Establishment method of medicinal rhubarb genetic transformation system

Abstract

The invention provides an establishment method of a medicinal rhubarb genetic transformation system, which comprises the following steps: callus induction, callus infection, co-culture, screening culture, multiplication culture, positive plant identification, rooting culture, acclimatization and transplantation and other processes, and the formula of the co-culture medium is MS + 2.0-3.0 mg / L 6-BA + 0.8-1.0 mg / L NAA + 0.3-0.5 g / L CH; the method has the advantages of short induction time, high infection efficiency, high proliferation speed, high survival rate and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of asexual propagation of seedlings, and particularly relates to a method for establishing a genetic transformation system of Rheum officinale Baill.. Background Art

[0002] Rheum officinale Baill., also known as Huangliang, Jiangjun, Western Rhubarb, Jinjun, is a perennial herb of the Polygonaceae family. Rheum officinale Baill. uses its rhizome as medicine, with the effects of purging heat and removing stagnation, clearing heat and purging fire, cooling blood and detoxifying, and removing stasis and dredging menstruation, and has extremely high economic value and development and utilization prospects. The main propagation methods of Rheum officinale Baill. are traditional seed propagation or budlet propagation. Due to continuous cropping obstacles, germplasm degradation, unreasonable introduction and fertilization, etc., diseases and pests such as rhubarb smut, rust, and aphids are becoming increasingly serious. Long-term budlet propagation leads to the fact that once infected with a virus, it will be transmitted to the offspring, seriously damaging the economic benefits and planting enthusiasm of farmers and being unfavorable to the sustainable development of the rhubarb industry. According to research, most viruses cannot be transmitted through pollen or seeds, but Rheum officinale Baill. is an often cross-pollinated plant with a high degree of outcrossing, is not easy to maintain its varietal characteristics, and has a relatively long propagation cycle, which greatly restricts the industrial development of Rheum officinale Baill.. Molecular breeding is the most effective way to cultivate new varieties of Rheum officinale Baill. to overcome the problems of small population size of cultivated varieties of Rheum officinale Baill., lack of disease-resistant resources, long breeding cycle, and difficulty in traditional breeding, and establishing an efficient genetic transformation system for Rheum officinale Baill. is an important prerequisite for realizing this way.

[0003] For a long time, a mature and stable genetic transformation system for Rheum officinale Baill. has not been established. "A method for rapid tissue culture and propagation of rhubarb" (CN201710965713.5) selects rhubarb seeds as explants and establishes a rapid tissue culture and propagation technology system for rhubarb through processes such as callus induction, proliferation, differentiation, rooting, and acclimatization transplantation. "A method for inducing callus of rhubarb" (CN202311027235.5) uses rhubarb explants as test materials and cultures them on a callus induction medium of MS + 30 g / L mannitol + 2 g / L phenylalanine + 7 g / L agar + 2.0 mg / L 6-BA + 0.2 mg / L NAA for 30 - 40 days to obtain callus. The methods for constructing genetic transformation systems for different plants are different. According to the genetic characteristics of different plants, differences in environmental adaptability, etc., it is necessary to optimize according to the construction of a conventional genetic transformation system and known tissue culture methods to solve the problems of long culture cycle, difficult plant regeneration, and low genetic transformation rate of Rheum officinale Baill. and meet the technical requirements for cultivating new varieties of Rheum officinale Baill..

[0004] Therefore, it is necessary to construct a genetic transformation system for Rheum officinale Baill. with a short induction time, high infection efficiency, fast proliferation rate, and high survival rate. Summary of the Invention

[0005] The object of the present invention is to overcome the defects of the prior art, and provides a method for establishing a genetic transformation system of Rheum officinale Baill., which has the advantages of short induction time, high infection efficiency, fast proliferation rate, high survival rate, etc., and provides a theoretical basis and technical guidance for establishing an efficient genetic transformation system of Rheum officinale Baill.

[0006] The present invention is realized as follows:

[0007] The present invention provides a method for establishing a genetic transformation system of Rheum officinale Baill., which specifically includes the following steps:

[0008] S1. Inoculate petioles as explants onto a callus induction medium for induction culture to obtain callus of Rheum officinale Baill.;

[0009] S2. Immerse the callus of Rheum officinale Baill. in an Agrobacterium liquid containing an expression vector, and then transfer it to a co-culture medium for co-culture. After the co-culture is completed, transfer it to a selection medium for screening culture to differentiate adventitious buds of Rheum officinale Baill.; the formula of the co-culture medium is MS + 2.0 - 3.0 mg / L 6-BA + 0.8 - 1.0 mg / L NAA + 0.3 - 0.5 g / L CH;

[0010] S3. Inoculate the differentiated adventitious buds into a proliferation medium for proliferation culture to obtain strong seedlings;

[0011] S4. Identify the strong seedlings through positive plant identification, and then inoculate them into a rooting medium for rooting culture to obtain rooted seedlings of Rheum officinale Baill.;

[0012] S5. Acclimatize the rooted seedlings of Rheum officinale Baill. in sterile water submerging the roots, and then transplant them into a substrate for culture to obtain tissue culture seedlings.

[0013] Preferably, in the above technical solution, the formula of the co-culture medium is MS + 2.0 mg / L 6-BA + 0.8 mg / L NAA + 0.5 g / L CH;

[0014] Further, the formula of the callus induction medium is: MS + 4.0 mg / L 6-BA + 0.8 mg / L NAA + 1.2 mg / L KT.

[0015] Further, the formula of the selection medium is: MS + 0.8 mg / L NAA + 25 mg / L Kan + 250 mg / L Carb + 250 mg / L Tim.

[0016] Further, the formula of the proliferation medium is: MS + 0.8 mg / L NAA + 1.6 mg / L KT.

[0017] Furthermore, the formula of the rooting medium is: 1 / 2MS + 1.6 mg / L IBA + 0.6 mg / L NAA + 1.6% activated carbon.

[0018] Furthermore, the conditions for induction culture include: dark culture at 23 ± 2 °C for 2 d, then transferred to light conditions at 23 ± 2 °C for 20 - 30 d, with a light intensity of 2200 lx and a daily light duration of 12 h / d.

[0019] Furthermore, the conditions for co - culture include: dark culture at 23 ± 2 °C for 1 - 3 d.

[0020] Furthermore, the conditions for screening culture include: light culture at 23 ± 2 °C for 20 - 30 d, with a light intensity of 3500 lx and a daily light duration of 15 h / d.

[0021] Furthermore, the conditions for proliferation culture include: light culture at 23 ± 2 °C for 10 - 15 d, with a light intensity of 3500 lx and a daily light duration of 15 h / d.

[0022] Furthermore, the conditions for rooting culture include: light culture at 23 ± 2 °C for 30 d, with a light intensity of 3500 lx and a daily light duration of 15 h / d.

[0023] The present invention has the following beneficial effects:

[0024] The present invention uses the young leaves and petioles of aseptic seedlings of Rheum officinale as explants for callus induction, and conducts genetic transformation by means of infecting with the plant expression vector pCAMBIA1301 and Agrobacterium liquid. A genetic transformation technology system for Rheum officinale has been successfully established, and the conversion rate of positive seedlings reaches 22%. The method provided by the invention has the advantages of short induction time, high infection efficiency, fast proliferation rate, high survival rate, etc., providing a theoretical basis and technical guidance for establishing an efficient genetic transformation system of Rheum officinale, and having important theoretical significance and development and utilization prospects for improving its medicinal material yield, gene function research and molecular breeding technology to cultivate new varieties of Rheum officinale. Description of the Drawings

[0025] Figure 1 is the overall process of genetic transformation of Rheum officinale; Note: A: Induction of callus from explants; B: Infection of callus; C: Co - culture of callus; D: Screening culture of callus for adventitious buds; E: Proliferation culture of adventitious buds; F: Identification of positive plants; G: Phenotype of GUS staining of positive plants; H: Rooting culture of positive plants; I: Hardening and transplanting of positive plants.

[0026] Figure 2 is the induction of callus from young leaves and petioles of aseptic seedlings; Note: A: Phenotype of callus induced from petioles; B: Phenotype of callus induced from young leaves;

[0027] Figure 3 is the vector map of pCAMBIA1301;

[0028] Figure 4 is the photo of the PCR detection result of positive colonies;

[0029] Figure 5 is the GUS staining phenotypes of the control and positive plants; Note: A: The staining phenotype of the leaves of the seedlings differentiated from the uninfected callus; B: The staining phenotype of the leaves of the positive plants differentiated from the infected callus. Detailed implementation manners

[0030] The present invention will be specifically described below in combination with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0031] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0032] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0033] The explanations of the relevant terms of the present invention are as follows:

[0034] 6-BA, 6-benzylaminopurine, alias: 6-benzylaminopurine, cytokinin, English common name: 6-Benzylaminopurine, molecular formula: C 12 H 11 N 5 .

[0035] NAA, naphthaleneacetic acid (1-Naphthaleneacetic acid), is an organic compound (chemical formula: C 10 H 7 CH 2 CO 2 H), and is a colorless solid that is easily soluble in organic solvents.

[0036] KT, kinetin, is an artificially synthesized cytokinin analogue. The chemical name of KT kinetin is 6-furfurylaminopurine (or N6-furfuryladenine), and the molecular formula is C 10 H 9N 5 O.

[0037] IBA, indole-3-butyric acid, is a synthetic substance with hormonal activity and belongs to plant growth regulators.

[0038] CH, Casein Hydrolysate (Oxiod), is a purified hydrolysate that provides nitrogenous substances for the medium.

[0039] Agar.

[0040] Sugar, sucrose.

[0041] Kan, kanamycin.

[0042] Carb, Carbenicillin disodium, is a semi-synthetic penicillin antibiotic with broad-spectrum antibacterial activity against Gram-negative bacteria.

[0043] Tim, Timentin, is a new and effective antibiotic for inhibiting Agrobacterium.

[0044] The Agrobacterium strain type in the examples of the present invention is specifically LBA4404.

[0045] The present application will be described in detail below in conjunction with examples and experimental data.

[0046] Example 1

[0047] A method for establishing a genetic transformation system of Rheum officinale Baill., specifically includes the following steps:

[0048] (1) Callus induction (see Figure 1 )

[0049] Using the petiole of sterile seedlings as explants, cut three times on the back of young leaves with a scalpel or cut the petiole into 1 cm long segments, and inoculate them into the callus induction medium (MS + 4.0 mg / L 6-BA + 0.8 mg / L NAA + 1.2 mg / L KT + 33 g / L Sugar + 6.5 g / L Agar, pH = 5.9). After dark culture at 23 ± 2 °C for 2 d, transfer them to the light condition at 24 °C for 20 - 30 d, with a light intensity of 2200 lx and a daily light time of 12 h / d, and then the callus of Rheum officinale Baill. can be obtained.

[0050] (2) Infecting the callus (see Figure 1 )

[0051] Immerse the obtained callus in the Agrobacterium liquid for 10 min, and intermittently shake the container during this period to make it fully contact.

[0052] (3) Co-culture (see Figure 1 )

[0053] After fully contacting the callus with sterilized filter paper to absorb the surface moisture, transfer it to the co-culture medium (MS + 2 mg / L 6-BA + 0.8 mg / L NAA + 0.5 g / L CH + 33 g / L Sugar + 6.5 g / L Agar), and incubate it in the dark at 23 ± 2 °C for 2 days.

[0054] (4) Screening culture (see Figure 1 )

[0055] After the co-culture is completed, transfer the callus to the selection medium (MS + 0.8 mg / L NAA + 33 g / L Sugar + 6.5 g / L Agar + 25 mg / L Kan + 250 mg / L Carb + 250 mg / L Tim, pH = 5.6) for screening culture. Incubate it under light at 23 ± 2 °C for 20 - 30 days, with a light intensity of 3500 lx and a daily light duration of 15 h / d, and adventitious buds of Rheum officinale can be differentiated.

[0056] (5) Proliferation culture (see Figure 1 )

[0057] Cut the adventitious buds obtained by differentiation from the base and inoculate them into the proliferation medium (MS + 0.8 mg / L NAA + 1.6 mg / L KT + 33 g / L Sugar + 6.5 g / L Agar, pH = 5.9) for propagation. Incubate it under light at 23 ± 2 °C for 10 - 15 days, with a light intensity of 3500 lx and a daily light duration of 15 h / d.

[0058] (6) Identification of positive plants (see Figure 1 )

[0059] Take the leaves of the robust seedlings obtained by proliferation culture, soak them in the GUS staining solution (0.1 mg / L X-Gluc + 0.1 M phosphate buffer + 5 mM potassium ferricyanide + 5 mM potassium ferrocyanide + 0.5 M EDTA + 40 mL methanol + 200 μM Triton X-100), incubate them in an incubator at 37 °C for 24 h, observe the staining effect and take pictures.

[0060] (7) Rooting culture (see Figure 1 )

[0061] The robust seedlings obtained from the proliferation culture are inoculated into a rooting medium (1 / 2MS+1.6mg / L IBA+0.6mg / L NAA+1.6% activated carbon+33g / L Sugar+6.5g / Agar, pH=5.9) to induce rooting, and cultured under light at 23±2°C for 30 days with a light intensity of 3500lx and a daily light time of 15h / d to obtain rooted medicinal rhubarb seedlings.

[0062] (8) Hardening and transplanting (see Figure 1 )

[0063] One month after rooting, pour a small amount of sterile water into the tissue culture bottle. After 2 days, wash the culture medium at the roots of the rooted seedlings, continue to harden the seedlings in the sterile water that submerges the roots for 2 days, and transplant them to a matrix with a ratio of peat soil: garden soil: river sand = 3:1:1 for cultivation. Cover them with a film to keep them warm and moist. After 7 days, remove the film, water them every 3 days, and count the survival rate 30 days after transplanting.

[0064] Example 2

[0065] In this embodiment, the co-culture formula is: MS+2mg / L 6-BA+0.1mg / L NAA+0.3g / LCH+33g / LSugar+6.5g / LAgar, and the other operation steps are the same as those in Example 1.

[0066] Example 3

[0067] In this embodiment, the co-culture formula is: MS+2mg / L 6-BA+0.8mg / L NAA+0.3g / LCH+33g / LSugar+6.5g / LAgar, and the other operation steps are the same as those in Example 1.

[0068] Example 4

[0069] In this embodiment, the co-culture formula is: MS+3mg / L 6-BA+1.0mg / L NAA+0.3g / LCH+33g / LSugar+6.5g / LAgar, and the other operation steps are the same as those in Example 1.

[0070] Example 5

[0071] In this embodiment, the co-culture formula is: MS+3mg / L 6-BA+0.8mg / L NAA+0.5g / LCH+33g / LSugar+6.5g / LAgar, and the other operation steps are the same as those in Example 1.

[0072] Example 6

[0073] In this example, the co-culture formula is: MS + 2.5 mg / L 6-BA + 0.9 mg / L NAA + 0.4 g / L CH + 33 g / L Sugar + 6.5 g / L Agar. All other operation steps are the same as those in Example 1.

[0074] Comparative Example 1

[0075] In this comparative example, young leaves of the aseptic seedlings of Rheum officinale were used as explants, and all other operation steps were the same as those in Example 1.

[0076] Comparative Examples 2 - 6

[0077] In Comparative Examples 2 - 6, the concentrations of BA, NAA, and CH in co-culture were adjusted (see Table 2 for details), and all other operation steps were the same as those in Example 1.

[0078] Comparative Examples 7 - 11

[0079] In Comparative Examples 7 - 11, the concentration of kan was adjusted (see Table 3 for details), and all other operation steps were the same as those in Example 1.

[0080] Experimental Example 1, Screening of Infection Materials

[0081] In Example 1, the petioles of aseptic seedlings of Rheum officinale were used as explants, and in Comparative Example 1, young leaves of aseptic seedlings of Rheum officinale were used as explants. With other conditions being the same, the induction rate and the texture of the callus were statistically analyzed as shown in Table 1.

[0082] Table 1 Effects of Leaves and Petioles on the Induction of Callus in Rheum officinale

[0083] Group Infection site Induction time (d) Induction rate (%) Callus texture Comparative example 1 Leaf 27±3 83.11±3.94 ++ Example 1 Petiole 22±2 92.22±4.01 +++

[0084] Note: “+” represents the compactness of the callus, “++” indicates that the callus texture is compact, and “+++” indicates that the callus texture is very compact.

[0085] As can be seen from Table 1, both leaves and petioles can induce callus, and the petioles induce callus with a shorter induction time, higher induction rate, and denser texture compared to leaves.

[0086] Experimental Example 2, Exploration of Co-Culture Medium Formula

[0087] After the infected callus was in full contact with the sterilized filter paper to absorb the surface moisture, it was transferred to the co-culture medium and dark-cultured at 27 ± 1°C for 2 d.

[0088] Table 2 Effects of Different Hormone Ratios on the Induction of Adventitious Buds

[0089]

[0090] As can be seen from Table 2, a suitable hormone application concentration is when the adventitious bud induction rate is higher than 70%. Therefore, Treatments 5, 6, 7, 8, and 10 all meet the requirements for adventitious bud induction. Among them, Treatment 5 (Example 1) has the highest adventitious bud induction rate, and the number of adventitious buds is higher than that of other treatments, and the induced adventitious buds grow the most robustly.

[0091] Therefore, when inducing callus after infection, we select the formulation range of the co-culture medium as MS + 2.0 - 3.0 mg / L 6-BA + 0.8 - 1.0 mg / L NAA + 0.3 - 0.5 g / L CH. Most preferably, MS + 2 mg / L 6-BA + 0.8 mg / L NAA + 0.5 g / L CH + 33 g / L Sugar + 6.5 g / L Agar is selected as the co-culture medium for adventitious bud induction.

[0092] Experimental Example 3, Kan Resistance Screening

[0093] Antibiotic concentration screening was carried out on non-infected callus. Kan concentrations of 0, 5, 10, 15, 20, and 25 mg / L were set and added to the selection medium without antibiotics respectively. After 25 days, the induction situation was observed.

[0094] Table 3 Effects of Different Concentrations of Antibiotics on Callus

[0095]

[0096] As can be seen from Table 3, no adventitious buds were produced in the medium with a Kan concentration of 25 mg / L in Treatment 6, and the callus could not grow normally and was basically dead. Therefore, in the selection medium, 25 mg / L Kan was used as the final screening concentration for positive seedling screening.

[0097] Application Example 1, Genetic Transformation of Medicinal Rhubarb with pCAMBIA1301 Vector

[0098] 1. Construction of pCAMBIA1301 Vector

[0099] The plasmid of the pCAMBIA1301 expression vector (Shanghai New Bio-Tech Co., Ltd., product number V010893) ( Figure 3 ) was transferred into Agrobacterium for bacterial liquid culture. The cultured bacterial liquid was spread on the LB plate medium with Kan resistance, and PCR reaction was carried out using the M13F and M13R primers in Table 4 (reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min; number of cycles 33) and DNA agarose gel electrophoresis screening ( Figure 4 ).

[0100] Table 4

[0101] Primer name Sequence (5’-3’) M13F TGTAAAACGACGGCCAGT(SEQ ID NO.1) M13R CAGGAAACAGCTATGAC(SEQ ID NO.2)

[0102] The obtained bacterial liquid is infected and cultured according to the genetic transformation technology system explored in the present invention.

[0103] 2. Identification of positive plants

[0104] Taking the seedlings differentiated from the uninfected callus as a control, GUS staining is performed on the positive plants differentiated from the callus infected with Agrobacterium tumefaciens carrying the pCAMBIA1301 expression vector.

[0105] The staining results are as Figure 5 shown. The leaves of the seedlings differentiated from the uninfected callus show light yellow, and the leaves of the positive plants differentiated from the infected callus show obvious blue, proving that the transferred vector is successfully expressed in the medicinal rhubarb seedlings after infection.

[0106] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for establishing a genetic transformation system for medicinal rhubarb, characterized in that: The method comprises: S1. Inoculating petioles as explants into callus induction medium for induction culture to obtain medicinal rhubarb callus; S2, soaking the medicinal rhubarb callus in an Agrobacterium solution containing an expression vector, and then transferring it to a co-culture medium for co-culture. After the co-culture is completed, transferring it to a selection medium for screening culture to differentiate and obtain medicinal rhubarb adventitious buds; the formula of the co-culture medium is MS+2.0-3.0mg / L 6-BA+0.8-1.0mg / L NAA+0.3-0.5g / L CH; S3, inoculating the differentiated adventitious buds into a proliferation medium for proliferation culture to obtain robust seedlings; S4, the healthy seedlings are identified as positive plants, and then inoculated into a rooting medium for rooting culture to obtain rooted medicinal rhubarb seedlings; S5, immersing the root of the rhubarb rooting seedlings in sterile water to harden the seedlings, and then transplanting them into a substrate for cultivation to obtain tissue culture seedlings.

2. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The formula of the callus induction medium is: MS+4.0 mg / L 6-BA+0.8 mg / L NAA+1.2 mg / L KT.

3. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The formula of the selection culture medium is: MS+0.8mg / LNAA+25mg / LKan+250mg / L Carb+250mg / L Tim.

4. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The formula of the proliferation culture medium is: MS+0.8mg / LNAA+1.6mg / LKT.

5. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The formula of the rooting medium is: 1 / 2MS+1.6mg / LIBA+0.6mg / LNAA+1.6% activated carbon.

6. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The induction culture conditions include: culturing in the dark at 23±2°C for 2 days, then transferring to light conditions at 23±2°C for culturing for 20-30 days, with a light intensity of 2200 lx and a light duration of 12 h / d.

7. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The co-cultivation conditions include: dark cultivation at 23±2° C. for 1-3 days.

8. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The screening culture conditions include: light culture at 23±2°C for 20-30 days, light intensity of 3500 lx, and light time of 15 h / d.

9. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The conditions of the proliferation culture include: culturing under light at 23±2° C. for 10-15 days, a light intensity of 3500 lx, and a light duration of 15 h / d.

10. The method for establishing the genetic transformation system of medicinal rhubarb according to claim 1, characterized in that: The rooting culture conditions include: light culture at 23±2°C for 30 days, light intensity of 3500 lx, and light time of 15 h / d.

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