CN114836466A - Agrobacterium-mediated genetic transformation method for stevia rebaudiana - Google Patents
Agrobacterium-mediated genetic transformation method for stevia rebaudiana Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8205—Agrobacterium mediated transformation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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Abstract
The invention provides an agrobacterium-mediated stevia rebaudiana genetic transformation method, which adopts a screening culture medium with a specific formula, not only can be suitable for resistance screening and induction culture of transgenic resistant callus of stevia rebaudiana, but also can be suitable for a subsequent differentiation and budding process of the resistant callus, so that a better budding rate of the resistant callus is obtained, and a more efficient agrobacterium-mediated stevia rebaudiana genetic transformation system is constructed.
Description
Technical Field
The invention relates to an agrobacterium-mediated genetic transformation method for stevia rebaudiana, belonging to the field of plant genetic engineering of plant biotechnology.
Background
Stevia rebaudiana (Bertoni) Hemsl.) is a perennial herb of the Stevia genus of the Compositae family. The stevioside rich in the stevia rebaudiana leaves is a natural sweetener with low calorie, no toxic or side effect and high sweetness, and is one of the raw materials in the food and medicine industries. China is one of the largest export countries of stevia raw materials and stevioside, and stevia has become an important economic crop for increasing the income of farmers.
The skilled person hopes to select a new variety of stevia rebaudiana with excellent quality and high content of stevioside by means of transgenic breeding.
At present, the conventional plant genetic transformation method mainly comprises an agrobacterium-mediated method, a gene gun transformation method, a pollen tube channel method, a flower dipping method and other methods; among them, the Agrobacterium tumefaciens mediated method is simple, easy, economical, efficient, and most widely used, and is an important means for improving traits by using molecular breeding technology in the field.
In the prior art, research reports about an agrobacterium-mediated genetic transformation method of stevia rebaudiana are that the technical problems of serious pollution of explants by agrobacterium, low survival rate of explants, low transformation rate, long transformation period, low callus budding rate, low adventitious bud differentiation efficiency and the like generally exist, and particularly the budding rate of transgenic resistant callus and the regeneration of stevia rebaudiana plants are severely restricted by the problems of low callus budding rate and low adventitious bud differentiation efficiency.
Therefore, those skilled in the art would expect to improve the existing agrobacterium-mediated genetic transformation method and construct a more efficient agrobacterium-mediated genetic transformation system for stevia rebaudiana and the method thereof.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides, in one aspect, an agrobacterium-mediated genetic transformation method of stevia rebaudiana, wherein,
the genetic transformation method of stevia rebaudiana comprises the following steps:
step 1), obtaining a stevia rebaudiana leaf explant as a genetic transformation receptor;
step 2), agrobacterium infection and co-culture: infecting the genetic transformation receptor of the step 1) with recombinant agrobacterium containing kanamycin resistance gene and target gene to obtain an infected explant; removing residual bacterial liquid on the surface of the infected explant, and then carrying out co-culture;
step 3), screening and culturing: transferring the explants obtained in the step 2) after co-culture to a screening culture medium for dark culture to obtain resistant callus; then continuing to culture the resistant callus in dark until adventitious buds are differentiated;
the screening culture medium is a solid MS culture medium containing cane sugar, and the formula of the screening culture medium further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin and 7-9mg/L of kanamycin;
step 4), rooting culture: cutting the adventitious bud obtained in the step 3) for rooting culture to obtain a transgenic stevia rebaudiana plant.
In a preferred embodiment of the present invention, in the step 3), after the co-cultured explant obtained in the step 2) is transferred to a screening medium for dark culture for 3-4 weeks, the resistant callus is excised and transferred to an adventitious bud differentiation medium for further dark culture until the resistant callus is differentiated into adventitious buds.
In a preferred embodiment of the present invention, the adventitious bud differentiation medium is a solid MS medium containing sucrose, and the formulation further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin, and 7-9mg/L of kanamycin.
In a specific embodiment of the present invention, the amount of 6-benzylaminopurine in the selection medium and/or the formulation of the adventitious bud differentiation medium is 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, or 1.60 mg/L.
In a specific embodiment of the present invention, the kinetin is contained in an amount of 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59 or 0.60mg/L in the formulation of the screening medium and/or the adventitious bud differentiation medium.
In a specific embodiment of the present invention, the indole-3-acetic acid content in the screening medium and/or the adventitious bud differentiation medium formulation is 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, or 0.60 mg/L.
In a specific embodiment of the present invention, the content of carbenicillin in the formulation of the screening medium and/or the adventitious bud differentiation medium is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg/L.
In a specific embodiment of the present invention, in the formulation of the screening medium and/or the adventitious bud differentiation medium, the kanamycin is contained in an amount of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0 mg/L.
In a preferred embodiment of the present invention, the selection medium and/or the adventitious bud differentiation medium is a solid MS medium containing sucrose, and the formulation further contains 1.5mg/L of 6-benzylaminopurine, 0.5mg/L of kinetin, 0.5mg/L of indole-3-acetic acid, 100mg/L of carbenicillin, and 8mg/L of kanamycin.
In a preferred embodiment of the present invention, the stevia leaf explant is taken from the superior leaf of a stevia plant seedling.
In another preferred embodiment of the present invention, in the step 2), the recombinant Agrobacterium is Agrobacterium tumefaciens, and the OD of the Agrobacterium infection solution 600 The infection time is 3-12 minutes when the infection time falls in the range of 0.4-0.6; and the co-culture time is 40-55 hours.
Preferably, in the step 2), the agrobacterium tumefaciens is strain EHA105, LBA4404, GV3101 or AGL 1.
Preferably, in the step 4), the formula of the culture medium adopted in the rooting culture in the step 4) is 1/2MS + 90-110 mg/L carbenicillin.
The invention also provides a screening culture medium applied to stevia rebaudiana genetic transformation, wherein the screening culture medium is a solid MS culture medium containing cane sugar, and the formula of the screening culture medium further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin and 7-9mg/L of kanamycin.
Preferably, the screening medium is a solid MS medium containing sucrose, and the formulation further contains 1.5mg/L of 6-benzylaminopurine, 0.5mg/L of kinetin, 0.5mg/L of indole-3-acetic acid, 100mg/L of carbenicillin and 8mg/L of kanamycin.
The invention provides an agrobacterium-mediated genetic transformation method for stevia rebaudiana, which adopts a screening culture medium with a specific formula, not only can be suitable for resistance screening and induction culture of transgenic resistant callus of the stevia rebaudiana, but also can be suitable for a subsequent differentiation and budding process of the resistant callus, obtains a better budding rate of the resistant callus, and improves the agrobacterium-mediated genetic transformation efficiency of the stevia rebaudiana.
Drawings
FIG. 1 is a photograph under a fluorescent microscope of the resistant callus obtained in example 1;
FIG. 2 is a photograph of adventitious buds differentiated from resistant callus in example 1;
FIG. 3 is a photograph of the regenerated plant obtained in example 1.
Detailed Description
In a specific embodiment of the present invention, there is provided an Agrobacterium-mediated genetic transformation method of stevia rebaudiana, wherein,
the genetic transformation method of stevia rebaudiana comprises the following steps:
step 1), obtaining a stevia rebaudiana leaf explant as a genetic transformation receptor;
step 2), agrobacterium infection and co-culture: infecting the genetic transformation receptor of the step 1) with recombinant agrobacterium containing kanamycin resistance gene and target gene to obtain an infected explant; removing residual bacterial liquid on the surface of the infected explant, and then carrying out co-culture;
step 3), screening and culturing: transferring the co-cultured explant obtained in the step 2) to a screening culture medium for culture to obtain a resistant callus; continuing to culture the resistant callus until adventitious buds are differentiated;
the screening culture medium is a solid MS culture medium containing cane sugar, and the formula of the screening culture medium further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin and 7-9mg/L of kanamycin;
step 4), rooting culture: cutting the adventitious bud obtained in the step 3) for rooting culture to obtain a transgenic stevia rebaudiana plant.
As explained in the background art, the agrobacterium-mediated genetic transformation method for stevia rebaudiana generally has the technical problems of serious pollution of an explant by agrobacterium, low survival rate of the explant, low transformation rate, long transformation period, low callus budding rate, low adventitious bud differentiation efficiency and the like, and particularly the problems of low callus budding rate and low adventitious bud differentiation efficiency seriously restrict the budding rate of transgenic resistant callus and the regeneration of stevia rebaudiana plants.
For those skilled in the art, if the technical problems of "inducing callus formation (callus induction rate)" and "adventitious bud differentiation growth (adventitious bud differentiation rate)" mentioned above, are to be solved, based on the basic theory and common general knowledge in the art, improvements of "the components of the growth regulator in the induction medium/differentiation medium and their concentrations" will be first conceived and focused, since these are technical characteristics directly associated with the above technical problems.
As the "auxin for callus induction", 2,4-D (2, 4-dichlorophenoxyacetic acid), IAA (indole-3-acetic acid), NAA (naphthylacetic acid) and the like are commonly used, 2,4-D (2, 4-D is selected for most plants) is most widely used, but NAA (naphthylacetic acid) is most effective and most commonly used for callus induction culture of stevia rebaudiana.
As the "auxin for promoting differentiation/induction of callus budding", 6-BA (6-benzylaminopurine) or KT (kinetin) is usually selected.
As for the combination of growth regulators used for tissue culture of stevia rebaudiana Bertoni, the combination of "NAA + 6-BA" is most commonly used in the induction medium in view of the protocols disclosed in the prior art, for example, in patent document CN103667343B, an Agrobacterium rhizogenes-mediated genetic transformation method of transgenic stevia rebaudiana Bertoni is disclosed, and the combination of growth regulators of "0.05 mg/LNAA +0.1 mg/L6-BA" is used in the pre-culture medium, co-culture medium, screening medium, etc. used in the method.
And (4) supplementary notes: the most commonly used rooting medium for the tissue culture of stevia rebaudiana is the combination of indolebutyric acid (IBA) +6-BA, and a few schemes of using the combination of IAA + NAA are available.
Therefore, from the prior art, IAA (indole-3-acetic acid) is known as a plant growth regulator, but because it is easily decomposed by light and the induction effect of the combination of "IAA + 6-BA" is poor, IAA has never been used in callus induction medium and adventitious bud differentiation medium of stevia rebaudiana; alternatively, for callus induction and adventitious bud differentiation of stevia rebaudiana, those skilled in the art prefer to use NAA instead of IAA.
In order to solve the technical problems of inducing callus formation (callus induction rate) and adventitious bud differentiation and growth (adventitious bud differentiation rate), the inventors of the present application made a great number of combination attempts, and surprisingly found that, although the induction effect of the combination of IAA +6-BA is poor, the combination of IAA +6-BA + KT is combined at a specific concentration and dark culture is applied to an explant, and the induction effect is significantly better than that of the combination of NAA +6-BA in the prior art; specifically, when a combination of 0.4-0.6 mg/L IAA, 1.4-1.6 mg/L6-BA and 0.4-0.6 mg/L KT (namely 0.4-0.6 mg/L indole-3-acetic acid, 1.4-1.6 mg/L6-benzylaminopurine and 0.4-0.6 mg/L kinetin) is adopted, the induction rate of the callus (non-transgenic callus) can reach about 95% at most, and the differentiation rate of the adventitious buds can reach about 67% at most.
However, the above-mentioned research only solved the technical problems of induction culture and differentiation of non-transgenic callus of stevia rebaudiana, and did not actually solve the technical problems of induction culture and differentiation growth of adventitious buds of transgenic and resistant callus to be solved by the present application. Specifically, on one hand, after a leaf explant of stevia rebaudiana is infected by agrobacterium, the induction rate and adventitious bud differentiation rate of transgenic resistant callus of the stevia rebaudiana are greatly reduced; on the other hand, the screening culture medium in the screening culture process needs to be added with a proper amount of bacteriostatic agent (controlling the explant to be polluted by agrobacterium) and a proper amount of selective antibiotic (realizing resistance screening of transgenic callus) besides the basic components of the induction culture medium; however, it is well known that the presence of bacteriostatic and selective antibiotics in the screening medium has an adverse effect on the induction culture of transgenic resistant callus, especially a certain inhibitory effect on the adventitious bud differentiation process (which affects the adventitious bud differentiation rate).
The inventors of the present application have made further studies in order to solve the technical problems of the induction culture of the transgenic resistant callus of stevia rebaudiana leaf and the differentiation and growth of adventitious buds.
Kanamycin (Kan) is the most commonly used antibiotic for selection in the selection medium, and kanamycin (Kan) is also used in the prior art for callus resistance selection of stevia rebaudiana. As the bacteriostatic agent in the screening medium, Plant Preservative Mix (PPM), carbenicillin (Car), cephamycin, hygromycin and the like are commonly used.
The inventor of the application finds that in the process of compounding and optimizing the formula of the screening culture medium, on the basis of the combination of '0.4-0.6 mg/L IAA + 1.4-1.6 mg/L6-BA + 0.4-0.6 mg/L KT' growth regulator optimized by the inventor, carbenicillin (bacteriostatic agent) and kanamycin (as antibiotics for resistance screening) with specific concentrations of 80-110mg/L and 7-9mg/L are added, so that the method is not only suitable for the resistance screening and induction culture of transgenic resistant callus of stevia rebaudiana (which is transferred with kanamycin resistance gene and target gene), but also suitable for the subsequent process of differentiation and sprouting of the resistant callus, namely, the screening culture medium formula can also be used as a subsequent adventitious bud differentiation culture medium, and the better budding rate of the resistant callus is obtained (the adventitious bud differentiation rate can reach about 12%).
In a preferred embodiment of the present invention, in the step 3), after the co-cultured explant obtained in the step 2) is transferred to a screening medium for dark culture for 3-4 weeks, the resistant callus is excised and transferred to an adventitious bud differentiation medium for further dark culture until the resistant callus is differentiated into adventitious buds.
In addition, the inventor of the present application has made improvement research on the formulation of the agrobacterium infection solution, the concentration and the infection time of agrobacterium, the culture medium and the co-culture time of co-culture, and as a result, found that the concentration, the infection time and the co-culture time of specific agrobacterium can further improve the technical problems of severe agrobacterium contamination of explants and low survival rate of explants. Specifically, in a preferred embodiment of the present invention, in the step 2), the recombinant Agrobacterium is Agrobacterium tumefaciens, and the OD of the Agrobacterium infection solution 600 The infection time is 3-12 minutes when the infection time falls in the range of 0.4-0.6; and the co-culture time is 40-55 hours.
In addition, the inventor of the application also optimizes the subsequent formulation of the rooting medium. In a preferred embodiment of the invention, in the step 4), the formula of the culture medium adopted by the rooting culture is 1/2MS + 90-110 mg/L carbenicillin. Preferably, gibberellin is gibberellin GA4+ 7.
Gibberellin, especially gibberellin GA4+7, is added into the rooting culture medium, so that the effect of strengthening seedlings can be achieved, the growth of stems and leaves can be remarkably promoted, and the survival rate of transgenic adventitious buds is improved.
The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to these embodiments are included in the scope of the present invention.
Example embodiments will now be described more fully with reference to the accompanying drawings. Example embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art.
The materials, reagents, and the like used in the following examples are commercially available (for example, those purchased from conventional biochemical reagent stores) unless otherwise specified.
The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, science Press, translated by Huang Petang et al) or according to the product instructions.
The plasmids and strains shown in the following examples are only used for further detailed description of the present invention, and do not limit the scope and spirit of the present invention.
Example 1
The agrobacterium-mediated genetic transformation method of stevia rebaudiana of example 1, generally, includes the steps of 1) obtaining a genetic transformation receptor, 2) agrobacterium infection and co-culture, 3) screening culture, and 4) rooting culture; the method comprises the following specific steps:
step 1), obtaining a genetic transformation receptor: obtaining a stevia rebaudiana leaf explant as a genetic transformation receptor.
Specifically, in the embodiment, 2 pairs of upper leaves of the stevia rebaudiana aseptic seedling are collected, and the leaves are cut into explants of 0.5cm multiplied by 0.5cm, which are used as genetic transformation receptors; the sterile stevia rebaudiana seedlings selected by the embodiment are from the laboratory of the inventor of the application (Ningxia university Life sciences college/Ningxia advantage characteristic crop modern Feizi breeding key laboratory).
Step 2), agrobacterium infection and co-culture: infecting the genetic transformation receptor of the step 1) by recombinant agrobacterium containing a target gene to obtain an infected explant; and removing residual bacterial liquid on the surface of the infected explant, and then carrying out co-culture.
The recombinant agrobacterium used in this example was agrobacterium strain EHA105 (the strain was transformed with a recombinant pnullpge 76G1 plasmid containing NPT II kanamycin resistance gene and the target gene UGT76G 1). Taking out the recombinant agrobacterium tumefaciens preservation solution from a refrigerator at the temperature of-80 ℃, thawing the solution on ice, inoculating the solution on an LB flat plate containing 50mg/L kanamycin, streaking the solution, and culturing the solution at the temperature of 28 ℃ for two days; thereafter, the obtained flat is pickedPlate monoclonal, inoculated into 20ml LB (containing 50mg/L kanamycin) liquid medium, 28 degrees C, 220rmp, shake bacteria to OD 600 0.6-1.0; centrifuging again to collect thallus (4000rmp, 4 deg.C, centrifuging for 10min), and suspending in 30-40ml infection culture solution (MS +30mg/L sucrose +0.25 mg/L2, 4-D +20mg/L acetosyringone +150mg/L dithiothreitol) to make OD 600 Falling in the range of 0.4-0.6 to obtain the activated recombinant agrobacterium infection liquid.
Transferring the stevia rebaudiana leaf explant obtained in the step 1) into the activated recombinant agrobacterium infection solution, and soaking for 5-10min (infection time); then, the infected explants are taken out, the residual bacterial liquid on the surfaces of the explants is sucked dry by sterile filter paper, and then the explants are transferred to a co-culture medium (a solid medium, MS +30mg/L sucrose +0.25 mg/L2, 4-D +0.7g/LAgar) to be co-cultured for 2 days under the dark culture condition at the temperature of 22 ℃, so as to obtain the co-cultured explants.
Step 3), screening and culturing: transferring the explants obtained in the step 2) after co-culture to a screening culture medium for dark culture until adventitious buds are differentiated from the resistant callus.
In a specific embodiment of the invention, the screening culture medium is a solid MS culture medium containing sucrose, and the formulation further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin and 7-9mg/L of kanamycin. Preferably, the screening medium is a solid MS medium containing sucrose, and the formulation further contains 1.5 mg/L6-benzylaminopurine, 0.5mg/L kinetin, 0.5mg/L indole-3-acetic acid, 100mg/L carbenicillin and 8mg/L kanamycin.
In a preferred embodiment of the present invention, in the step 3), after the co-cultured explant obtained in the step 2) is transferred to a screening medium for dark culture for 3-4 weeks, the resistant callus is excised and transferred to a newly prepared screening medium for further dark culture until the resistant callus is differentiated into adventitious buds.
Specifically, in this example, the explants obtained in step 2) after co-culture were transferred to a selection medium (MS +30mg/L sucrose +0.5mg/L indole-3-acetic acid +1.5 mg/L6-benzylaminopurine +0.5mg/L kinetin +7g/L agar +100mg/L carbenicillin +8mg/L kanamycin), and the leaf faces were raised and cultured in the dark for about 4 weeks to obtain resistant calli (the inventors observed the resistant calli obtained by selection and culture under a fluorescent microscope, observed green fluorescence, see fig. 1, which indicates that the differentiated calli are positive calli, i.e., transgenic calli into which the desired gene was transferred); the resistant callus was excised and transferred to a newly prepared selection medium (formula as above) with the leaf facing up, and dark culture was continued (about 15 days) until the resistant callus differentiated adventitious buds (see FIG. 2).
Step 4), rooting culture: cutting the adventitious bud obtained in the step 3) for rooting culture to obtain a transgenic stevia rebaudiana plant.
In a specific embodiment of the application, in the step 4), the formula of the culture medium adopted by the rooting culture is 1/2MS + 90-110 mg/L carbenicillin.
In a preferred embodiment of the present application, the gibberellin is gibberellin GA4+ 7.
Specifically, in this embodiment, the rooting medium used for rooting culture has the following formula: 1/2MS +2mg/L gibberellin (GA4+7) +100mg/L carbenicillin, and the rooting culture conditions are as follows: irradiating at 25 ℃ by 4000 lx; after rooting culture for about 15 days, until the roots and the buds grow to be strong, obtaining a regeneration plant shown in figure 3; the inventor utilizes a PCR amplification method to identify the regeneration plant as a transgenic stevia rebaudiana plant carrying a target gene.
Effect data
1. Selection of concentrations of Components with respect to growth regulators
Aiming at callus induction culture and adventitious bud differentiation culture of stevia rebaudiana leaf explant, after a great amount of combination attempts are made on the conventional ' growth regulator for inducing callus ' and ' growth regulator for promoting callus differentiation/inducing budding ', the inventor of the application unexpectedly discovers that the IAA +6-BA + KT ' are combined at a specific concentration, and the induction effect of the combination is remarkably superior to that of the ' NAA +6-BA ' combination in the prior art.
To further verify and screen out the combination scheme of the optimal concentration range, the inventor inoculates stevia rebaudiana leaf explants (explants obtained in step 1 of the above example 1) to culture media (MS +30mg/L sucrose +7g/L agar) with different concentrations and mixture ratios of IAA, 6-BA and KT, and calculates the callus induction rate and adventitious bud differentiation rate after dark culture for 4 weeks.
For the statistical method of callus induction rate and adventitious bud differentiation rate, see the literature "establishment of regeneration system of leaf explant of stevia rebaudiana Bertoni".
The results are shown in Table 1 below.
TABLE 1
And (4) supplementary notes: the different lower case letters in the table indicate that the mean values differ significantly at a level of p < 0.05.
Each data in table 1 was calculated by 3 sets of parallel experiments by the SPSS statistical method. The statistical methods of the data in tables 2-4 below are the same as those in table 1, and are not repeated.
As can be seen from Table 1 above, when the culture medium was treated with the combination of treatment 2(0.5mg/L indole-3-acetic acid +1.5 mg/L6-benzylaminopurine +0.5mg/L kinetin) shown in Table 1, the results of callus induction rate and adventitious bud differentiation rate were optimized.
Based on this optimum combination concentration, the inventors made the following combination of concentrations and the results are shown in table 2 below.
TABLE 2
By combining the results in tables 1 and 2, it was confirmed that the callus induction rate and adventitious bud differentiation rate of the selection medium for the resistant callus of stevia rebaudiana leaf blade were both at optimum levels by using a combination of 0.4 to 0.6mg/L of indole-3-acetic acid, 1.4 to 1.6mg/L of 6-benzylaminopurine, and 0.4 to 0.6mg/L of kinetin.
2. Selection of concentration of bacteriostatic and selective agents in screening medium and adventitious bud differentiation medium
For the technical problems of the induction culture and the differentiation and growth of adventitious buds of the transgenic resistant callus of the stevia rebaudiana leaf, the inventor finds that: on the basis of the combination of the growth regulators, the screening culture medium is added with carbenicillin (bacteriostatic agent) and kanamycin (resistance screening agent) with specific concentrations, which not only can be suitable for resistance screening and induction culture of stevia rebaudiana transgenic resistant callus (with kanamycin resistance gene and target gene transferred), but also can be suitable for the subsequent process of differentiation and sprouting of the resistant callus, namely, the screening culture medium formula can also be used as a subsequent adventitious bud differentiation culture medium, and the better sprouting rate of the resistant callus is obtained.
To further verify and screen out the optimal concentration range of the combination scheme, the inventor inoculates the stevia rebaudiana leaf explant (obtained by the same step 2) of the above example 1) after being infected and co-cultured onto the culture medium (the basic formula is MS +30mg/L sucrose +7g/L agar +0.5mg/L indole-3-acetic acid +1.5 mg/L6-benzylaminopurine +0.5mg/L kinetin) with different concentrations of bacteriostat and screening agent ratios, and after dark culture for 4 weeks, the bacteriostat effect is observed by naked eyes; the resistant callus was cut out and transferred to a newly prepared medium, dark cultured for about 15 days, and the adventitious bud differentiation rate was calculated.
See table 3 below for experimental results. And (4) supplementary notes: in Table 3, PPM represents Plant Preservative mix, Car represents carbenicillin, Hyg represents hygromycin, and Kan represents kanamycin.
TABLE 3
As can be seen from Table 3, with the combination of PPM + Kan, the stevia rebaudiana transgenic resistant callus did not sprout substantially; by adopting the combination of Car and Hyg, the adventitious bud differentiation rate of the stevia rebaudiana transgenic resistant callus is 6 percent at most; with a specific combination of 100mg/L carbenicillin and 8mg/L kanamycin (treatment 6 in Table 3), the adventitious bud differentiation rate could reach 12%; furthermore, it can be seen from the results of comparing treatment 7 and treatment 8 that Kan concentration control is critical.
Based on this optimum combination concentration, the inventors made the following combination of concentrations and the results are shown in table 4 below.
TABLE 4
By combining the results in tables 1-4, it was determined that the selection medium and the adventitious bud differentiation medium were combined with a growth regulator comprising indole-3-acetic acid at a concentration of 0.4-0.6 mg/L, 6-benzylaminopurine at a concentration of 1.4-1.6 mg/L, and kinetin at a concentration of 0.4-0.6 mg/L, and carbenicillin at a concentration of 80-110mg/L and kanamycin at a concentration of 7-9mg/L, and that the differentiation and germination rates of the transgenic resistant callus of stevia leaf could reach an optimal level.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. An agrobacterium-mediated genetic transformation method of stevia rebaudiana, which is characterized in that:
the genetic transformation method of stevia rebaudiana comprises the following steps:
step 1), obtaining a stevia rebaudiana leaf explant as a genetic transformation receptor;
step 2), agrobacterium infection and co-culture: infecting agrobacterium containing kanamycin resistance gene and target gene with the genetic transformation receptor in the step 1) to obtain an infected explant; removing residual bacterial liquid on the surface of the infected explant, and then carrying out co-culture;
step 3), screening and culturing: transferring the co-cultured explant obtained in the step 2) to a screening culture medium for culture to obtain a resistant callus; continuing to culture the resistant callus until adventitious buds are differentiated;
the screening culture medium is a solid MS culture medium containing cane sugar, and the formula of the screening culture medium further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin and 7-9mg/L of kanamycin;
step 4), rooting culture: cutting the adventitious bud obtained in the step 3) for rooting culture to obtain a transgenic stevia rebaudiana plant.
2. The method of genetic transformation of stevia rebaudiana as claimed in claim 1, wherein:
in the step 3), the explants obtained in the step 2) after co-culture are transferred to a screening culture medium for dark culture for 3-4 weeks, then the resistant callus is cut off, and then the explants are transferred to an adventitious bud differentiation culture medium for continuing dark culture until the resistant callus differentiates adventitious buds.
3. The method of genetic transformation of stevia rebaudiana as claimed in claim 2, wherein:
the adventitious bud differentiation culture medium is a solid MS culture medium containing cane sugar, and the formula of the culture medium also contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 80-110mg/L of carbenicillin and 7-9mg/L of kanamycin.
4. The method of genetic transformation of stevia rebaudiana as claimed in claim 1, wherein:
the screening culture medium is a solid MS culture medium containing sucrose, and the formula of the solid MS culture medium also contains 1.5 mg/L6-benzylaminopurine, 0.5mg/L kinetin, 0.5mg/L indole-3-acetic acid, 100mg/L carbenicillin and 8mg/L kanamycin.
5. A method of genetic transformation of stevia rebaudiana as claimed in any of claims 1 to 4, wherein: the stevia rebaudiana leaf explant is taken from the upper leaves of a stevia rebaudiana plant seedling.
6. A method of genetic transformation of stevia rebaudiana as claimed in any of claims 1 to 4, wherein: in the step 2), the recombinant agrobacterium is agrobacterium tumefaciens, and the OD of the agrobacterium infection solution 600 The infection time is 3-12 minutes when the infection time falls in the range of 0.4-0.6; and the co-culture time is 40-55 hours.
7. A method of genetic transformation of stevia rebaudiana as claimed in any of claims 1 to 4, wherein: in the step 2), the agrobacterium tumefaciens is strain EHA105, LBA4404, GV3101 or AGL 1.
8. A method of genetic transformation of stevia rebaudiana as claimed in any of claims 1 to 4, wherein: in the step 4), the formula of the culture medium adopted by the rooting culture is 1/2MS + 1.5-2.5 mg/L gibberellin + 90-110 mg/L carbenicillin.
9. A screening culture medium applied to stevia rebaudiana genetic transformation is characterized in that:
the screening culture medium is a solid MS culture medium containing cane sugar, and the formula of the screening culture medium further contains 1.4-1.6 mg/L of 6-benzylaminopurine, 0.4-0.6 mg/L of kinetin, 0.4-0.6 mg/L of indole-3-acetic acid, 90-110 mg/L of carbenicillin and 7-9mg/L of kanamycin.
10. The screening medium of claim 9, wherein:
the screening culture medium is a solid MS culture medium containing sucrose, and the formula of the solid MS culture medium also contains 1.5 mg/L6-benzylaminopurine, 0.5mg/L kinetin, 0.5mg/L indole-3-acetic acid, 100mg/L carbenicillin and 8mg/L kanamycin.
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| CN103667343A (en) * | 2013-12-06 | 2014-03-26 | 安徽大学 | Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method |
| CN103858757A (en) * | 2012-12-11 | 2014-06-18 | 丰益(上海)生物技术研发中心有限公司 | Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof |
| US20210123066A1 (en) * | 2018-01-19 | 2021-04-29 | Temasek Life Sciences Laboratory Limited | Methods of regeneration and transformation of stevia plant and transgenic stevia plants having enhanced steviol glycosides content |
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| CN103858757A (en) * | 2012-12-11 | 2014-06-18 | 丰益(上海)生物技术研发中心有限公司 | Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof |
| CN103667343A (en) * | 2013-12-06 | 2014-03-26 | 安徽大学 | Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method |
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