Mulberry positive embryogenic callus and preparation method thereof

Abstract

The invention relates to a genetic transformation method of mulberry positive embryogenic calluses and a preparation method of the mulberry positive embryogenic calluses by researching the infection mode, infection strain, infection liquid concentration and the like of mulberry. An effective transgenic platform is provided for subsequent research of mulberry, and a solid foundation is laid for cultivation from mulberry positive calluses to positive seedlings.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a positive embryogenic callus of Morus notabilis C.K.Schneid. and a preparation method thereof. Background Art

[0002] Morus notabilis C.K.Schneid. is a deciduous tree or shrub in the order Rosales, family Moraceae, and genus Morus, and is an important economic crop. Mulberry leaves are the main food source of silkworms, and the whole mulberry tree is full of treasures. Mulberry leaves, mulberry bark, mulberries, mulberry branches, etc. all have certain medicinal functions.

[0003] Genetically modified breeding (GMB) refers to a breeding method in which one or more genes are added to a biological genome through modern molecular biology techniques to produce organisms with improved characteristics. Genetically modified breeding has the advantages of increasing crop yield and quality, improving nutritional value, reducing the use of pesticides and fertilizers, and shortening the breeding time. However, there are relatively few reports on the genetic transformation of mulberry trees in the existing literature. Although there are several Indian literatures reporting the genetic transformation of mulberry trees, the results show poor repeatability when experiments are carried out with their materials. There are also some experiments that obtained positive callus by transforming hypocotyls, but the method of using hypocotyls for transformation is cumbersome and has poor repeatability (because each explant comes from different seeds and has different genetic backgrounds), and the positive transformation efficiency is low. Therefore, most current mulberry research teams are trapped in the genetic transformation of mulberry trees.

[0004] The basic chromosome number of most cultivated mulberry trees is 14, while Morus notabilis C.K.Schneid. is a diploid material with a basic chromosome number of 7. There are currently no reports on any genetic transformation related to mulberry tree materials with a basic chromosome number of 7. Summary of the Invention

[0005] Based on the above problems, the purpose of the present invention is to provide a positive embryogenic callus of Morus notabilis C.K.Schneid. and a preparation method thereof. This method is simple, efficient, and has high repeatability, can provide an effective transgenic platform for the research of Morus notabilis C.K.Schneid., and lay a solid foundation for the transformation of positive callus of Morus notabilis C.K.Schneid. into positive seedlings.

[0006] The technical solutions to achieve the above purpose are as follows.

[0007] In the first aspect of the present invention, a preparation method of a positive embryogenic callus of Morus notabilis C.K.Schneid. is provided, including the following steps:

[0008] (1) Take healthy Morus notabilis C.K.Schneid. leaves that are fully expanded and place them in a pre-culture medium for culture to obtain explant leaves;

[0009] (2) Integrate the betacyanin synthesis gene cluster RUBY into the binary expression vector, transfer the vector into Agrobacterium tumefaciens K599, culture Agrobacterium tumefaciens K599 with LB medium until OD 600 = 1 - 1.4, collect the bacteria, suspend the bacteria with the infection solution until the bacterial liquid concentration OD 600 = 0.10 - 0.30, let it stand to obtain a bacterial suspension;

[0010] (3) Put the explant leaves into the bacterial suspension, culture them in the dark on a shaker, then infect them in a syringe until there is obvious liquid infiltration at the leaf incision, take out the explants, blot dry the water, and place them on the co-culture medium for culture;

[0011] (4) Take out the explants after co-culture, place them on the selection medium, change the medium once every 2 - 3 weeks, culture until red callus points appear to obtain positive embryogenic callus.

[0012] In some of the embodiments, step (3) is: Take out the syringe piston, put the explant leaves into the syringe barrel, install the piston, suck in the bacterial suspension, expel the gas in the syringe barrel, cover the screw seal of the liquid suction port, culture in the dark on a shaker, take out, gently push the syringe piston until there is obvious liquid infiltration at the leaf incision, take out the piston, blot dry the explants with sterile filter paper, place them on the co-culture medium, cover with a layer of sterile filter paper, and conduct co-culture;

[0013] In some of the preferred embodiments, the specification of the syringe is 50 mL.

[0014] In some of the embodiments, the co-culture medium is composed of the following components: sucrose (18 - 22) g / L, agar (8 - 10) g / L, ZR (1.8 - 2.2) mg / L, 2,4-D (0.08 - 0.12) mg / L, AS (35 - 45) mg / L, MS, pH (5.2 - 5.6).

[0015] In some of the embodiments, step (2) is: Integrate the betacyanin synthesis gene cluster RUBY into the binary expression vector, transfer the vector into Agrobacterium tumefaciens K599, culture Agrobacterium tumefaciens K599 with LB medium until OD 600 = 1.15 - 1.25, collect the bacteria, suspend the bacteria with the infection solution until the bacterial liquid concentration OD 600 = 0.1 - 0.25, let it stand for 1.8 - 2.2 hours to obtain a bacterial suspension.

[0016] In some of the preferred embodiments, suspend the bacteria with the infection solution until the bacterial liquid concentration 0D 600 = 0.10 - 0.20, more preferably 0.195 - 0.215.

[0017] In some of these embodiments, the infection solution consists of the following components: MES 1.8 g / L - 2.2 g / L, MgCl2 1.8 g / L - 2.2 g / L, AS 15 mg / L - 25 mg / L, pH 5.2 - 5.6.

[0018] In some of the preferred embodiments among them, the infection solution consists of the following components: MES 2 g / L, MgCl2 2 g / L, AS 20 mg / L, pH 5.4.

[0019] In some of these embodiments, in step (1), the Morus notabilis leaves are obtained by subculture of Morus notabilis under tissue culture conditions.

[0020] In some of these embodiments, the subculture is to subculture Morus notabilis under tissue culture conditions for 15 - 35 days, with subculture once every 2 - 4 weeks. The medium for the subculture consists of the following components: sucrose (25 - 35) g / L, agar (5 - 8) g / L, 6 - BA (0.4 - 0.6) mg / L, MS, pH (5.5 - 5.9).

[0021] The medium for the subculture is: sucrose (25 - 35) g / L + agar (5 - 8) g / L + 6 - BA (0.4 - 0.6) mg / L + pH (5.5 - 5.9) + MS, with subculture once every 2 - 4 weeks and subculture for 30 days. The materials subcultured for about 30 days have the best growth and are most suitable for subsequent culture operations.

[0022] In some of these embodiments, the LB medium in step (2) is: Tryptone (8 - 12) g / L, Yeast extract (4 - 6) g / L, Sodium chloride (NaCl) (8 - 12) g / L, Kanamycin (40 - 60) mg / L.

[0023] In some of these embodiments, the screening medium in step (4) is: sucrose (25 - 35) g / L, agar (6 - 10) g / L, ZR (1.8 - 2.2) mg / L, 2,4 - D (0.08 - 0.12) mg / L, Tim (250 - 350) mg / L, Kan (45 - 55) mg / L, MS, pH (5.5 - 6).

[0024] The second aspect of the present invention is to provide a positive embryogenic callus of Morus notabilis obtained by the aforementioned preparation method.

[0025] Through research on aspects such as the infection mode, infecting strains, and concentration of the infection solution of *Morus notabilis*, a positive embryogenic callus of *Morus notabilis* and its preparation method are obtained. This method can obtain a high frequency of positive embryogenic callus, provide an effective transgenic platform for subsequent research on *Morus notabilis*, and lay a solid foundation for the cultivation of positive seedlings from positive callus of *Morus notabilis*. Brief Description of the Drawings

[0026] Figure 1 is the embryogenic callus of *Morus notabilis*.

[0027] Figure 2 is the operation diagram of the improved infection transformation method of the present invention.

[0028] Figure 3 is the growth situation of *Morus notabilis* at different times during one subculture.

[0029] Figure 4 is the difference between the explant leaves during traditional infection and the improved infection of the present invention.

[0030] Figure 5 is the positive embryogenic callus of *Morus notabilis*. Detailed Embodiments

[0031] For ease of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0032] The experimental methods without specific conditions noted in the following examples are generally in accordance with conventional conditions, for example, the fourth edition of *Molecular Cloning: A Laboratory Manual* edited by Green and Sambrook was published in 2013, or in accordance with the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0034] The following further elaborates on the present invention in conjunction with specific embodiments.

[0035] Example 1: Medium Screening

[0036] Based on the basic formula (MS, 30 g / L sucrose, 6 g / L agar, pH 5.7), different hormones were added to prepare the culture medium. After cutting the leaves of Morus notabilis explants, they were directly placed on the culture medium to induce embryogenic callus. The results are shown in Table 1. From the callus state, it can be seen that the callus effect of medium No. 9 is the best( Figure 1 ). The culture media used later were all prepared by adding the drugs required during the transformation process (such as AS) to medium No. 9.

[0037] Table 1 Callus states of culture media with different formulations (unit: mg / L)

[0038]

[0039]

[0040] Example 2

[0041] 1. The method for preparing positive embryogenic callus of Morus notabilis described in this example includes the following steps:

[0042] (1) Explant pre-culture

[0043] The subculture medium is: 30 g / L sucrose, 6.5 g / L agar, MS, pH 5.6.

[0044] Take the leaves of Morus notabilis, place them on the subculture medium, and subculture them under tissue culture conditions (room temperature 25 ± 1°C, humidity 40 - 50%, LED light illumination for 16 hours, darkness for 8 hours). Replace the subculture medium every 3 weeks. After 30 days of culture, take the fully expanded and healthy leaves of Morus notabilis and put them into the pre-culture medium (the pre-culture medium used here is the subculture medium) for culture to retain moisture and obtain explant leaves.

[0045] (2) Preparation of bacterial suspension

[0046] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 mg / L kanamycin.

[0047] The composition of the infection solution is: 2 g / L MES, 2 g / L MgCl2, 20 mg / L AS, pH 5.4.

[0048] Agrobacterium tumefaciens GV3101 bacterial suspension: Integrate the betacyanin synthesis gene cluster RUBY into a binary expression vector (for the specific method, see A reporter for noninvasively monitoring gene expression and plant transformation Yubing He, Tao Zhang, Hui Sun, Huadong Zhan and Yunde Zhao). Transfer the vector into Agrobacterium tumefaciens GV3101, culture Agrobacterium tumefaciens GV3101 in LB medium until OD 600 = 1.2, collect the bacterial cells, suspend the bacterial cells in the infection solution until the concentration of the bacterial suspension is OD 600 = 0.2, and let it stand for 2 hours for later use.

[0049] Agrobacterium rhizogenes K599 bacterial suspension: Integrate the betacyanin synthesis gene cluster RUBY into a binary expression vector, transfer the vector into Agrobacterium rhizogenes K599, culture Agrobacterium rhizogenes K599 until OD 600 = 1.2, collect the bacterial cells, suspend the bacterial cells in the infection solution to different concentrations (Table 1), and let it stand for 2 hours for later use.

[0050] (3) Infection method and co-culture

[0051] The co-culture medium is: 20 g / L sucrose + MS + 9 g / L agar + 2 mg / L ZR + 0.1 mg / L 2,4-D + 40 mg / L AS + pH 5.4.

[0052] The improved infection method of the present invention: Take a 50 mL syringe, remove the piston of the syringe, put the explant leaf into the syringe barrel, install the piston, suck in the Agrobacterium tumefaciens GV3101 bacterial suspension and the Agrobacterium rhizogenes K599 bacterial suspension with different concentrations respectively, expel the gas in the syringe barrel, cover the screw seal of the liquid suction port, place it on a shaker and culture it in the dark for 20 min, take it out, and gently push the piston of the syringe until there is obvious liquid infiltration at the cut of the leaf. The operation process is as Figure 1 shown.

[0053] This study also explored the differences from traditional infection: Each step is the same as that of the present invention, except that the infection method is different. Traditional infection: The explant leaf is soaked in the bacterial suspension with each concentration for 40 min for infection.

[0054] Co-culture: Take out the explant leaf, dry it with sterile filter paper, place it on the co-culture medium, cover it with a layer of sterile filter paper, place it in an incubator, and co-culture it in the dark for 3 days under the conditions of indoor temperature 25 ± 1°C and humidity 40 - 50%.

[0055] (4) Screening culture

[0056] The screening medium is: sucrose 30 g / L + MS + agar 8 g / L + ZR 2 mg / L + 2,4-D 0.1 mg / L + Tim 300 mg / L + Kan 50 mg / L + pH 5.8.

[0057] After co-culture, the explants are taken out and placed on the screening medium. The medium is changed every 2 - 3 weeks until red callus points appear, and embryogenic callus after genetic transformation is obtained.

[0058] 2. Result analysis

[0059] 2.1 Subculture time

[0060] The results show ( Figure 3 ) that the best time for subculture once is 2 - 4 weeks. If it exceeds 5 weeks, the materials will necrose. And the materials subcultured for about 30 days grow best and are most suitable for subsequent experiments.

[0061] 2.2 Infection method

[0062] Using the betacyanin synthesis gene cluster RUBY as the reporter system, in order to more conveniently observe the test results, in this experiment, the mulberry material YH with obvious transient transformation phenotype is used. This material is the seeds obtained from the hybridization of Yun 7 (2n = 8x = 56) and Morus cathayana (2n = 4x = 28). The seeds are sterilized to obtain sterile seedlings.

[0063] When using the traditional infection method, only sporadic red signals can be seen on the explant leaves ( Figure 4 A), and when infecting with the improved infection method of the present invention, red signals can be seen in the whole explant leaves ( Figure 4 B), and the transformation efficiency is significantly improved.

[0064] 2.3 Agrobacterium species, concentration of bacterial suspension and infection method (Table 1)

[0065] As shown in Table 1, when Agrobacterium tumefaciens GV3101 is used, there are no positive leaves, indicating that its infection rate is extremely low.

[0066] When the concentration of the bacterial suspension of Agrobacterium K599 reaches 0.4 and in the improved infection, due to the too high concentration, over-infection of the leaves occurs, so the positive rate is only 6.25%. While at a low concentration of 0.1, the positive rate is only 10% because the bacterial solution is too little.

[0067] It can also be seen from Table 1 that when using the traditional infection, the positive conversion rate of the leaves is extremely low, while the highest positive rate of the improved infection method of the present invention can reach 31%. The positive embryogenic callus of M. notabilis is as Figure 5 shown.

[0068] Table 1 Statistical results of different Agrobacterium species, concentration of bacterial suspension, and infection methods

[0069]

[0070] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for preparing positive embryogenic callus of Morus notabilis Schneid., characterized in that It includes the following steps: (1) Take fully expanded healthy Morus notabilis leaves, place them in a pre-culture medium for culture to obtain explant leaves; (2) Integrate the betacyanin synthesis gene cluster RUBY into a binary expression vector, transfer the vector into Agrobacterium tumefaciens K599, culture Agrobacterium tumefaciens K599 in LB medium until OD 600 = 1 - 1.4, collect the bacterial cells, suspend the bacterial cells in an infection solution until the bacterial liquid concentration OD 600 = 0.10 - 0.30, and obtain a bacterial suspension after standing; (3) Put the explant leaves into the bacterial suspension, culture them in the dark on a shaker, and then infect them in a syringe until there is obvious liquid infiltration at the leaf incision. Take out the explants, blot them dry, and place them on a co-culture medium for culture; (4) After co-culture, take out the explants, place them on a selection medium, change the medium once every 2 - 3 weeks, and culture until red callus points appear to obtain positive embryogenic callus.

2. The preparation method of positive embryogenic callus of *Morus notabilis* as described in claim 1, characterized in that: The step (3) is as follows: Take out the piston of the syringe, put the explant leaves into the syringe barrel, install the piston, suck in the bacterial suspension, expel the gas in the syringe barrel, cover the screw seal of the liquid suction port, place it on a shaker for dark culture, take it out, gently push the syringe piston until there is obvious liquid infiltration at the leaf incision, take out the piston, blot the explants dry with sterile filter paper, place them on a co-culture medium, cover them with a layer of sterile filter paper, and conduct co-culture. Preferably, the syringe is 50 mL.

3. The preparation method of positive embryogenic callus of *Morus notabilis* as described in claim 1, characterized in that: The co-culture medium consists of the following components: sucrose (18 - 22) g / L, agar (8 - 10) g / L, ZR (1.8 - 2.2) mg / L, 2,4-D (0.08 - 0.12) mg / L, AS (35 - 45) mg / L, MS, pH (5.2 - 5.6).

4. The preparation method of positive embryogenic callus of *Morus notabilis* as described in any one of claims 1-3, characterized in that: The step (2) is: integrating the betacyanin synthesis gene cluster RUBY into a binary expression vector, transferring the vector into Agrobacterium tumefaciens K599, culturing Agrobacterium tumefaciens K599 with LB medium until OD 600 = 1.15 - 1.25, collecting the bacterial cells, suspending the bacterial cells with an infection solution until the bacterial liquid concentration OD 600 = 0.1 - 0.25, standing for 1.8 - 2.2 hours to obtain a bacterial suspension. Preferably, the bacterial cells are suspended with an infection solution until the bacterial liquid concentration OD 600 = 0.10 - 0.20, more preferably 0.195 - 0.

215.

5. The preparation method of positive embryogenic callus of *Morus notabilis* as described in claim 4, characterized in that: The infection solution consists of the following components: MES 1.8 g / L - 2.2 g / L, MgCl2 1.8 g / L - 2.2 g / L, AS 15 mg / L - 25 mg / L, pH 5.2 - 5.

6. Preferably, the infection solution consists of the following components: MES 2 g / L, MgCl2 2 g / L, AS 20 mg / L, pH 5.

4.

6. The preparation method of positive embryogenic callus of *Morus notabilis* as described in any one of claims 1-3, characterized in that: In step (1), the Morus notabilis leaves are obtained by subculture of Morus notabilis under tissue culture conditions.

7. The preparation method of positive embryogenic callus of *Morus notabilis* as described in claim 6, characterized in that: The subculture is to subculture Morus notabilis under tissue culture conditions for 15 - 35 days, with subculture once every 2 - 4 weeks. The medium for subculture consists of the following components: sucrose (25 - 35) g / L, agar (5 - 8) g / L, 6-BA (0.4 - 0.6) mg / L, MS, pH (5.5 - 5.9).

8. The method for preparing positive embryogenic callus of *Morus notabilis* as described in any one of claims 1 to 3, characterized in that: The LB medium in step (2) is: Tryptone (8 - 12) g / L, Yeast extract (4 - 6) g / L, Sodium chloride (NaCl) (8 - 12) g / L, Kanamycin (40 - 60) mg / L.

9. The method for preparing positive embryogenic callus of *Morus notabilis* as described in any one of claims 1-3, characterized in that: The selection medium in step (4) is: sucrose (25 - 35) g / L, agar (6 - 10) g / L, ZR (1.8 - 2.2) mg / L, 2,4-D (0.08 - 0.12) mg / L, Tim (250 - 350) mg / L, Kan (45 - 55) mg / L, MS, pH (5.5 - 6).

10. The positive embryogenic callus of Morus notabilis obtained by the preparation method according to any one of claims 1 - 9.

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