) regenerated bud subculture medium is: WPM+6-BA0.6mg / L+NAA0.1mg / L+GA30.05mg / L+sucrose 30g / L+agar 6g / L, with a pH of 5.8.

[0024] As one of the preferred embodiments of the present invention, the culture environment (3) is a light intensity of 3000 lx, a light duration of 16 h / d, and a culture temperature of 25±2°C.

[0025] As one of the preferred embodiments of the present invention, the rooting culture (4) is: 1 / 2MS+IBA1.0mg / L+sucrose 30g / L+agar 6g / L, pH 6.0.

[0026] As one of the preferred embodiments of the present invention, the culture environment (4) is first cultured in the dark for 10 days at a culture room temperature of 25±2°C, and then cultured in light with a light intensity of 2500 lx and a light duration of 16 h / d; the culture room temperature is 25±2°C.

[0027] 3. Beneficial effects

[0028] Compared with the prior art, the present invention provides a direct organogenesis regeneration pathway for blackberry leaves, using sterile blackberry seedling leaves as explants to achieve regeneration without an indirect callus approach. This method has a short cycle, low mutation rate, and genetic stability, and is more suitable for genetic transformation. The method of the present invention solves the problem of difficulty in regenerating adventitious buds from blackberry leaves, and the adventitious bud induction rate reaches more than 85%, which is much higher than the leaf regeneration efficiency reported previously. The method of the present invention provides technical support for research on genetic transformation, gene editing, and gene function of blackberries. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The invention relates to obtaining the sterile blackberry material provided by the embodiment of the present invention.

[0031] Figure 2 The invention provides a method for directly inducing adventitious bud regeneration from blackberry leaves. Figure 1 .

[0032] Figure 3 The invention provides a method for directly inducing adventitious bud regeneration from blackberry leaves by microscope observation. Figure 2 .

[0033] Figure 4 The invention provides a method for indirectly inducing adventitious bud regeneration using blackberry leaves. Figure 3 .

[0034] Figure 5 The invention relates to the blackberry adventitious bud proliferation provided by the embodiment of the present invention.

[0035] Figure 6 The blackberry rooting method provided by the embodiment of the present invention Figure 1 .

[0036] Figure 7 The blackberry rooting method provided by the embodiment of the present invention Figure 2 .

[0037] Figure 8 The blackberry transplanting method provided by the embodiment of the present invention is Figure 1 .

[0038] Fig. 9 The blackberry transplanting method provided by the embodiment of the present invention is Figure 2 .

[0039] Fig.10 The blackberry transplanting method provided by the embodiment of the present invention is Figure 3

[0040] Example 1

[0041] A direct organogenesis regeneration pathway for blackberry-190 leaves, comprising the following steps:

[0042] (1) Collection of explants: On sunny days for more than 3 consecutive days, young shoots with buds of the current year were cut from the field of Zibo Academy of Agricultural Sciences. The shoots should be healthy, free of pests and diseases, and 5 to 8 cm in length. Remove the leaves, soak in a dilute detergent solution, and rinse with tap water. Then cut the new shoots into single axillary bud stem segments of about 1 to 1.5 cm. Sterilize on a clean bench, sterilize the surface with 75% alcohol for 30 seconds, rinse with sterile water 3 times, soak and shake in 2% NaClO solution for 7 minutes, and rinse with sterile water 5 times. The culture medium is: MS + 6-BA 2.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. Then light culture, the light intensity is 2000 lx, the light time is 16 h / d; the culture room temperature is 25 ± 2 °C.

[0043] (2) Leaf regeneration culture: Select the leaves of tissue culture seedlings, make 2 to 4 cuts perpendicular to the main veins, and then place them with the back of the leaves facing down on MS medium to induce regeneration buds. The medium for leaf regeneration culture is MS + TDZ 4.0 mg / L + 6-BA 1.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. The environmental conditions for leaf regeneration culture are dark culture for 10 days, and the culture room temperature is 25 ± 2 ° C. Then light culture, the light intensity is 2000 lx, the light time is 16 h / d; the culture room temperature is 25 ± 2 ° C.

[0044] (3) Subculture of regenerated buds: The regenerated buds are cultured in WPM medium and subcultured multiple times to obtain regenerated plants. The subculture medium for regenerated buds (3) is:

[0045] WPM+6-BA0.6mg / L+NAA0.1mg / L+GA30.05mg / L+sucrose 30g / L+agar 6g / L, pH 5.8. The culture environment is a light intensity of 3000lx, a light time of 16h / d, and a culture temperature of 25±2℃.

[0046] (4) Rooting culture of regenerated seedlings: The regenerated plants were inoculated into 1 / 2MS medium for rooting culture. The rooting culture was: 1 / 2MS + IBA 1.0 mg / L + sucrose 30 g / L + agar 6 g / L, pH 6.0. The culture environment was first cultured in the dark for 10 days, and the culture room temperature was 25±2℃. Then the light culture was carried out, with a light intensity of 2500 lx and a light time of 16 h / d; the culture room temperature was 25±2℃.

[0047] Example 2

[0048] In the embodiments of the present invention, a process for screening the amount of hormone added for the direct organogenesis regeneration pathway of blackberry leaves is provided, which is as follows:

[0049] Select the leaves of tissue culture seedlings, cut 2 to 4 times vertically to the main veins, and then place the leaves with the back side facing down on MS culture medium to induce regeneration buds. In order to further optimize the treatment method of hormone addition method for the direct organogenesis regeneration pathway of blackberry leaves, an orthogonal experiment was designed. The present invention designed a total of 9 treatments as shown in Table 1. The environmental conditions for leaf regeneration culture were dark culture for 10 days, and the culture room temperature was 25±2°C. Then light culture was carried out, with a light intensity of 2000lx and a light time of 16h / d; the culture room temperature was 25±2°C. 30 leaves were treated for each treatment, and each treatment was repeated 3 times.

[0050] Table 1 Effects of different concentrations of plant growth regulators on blackberry bud induction

[0051]

[0052] From the above experimental data, it can be seen that, as shown in Table 1. The optimal induction medium for adventitious buds was screened as MS basic medium with the addition of MS + TDZ 4.0 mg / L + 6-BA 1.5 mg / L + NAA 0.4 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. The adventitious bud induction rate under this condition reached more than 83.33%. On this basis, the adventitious bud induction medium was fine-tuned, and it was found that when MS + TDZ 4.0 mg / L + 6-BA 1.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 6 g / L was added to the MS basic medium, the pH was 5.8, and the adventitious bud induction rate reached more than 85%.

[0053] Example 3

[0054] In the examples of the present invention, experiments on the effects of different culture media and hormone concentrations on the rooting of blackberry tissue culture seedlings are given. The details are as follows:

[0055] The experiment was based on the basic culture medium. When the cluster buds grew to 2.5 cm in height, the proliferated buds were cut and transferred to the rooting culture medium on the clean bench. The added plant hormone was auxin IBA, with concentrations of 0 mg / L, 0.5 mg / L, 1.0 mg / L, and 1.5 mg / L. Three buds were inoculated in each bottle, and 30 buds were inoculated in each treatment. The experiment was repeated three times. The dark culture was first carried out for 10 days, and the temperature of the culture room was 25±2℃. Then the light culture was carried out, with a light intensity of 2500lx and a light time of 16h / d; the temperature of the culture room was 25±2℃. After 30 days, the effects of different concentrations of hormone IBA and culture medium on the rooting of sterile blackberry seedlings were compared.

[0056] Table 2 Screening of blackberry adventitious root induction medium and hormone concentration

[0057]

[0058] The effect of different culture media and hormone concentrations on the rooting of blackberry tissue culture seedlings. As shown in Table 2, 1 / 2MS culture medium is better than MS culture medium in inducing rooting of blackberry. Under IBA treatment, blackberry has obvious rooting phenomenon. When the rooting induction culture is about 30 days, when the IBA concentration is 1.0mg / L, the rooting rate in 1 / 2MS culture medium reaches the maximum value, which is 100%, with a large number of roots and capillary roots, and the best rooting effect; in summary, the rooting culture medium is: 1 / 2MS+IBA1.0mg / L+I sucrose 30g / L+agar 6g / L, pH 5.8.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for constructing a direct organogenic regeneration pathway of blackberry leaves, characterized in that: The following steps are involved: (1) Acquisition of sterile materials: Cut young shoots with buds of the current year in the field, with a length of 5 to 8 cm. Remove the leaves, wash and trim them into stem segments with axillary buds, disinfect them, and inoculate them into MS medium to obtain sterile in vitro plants. (2) Leaf regeneration culture: Select the sterile in vitro plant leaves, scratch them, and place them in MS medium to induce the production of regenerated buds; (3) Subculture of regenerated buds: The regenerated buds were cultured in WPM medium and subcultured multiple times to obtain regenerated plants. (4) Rooting culture of regenerated plants: The regenerated plants were inoculated into 1 / 2 MS medium for rooting culture. (5) Hardening and transplanting: Remove the bottle caps of the rooted tissue culture seedlings about 3 to 5 cm in height and place them under natural light for 5 to 7 days. Then, take the tissue culture seedlings out of the culture bottles, wash off the root culture medium, and plant them in a matrix mixed with peat and vermiculite (3:1).

2. A method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: When collecting explants in (1) above, sunny days for more than 3 consecutive days should be selected, and the new buds should be the stem segments with buds at the top of branches that are healthy and free of diseases and insect pests.

3. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The cleaning method in (1) is: soaking in a dilute detergent solution and rinsing with tap water.

4. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The method for pruning into stem segments with axillary buds in (1) is: cutting the new shoots into single axillary bud stem segments of about 1 to 1.5 cm.

5. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The (1) disinfection treatment is as follows: sterilize on a clean bench, sterilize the surface with 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak and shake in 2% NaClO solution for 7 minutes, and rinse with sterile water for 5 times.

6. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The culture medium in (1) is: MS+6-BA 2.0 mg / L+NAA 0.1 mg / L+sucrose 30 g / L+agar 6 g / L, pH 5.

8. Then, the culture is illuminated, the illumination intensity is 2000 lx, the illumination time is 16 h / d, and the culture room temperature is 25±2°C.

7. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The culture medium for leaf regeneration culture in (2) is: MS+TDZ 4.0 mg / L+6-BA 1.5 mg / L+NAA 0.3 mg / L+sucrose 30 g / L+agar 6 g / L, with a pH of 5.

8.

8. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The scratching method in (2) is to make 2 to 4 cuts perpendicular to the main veins of the leaf, and then place the leaf with the back side facing downward.

9. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The leaf regeneration culture environment conditions in (2) are dark culture for 10 days at a culture room temperature of 25±2°C, followed by light culture at a light intensity of 2000 lx and a light duration of 16 h / d at a culture room temperature of 25±2°C.

10. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The (3) regenerated bud subculture medium is: WPM+6-BA0.6mg / L+NAA0.1mg / L+GA30.05mg / L+sucrose 30g / L+agar 6g / L, pH is 5.

8.

11. A method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The (3) culture environment is as follows: the light intensity is 3000 lx, the light time is 16 h / d, and the culture temperature is 25±2°C.

12. A method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The rooting culture (4) is: 1 / 2MS+IBA1.0mg / L+sucrose 30g / L+agar 6g / L, pH is 6.

0.

13. The method for constructing a direct organogenic regeneration pathway of blackberry leaves according to claim 1, characterized in that: The culture environment (4) is first dark cultured for 10 days, with the culture room temperature at 25±2°C, and then light cultured, with a light intensity of 2500 lx and a light duration of 16 h / d; the culture room temperature is 25±2°C.

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