A positive embryogenic callus of morus notabilis and a preparation method thereof

Abstract

The present application relates to a kind of genetic transformation methods of morus notabilis embryonic callus, which is obtained by the research of the infection mode, infection species, infection liquid concentration and other aspects of morus notabilis, and a kind of morus notabilis positive embryonic callus and its preparation method, which can obtain high-frequency positive embryonic callus, provide effective transgenic platform for the subsequent morus notabilis research, and lay a solid foundation for the cultivation of morus notabilis positive callus to positive seedling.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a positive embryogenic callus of *Morus alba* and its preparation method. Background Technology

[0002] Mulberry (Morus notabilis CK Schneid..) is a deciduous tree or shrub belonging to the order Rosales, family Moraceae, and genus Morus. It is an important economic crop. Mulberry leaves are the main food source for silkworms, and the mulberry tree is valuable in many ways; its leaves, bark, fruit, and branches all have certain medicinal properties.

[0003] Genetically modified breeding (GMB) refers to breeding methods that use modern molecular biology techniques to add one or more genes to the genome of an organism, thereby producing organisms with improved characteristics. GMB has advantages such as increasing crop yield and quality, improving nutritional value, reducing the use of pesticides and fertilizers, and shortening breeding time. However, there are relatively few reports on mulberry GM research. Although several papers from India have reported on mulberry GM, experiments using their materials showed very poor reproducibility. Some experiments have also attempted to transform hypocotyls to obtain positive callus tissue, but this method is cumbersome and has poor reproducibility (because each explant comes from a different seed with a different genetic background), resulting in low positive transformation efficiency. Therefore, most mulberry research teams are currently stuck on the genetic transformation of mulberry.

[0004] Most cultivated mulberry trees have a chromosome number of 14, while Sichuan mulberry is a diploid material with a chromosome number of 7. There are currently no reports on genetic transformation of mulberry materials with a chromosome number of 7. Summary of the Invention

[0005] Based on the above problems, the purpose of this invention is to provide a positive embryogenic callus of *Malus chuanxiong* and its preparation method. This method is simple, efficient, and highly reproducible, and can provide an effective transgenic platform for the study of *Malus chuanxiong*, laying a solid foundation for the transformation of positive callus of *Malus chuanxiong* into positive seedlings.

[0006] The technical solution to achieve the above objectives is as follows.

[0007] The first aspect of this invention is to provide a method for preparing positive embryogenic callus tissue from *Lonicera japonica*, comprising the following steps:

[0008] (1) Take fully expanded healthy leaves of *Morus alba* and place them in a pre-culture medium to obtain explant leaves;

[0009] (2) The betaine synthesis gene cluster RUBY was integrated into a binary expression vector, which was then transformed into Agrobacterium K599. Agrobacterium K599 was cultured to OD using LB medium. 600 =1-1.4, collect bacterial cells, and suspend the bacterial cells in the infection solution until the bacterial concentration reaches OD100. 600 =0.10-0.30, after standing, a bacterial suspension is obtained;

[0010] (3) Place the explant leaves into the bacterial suspension, culture them in the dark on a shaker, and then infect them with a syringe until there is obvious liquid seepage at the cut of the leaf. Remove the explant, absorb the water, and place it on a co-culture medium for culture.

[0011] (4) After co-culture, the explants are removed and placed on the selection medium. The medium is changed every 2-3 weeks. The culture is continued until red callus points appear, and positive embryogenic callus tissue is obtained.

[0012] In some of the embodiments, step (3) is as follows: take out the syringe plunger, put the explant leaf into the syringe, attach the plunger, aspirate the bacterial suspension, expel the gas from the syringe, cover the spiral seal cap of the liquid aspiration port, place it on a shaker for dark culture, take it out, gently push the syringe plunger until there is obvious liquid seepage at the leaf cut, take out the plunger, dry the explant with sterile filter paper, place it on the co-culture medium, cover it with a layer of sterile filter paper, and co-culture.

[0013] In some preferred embodiments, the syringe has a capacity of 50 mL.

[0014] In some of these embodiments, 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, and pH (5.2-5.6).

[0015] In some embodiments, step (2) involves: integrating the betaine synthesis gene cluster RUBY into a binary expression vector, transforming the vector into Agrobacterium K599, and culturing Agrobacterium K599 to OD using LB medium. 600 =1.15-1.25, collect the bacterial cells, and suspend the bacterial cells in the infection solution until the bacterial concentration reaches OD0.05. 600 =0.1-0.25, let stand for 1.8-2.2 hours to obtain bacterial suspension.

[0016] In some preferred embodiments, the bacterial cells are suspended in an infection solution to a bacterial concentration of 0D. 600 =0.10-0.20, more preferably 0.195-0.215.

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

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

[0019] In some of these embodiments, in step (1), the leaves of *Malus baccata* are obtained by subculturing *Malus baccata* under tissue culture conditions.

[0020] In some of the embodiments, the subculture involves subculturing *Lonicera japonica* under tissue culture conditions for 15-35 days, with subculture occurring every 2-4 weeks. The culture 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, and pH (5.5-5.9).

[0021] The culture medium for subculture was: sucrose (25-35) g / L + agar (5-8) g / L + 6-BA (0.4-0.6) mg / L + pH (5.5-5.9) + MS. Subculture was performed every 2-4 weeks for 30 days. The material subcultured for about 30 days showed the best growth and was 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, and kanamycin (40-60) mg / L.

[0023] In some of these embodiments, the screening medium described 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] A second aspect of the present invention is to provide a positive embryogenic callus tissue of *Chuansang* obtained by the aforementioned preparation method.

[0025] This invention, through research on the infection mode, infecting fungal species, and concentration of the infection solution of *Lonicera japonica*, obtains a positive embryogenic callus of *Lonicera japonica* and its preparation method. This method can obtain a high frequency of positive embryogenic callus, providing an effective transgenic platform for subsequent research on *Lonicera japonica* and laying a solid foundation for the cultivation of positive callus into positive seedlings. Attached Figure Description

[0026] Figure 1 It is a mulberry embryonic callus.

[0027] Figure 2 This is an operational diagram of the improved infection and transformation method of the present invention.

[0028] Figure 3 This describes the growth of the Sichuan mulberry at different times during its successive generations.

[0029] Figure 4 This is the difference between traditional infection of explant leaves and the improved infection method of this invention.

[0030] Figure 5 It is a positive embryogenic callus tissue of *Chuansang*. Detailed Implementation

[0031] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0032] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0034] The present invention will be further described in detail below with reference to specific embodiments.

[0035] Example 1: Culture medium screening

[0036] Different hormones were added to the basic formulation (MS, 30 g / L sucrose, 6 g / L agar, pH 5.7) to prepare culture media. Leaves from *Morus alba* explants were cut and placed directly on the culture media to induce embryogenic callus. The results are shown in Table 1. The callus state shows that medium No. 9 had the best callus formation effect. Figure 1 The culture media used later were all made by adding the necessary reagents (such as AS) to the No. 9 culture medium.

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

[0038]

[0039]

[0040] Example 2

[0041] 1. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in this embodiment includes the following steps:

[0042] (1) Pre-culture of explants

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

[0044] Leaves of *Morus alba* were taken and placed on a subculture medium. They were then subcultured under the following conditions: indoor temperature 25±1℃, humidity 40-50%, LED light for 16 hours, and darkness for 8 hours. The subculture medium was changed every 3 weeks. After 30 days of culture, fully expanded healthy leaves of *Morus alba* were taken and placed in a pre-culture medium (the subculture medium used here) to retain moisture and obtain explant leaves.

[0045] (2) Preparation of bacterial suspension

[0046] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L, Kanamycin 50 mg / L.

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

[0048] Agrobacterium tumefaciens GV3101 bacterial suspension: The betaine synthesis gene cluster RUBY was integrated into a binary expression vector (for details, see A reporter for noninvasively monitoring gene expression and plant transformation Yubing He, Tao Zhang Hui Sun, Huadong Zhan and YundeZhao), and the vector was transformed into Agrobacterium tumefaciens GV3101. Agrobacterium tumefaciens GV3101 was cultured in LB medium to OD. 600 =1.2, collect bacterial cells, and suspend the bacterial cells in the infection solution until the bacterial concentration reaches OD100. 600 =0.2, let stand for 2 hours before use.

[0049] Agrobacterium rhizogenes K599 bacterial suspension: The betaine synthesis gene cluster RUBY was integrated into a binary expression vector, which was then transformed into Agrobacterium rhizogenes K599. Agrobacterium rhizogenes K599 was cultured to OD200. 600 =1.2, collect bacterial cells, suspend the bacterial cells in the infection solution to different bacterial concentrations (Table 2), and let stand for 2 hours before use.

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

[0051] The co-culture medium was: 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 this invention is as follows: Take a 50mL syringe, remove the syringe plunger, place the explant leaf into the syringe, attach the plunger, and draw in Agrobacterium tumefaciens GV3101 bacterial suspension and Agrobacterium rhizogenes K599 bacterial suspension at different concentrations, respectively. Expel the gas from the syringe, cover the liquid aspiration port with the screw-on seal cap, and place it on a shaker for incubation in the dark for 20 minutes. Remove it, and gently push the syringe plunger until there is obvious liquid seepage at the leaf cut. The operation procedure is as follows: Figure 1 As shown.

[0053] This study also explored the differences from traditional infection: the steps are the same as those of this invention, but the infection method is different. Traditional infection: explant leaves are soaked in bacterial suspensions of various concentrations for 40 minutes for infection.

[0054] Co-culture: Remove the explant leaves, blot them dry with sterile filter paper, place them on the co-culture medium, cover them with a layer of sterile filter paper, and place them in the culture room. Co-culture in the dark for 3 days under the conditions of room temperature 25±1℃ and humidity 40-50%.

[0055] (4) Screening and cultivation

[0056] The screening medium was: 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 removed and placed on a selection medium. The medium is changed every 2-3 weeks. The culture is continued until red callus points appear, thus obtaining embryogenic callus tissue after genetic transformation.

[0058] 2. Results Analysis

[0059] 2.1 Subculture time

[0060] The results show that ( Figure 3 The optimal time for a single subculture is 2-4 weeks; materials will die after more than 5 weeks. Materials subcultured for about 30 days exhibit the best growth and are most suitable for subsequent experiments.

[0061] 2.2 Infection Methods

[0062] Using the betaine red synthesis gene cluster RUBY as the reporter system, and to facilitate the observation of experimental results, this experiment used the mulberry material YH with a transiently transformed phenotype. This material was obtained by crossing Yun 7 (2n=8x=56) with Huasang (2n=4x=28) seeds. After the seeds were disinfected, sterile seedlings were obtained.

[0063] When using traditional infection methods, only scattered red signals can be seen on the explant leaves. Figure 4 A), when infected using the improved infection method of the present invention, a red signal can be seen throughout the entire explant leaf. Figure 4 (B) The conversion efficiency is significantly improved.

[0064] 2.3 Agrobacterium species, bacterial suspension concentration, and infection method (Table 2)

[0065] As shown in Table 2, when Agrobacterium tumefaciens GV3101 was used, no positive leaves were observed, indicating that its infection rate was extremely low.

[0066] When the concentration of Agrobacterium K599 bacterial suspension reached 0.4, the leaves were over-infected due to the high concentration during improved infection, resulting in a positive rate of only 6.25%. At a low concentration of 0.1, the positive rate was only 10% due to insufficient bacterial suspension.

[0067] As can also be seen from Table 2, the positive conversion rate of leaves is extremely low when using traditional infection methods, while the improved infection method of this invention can achieve a positive rate of up to 31%. Positive embryogenic callus tissue from *Morus alba* is shown in Table 2. Figure 5 As shown.

[0068] Table 2. Statistical results of different Agrobacterium species, bacterial suspension concentrations, and infection methods.

[0069]

[0070] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing positive embryogenic callus from *Morus alba*, characterized in that... Includes the following steps: (1) Take fully expanded healthy leaves of *Morus alba* and place them in a pre-culture medium to obtain explant leaves; (2) The betaine synthesis gene cluster RUBY was integrated into a binary expression vector, which was then transformed into Agrobacterium K599. Agrobacterium K599 was cultured to OD using LB medium. 600 =1-1.4, collect bacterial cells, and suspend the bacterial cells in the infection solution until the bacterial concentration reaches OD100. 600 =0.10-0.30, after standing, a bacterial suspension is obtained; (3) Place the explant leaf into the bacterial suspension, culture it in the dark on a shaker, then remove the syringe plunger, put the explant leaf into the syringe, attach the plunger, draw in the bacterial suspension, expel the gas from the syringe, cover the spiral seal cap of the liquid inlet, place it on a shaker for dark culture, take it out, gently push the syringe plunger until there is obvious liquid seepage at the leaf cut, remove the plunger, dry the explant with sterile filter paper, place it on the co-culture medium, cover it with a layer of sterile filter paper, and co-culture. (4) After co-culture, the explants are removed and placed on the selection medium. The medium is changed every 2-3 weeks. The culture is continued until red callus points appear, and positive embryogenic callus tissue is obtained. 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). The screening medium consists of the following components: 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).

2. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in claim 1, characterized in that: The syringe has a capacity of 50 mL.

3. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in claim 1, characterized in that: The co-culture medium consists of the following components: 20 g / L sucrose, 9 g / L agar, 2.0 mg / L ZR, 0.1 mg / L 2,4-D, 40 mg / L AS, MS, pH 5.

4.

4. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in any one of claims 1-3, characterized in that: Step (2) involves: integrating the betaine synthesis gene cluster RUBY into a binary expression vector, transforming the vector into Agrobacterium K599, and culturing Agrobacterium K599 to OD using LB medium. 600 =1.15-1.25, collect the bacterial cells, and suspend the bacterial cells in the infection solution until the bacterial concentration reaches OD0.

05. 600 =0.1-0.25, let stand for 1.8-2.2 hours to obtain bacterial suspension.

5. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in claim 4, characterized in that: The bacterial cells were suspended in the infection solution until the bacterial concentration was 0 D. 600 =0.10-0.

20.

6. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in claim 4, characterized in that: The inoculation solution consists of the following components: MES 1.8g / L-2.2g / L, MgCl2 1.8g / L-2.2g / L, AS 15mg / L-25mg / L, pH 5.2-5.

6.

7. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in claim 6, characterized in that: The inoculation solution consists of the following components: MES 2g / L, MgCl2 2g / L, AS 20mg / L, pH 5.

4.

8. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in any one of claims 1-3, characterized in that: In step (1), the leaves of the Sichuan mulberry are obtained by subculturing Sichuan mulberry under tissue culture conditions.

9. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in any one of claims 1-3, characterized in that... The LB medium mentioned in step (2) is: tryptone (8-12) g / L, yeast extract (4-6) g / L, sodium chloride (8-12) g / L, and kanamycin (40-60) mg / L.

10. The method for preparing positive embryogenic callus of *Lonicera japonica* as described in any one of claims 1-3, characterized in that: The screening medium described in step (4) consists of the following components: sucrose 30 g / L, agar 8 g / L, ZR 2.0 mg / L, 2,4-D 0.1 mg / L, Tim 300 mg / L, Kan 50 mg / L, MS, pH 5.

8.

11. The positive embryogenic callus of *Chuansang* obtained by any one of the preparation methods according to claims 1-10.

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