CN113215191B - Agrobacterium-mediated genetic transformation method for toona sinensis - Google Patents
Agrobacterium-mediated genetic transformation method for toona sinensis Download PDFInfo
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Abstract
The invention relates to an agrobacterium-mediated genetic transformation method for toona sinensis, which comprises the following steps: taking leaves of the toona sinensis as explants, and infecting the explants with a bacterial solution of agrobacterium tumefaciens carrying a plant expression vector; inoculating the infected explants to a co-culture medium for co-culture; subjecting the co-cultured explants to an induction culture comprising: induction culture of callus, induction culture of adventitious buds, induction culture of buds and induction culture of roots; the bacterial liquid contains acetosyringone, and the co-culture medium contains acetosyringone. The method successfully realizes the agrobacterium tumefaciens-mediated genetic transformation of the toona sinensis, keeps the genetic transformation rate at about 10 percent, has good repeatability and stability, is suitable for batch seedling culture, and is beneficial to the development of agricultural industrialization.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic engineering, and relates to an agrobacterium-mediated genetic transformation method for toona sinensis.
Background
Toonae sinensis (Toona ciliata) is a species of broad-leaved tree of Toona of Meliaceae (Meliaceae). In China, the natural population of the toona sinensis has the characteristic of sporadic distribution, is mainly distributed in areas of south China, east China and southwest, and is an excellent furniture material because the material is good, the color is bright and beautiful, the patterns are beautiful, the core material is raw reddish brown, and the toona sinensis is covered by a Chinese mahogany, and is widely applied to the fields of furniture design, vehicle and ship manufacturing and indoor decoration; the tree shape is beautiful, and the tree can also be planted as a street tree; the toona sinensis is also a medicinal plant which generally pays attention in recent years, the root, stem and leaf of the toona sinensis can be used as a medicine, and the branch and leaf extract has biological activities of resisting virus, resisting bacteria and the like and has good medicinal efficacy; the tree species has the characteristics of rapid early growth and the like, and the growth amount of the tree species exceeds that of the evergreen broad-leaved tree species which are generally artificially cultured. Therefore, in southern areas of China, the Chinese toon becomes a high-quality fast-growing timber tree species which is intensively developed and utilized.
At present, researches find that the toona sinensis is extremely easy to be damaged by taking food by pests in the early growth process, branches are directly hollow and broken, so that a multi-head tree is formed, even young trees die, and the toona sinensis is difficult to be grown and even becomes fatal damage to the toona sinensis planting. The pest with great harm to the toona sinensis is the toonapha molesta borer, and if the damage of the toonapha molesta borer to the toona sinensis cannot be effectively solved, the tree species cannot be popularized in a large range, and the sustainable development of the fast-growing broad-leaved tree species is severely restricted. Meanwhile, in part of forest stand test fields, the toona sinensis is extremely easy to suffer repeated freezing injury, so that the toona sinensis cannot grow normally.
By genetic transformation technology, ideal genes are transferred into the genome of a receptor plant, so that the aim of targeted improvement is achieved, and a new way is provided for tree breeding. At present, with the progress of the isolation and identification of related genes and in vitro culture regeneration technology, it has become practical to breed new varieties with excellent traits by genetic transformation methods. With the continuous and deep research of transgenosis, the changed traits are not only limited to insect resistance or disease resistance, but also extend to the aspects of resistance to environmental stress and the like. However, the toona sinensis as a woody plant has the characteristics of long growth period, high heterozygosity, poor regeneration capacity and the like, so that genetic transformation has certain difficulty and specificity, and the biological characteristics of the toona sinensis cause that the toona sinensis is difficult to be disinfected thoroughly in the in vitro tissue culture process, so that the technical problems of endophyte pollution, explant browning difficult to overcome and the like are easily generated, so that the successful construction of a toona sinensis genetic transformation system is seriously influenced, and the research on the toona sinensis genetic transformation system is not reported yet. Based on the above, at present, no suitable genetic transformation system can be used for transferring resistance genes and the like into the toona sinensis so as to achieve the purpose of promoting the directional genetic improvement of the toona sinensis.
Disclosure of Invention
Based on the situation, the invention aims to provide an agrobacterium-mediated genetic transformation method of the toona sinensis, which realizes the successful application of the genetic transformation technology in the toona sinensis and obtains higher transformation rate.
The main purpose of the invention is realized by the following technical scheme:
an agrobacterium-mediated genetic transformation method of toona sinensis, the method comprising the steps of:
taking leaves of the toona sinensis as explants, and infecting the explants with a bacterial solution of agrobacterium tumefaciens carrying a plant expression vector;
inoculating the infected explants to a co-culture medium for co-culture;
subjecting the co-cultured explants to an induction culture comprising: induction culture of callus, induction culture of adventitious buds, induction culture of buds and induction culture of roots;
in one embodiment, the bacterial liquid contains 45 mmol/L-155 mmol/L acetosyringone, and the co-culture medium contains 145 mmol/L-155 mmol/L acetosyringone.
In one embodiment, the co-culture medium is an MS culture medium containing 2.8-3.2 mg/L6-BA, 0.8-1.2 mg/L KT, 0.03-0.06 mg/L NAA, 28-32 g/L sucrose, 4.8-5.2 g/L agar and 145-155 mmol/L acetosyringone.
In one embodiment, the co-cultivation conditions include: and keeping away from light, wherein the temperature is 23-27 ℃, and the time duration is 22-26 h.
In one embodiment, the explants are subjected to ultrasonic treatment before infection, wherein the ultrasonic treatment lasts for 25-35 s, and the power of ultrasonic treatment is 4.5-5.5 kHz.
In one embodiment, the plant expression vector is pcambia1305.2 and the agrobacterium tumefaciens is agrobacterium tumefaciens EHA105.
In one embodiment, the step of inducing culture of adventitious buds comprises:
the callus induction culture adopts a T1 culture medium, wherein the T1 culture medium is an MS culture medium containing 2.8-3.2 mg/L6-BA, 0.8-1.2 mg/L KT, 0.03-0.06 mg/L NAA, 28-32 g/L sucrose, 4.8-5.2 g/L agar and 90-110 mg/L cefotaxime sodium;
the induction culture of the adventitious bud adopts a T2 culture medium, wherein the T2 culture medium is an MS culture medium containing 2.8-3.2 mg/L6-BA, 0.8-1.2 mg/L KT, 0.03-0.06 mg/L NAA, 28-32 g/L sucrose, 4.8-5.2 g/L agar, 90-110 mg/L cefotaxime sodium and 9-11 mg/L kanamycin;
the induction culture of the bud adopts a T3 culture medium, and the T3 culture medium is an MS culture medium containing 0.25-0.35 mg/L of 6-BA, 0.18-0.22 mg/L of NAA, 28-32 g/L of cane sugar, 4.8-5.2 g/L of agar, 90-110 mg/L of cefotaxime sodium and 9-11 mg/L of kanamycin.
The root is induced by a T4 culture medium, wherein the T4 culture medium is an MS culture medium containing 0.08-0.12 mg/L NAA, 13-17 g/L sucrose, 4.8-5.2 g/L agar, 90-110 mg/L cefotaxime sodium and 9-11 mg/L kanamycin.
In one embodiment, the conditions for inducing culture include: the temperature is 23-27 ℃, the illumination intensity is 2300 lx-2700 lx, the illumination time is 10-14 h every day, and the replacement frequency of the culture medium is changed once every 13-16 d.
In one embodiment, the OD of the bacterial liquid 600 0.4 to 0.8.
In one embodiment, the method further comprises the step of identifying the products of the genetic transformation.
In one embodiment, the identification method comprises one or more of PCR molecular detection, reporter gene detection and laser confocal fluorescence microscopy.
In one embodiment, the method further comprises the step of hardening off and transplanting the identified positive plants.
In one embodiment, the explant is pre-cultured and then infected with a bacterial solution of Agrobacterium tumefaciens carrying a plant expression vector; the pre-culture duration is 1 d-3 d, the pre-culture medium is an MS culture medium containing 2.8 mg/L-3.2 mg/L6-BA, 0.8 mg/L-1.2 mg/L KT, 0.03 mg/L-0.06 mg/L NAA, 28 g/L-32 g/L sucrose and 4.8 g/L-5.2 g/L agar, and the pre-culture conditions comprise: and keeping out of light at the temperature of 23-27 ℃.
Compared with the prior art, the invention has the following beneficial effects:
aiming at the toona sinensis, the leaves are taken as explants, and in the process of infecting the explants by using an infecting liquid of agrobacterium tumefaciens carrying a plant expression vector and co-culturing the infected explants, acetosyringone is added into the infecting liquid and the co-culture medium at the same time, particularly the explants are further matched with proper ultrasonic treatment and optimization of tissue culture conditions, so that the toona sinensis genetic transformation mediated by agrobacterium is successfully realized for the first time, and the genetic transformation rate is higher (the genetic transformation rate is kept at about 10%). Meanwhile, repeated tests show that the method has good repeatability and stability, is not influenced by seasonal environments and the like, is suitable for batch seedling culture, and is favorable for promoting the industrial development of the toona sinensis. Overall, the method provided by the invention lays a technical foundation for improving the toona sinensis by the exogenous gene and provides an effective platform.
Drawings
FIG. 1 is a diagram of preculture in one example of the invention;
FIG. 2 is a diagram of co-cultivation in one embodiment of the present invention;
FIG. 3 is a diagram of the induction culture of buffered callus according to an embodiment of the present invention;
FIG. 4 is a diagram showing induction culture (culture 30 d) of a positive callus according to an example of the present invention;
FIG. 5 is a diagram showing induction culture (culture 45 d) of positive callus according to an example of the present invention;
FIG. 6 is a drawing showing elongation culture (culture 60 d) of a positive adventitious bud in one example of the present invention;
FIG. 7 is a drawing showing rooting culture (culture 90 d) of a positive seedling in one embodiment of the present invention;
FIG. 8 is a graph showing the results of detection of PCR molecules in one embodiment of the present invention; in the figure, m.maeker; CK-. Negative control; CK + positive control; 1 to 5, 7 to 8 and 10 to 12 are all positive Miao Yangpin; 6.9 is an untransformed shoot sample;
FIG. 9 is a graph showing the result of GUS staining in one example of the present invention; in the figure, a negative control; B. positive seedlings;
FIG. 10 is a confocal laser fluorescence microscope in accordance with an embodiment of the present invention;
FIG. 11 is a graph of the effect of different concentrations of kanamycin on the induction rate of implants;
figure 12 is a graph of the effect of different concentrations of cefotaxime sodium on explant induction rate.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention 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.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides an agrobacterium-mediated genetic transformation method for toona sinensis, which comprises the following steps:
taking leaves of the toona sinensis as explants, and infecting the explants with a bacterial solution of agrobacterium tumefaciens carrying a plant expression vector;
inoculating the infected explants to a co-culture medium for co-culture;
subjecting the co-cultured explants to an induction culture comprising: induction culture of callus, induction culture of adventitious buds, induction culture of buds and induction culture of roots;
the bacterial liquid contains acetosyringone, and the co-culture medium contains acetosyringone;
in the embodiment of the invention, the explant is pre-cultured before infection, and the pre-culture medium and the culture conditions are basically consistent with those of the co-culture medium after infection, so that the explant can adapt to the medium and the culture conditions in advance, and the conversion rate is improved. The pre-culture duration of the embodiment of the invention is 1 d-3 d, the pre-culture medium is an MS culture medium containing 2.8 mg/L-3.2 mg/L6-BA, 0.8 mg/L-1.2 mg/L KT, 0.03 mg/L-0.06 mg/L NAA, 28 g/L-32 g/L sucrose and 4.8 g/L-5.2 g/L agar, and the pre-culture conditions comprise: and keeping out of light at the temperature of 23-27 ℃.
It is understood that the explant is cut to produce a wound and then infected.
According to the embodiment of the invention, acetosyringone (AS) is added into the infection bacterial liquid and the co-culture medium, and the AS can promote the processing and transfer of T-DNA by inducing the activation and expression of the Vir region gene of the agrobacterium, so that the T-DNA of the agrobacterium can more easily enter the plant genome and be integrated with the plant genome, and the transformation rate is increased.
The callus induction culture is the callus induction culture from the wound of the explant and is buffer culture, and the buffer culture is performed before the screening culture (adventitious bud induction culture, bud induction culture and root induction culture) in the embodiment of the invention, because the explant is very sensitive to kanamycin, the explant is prevented from direct browning death after contacting kanamycin, and the buffer culture is performed before the screening culture, so that the transformation rate is improved. Preferably, the duration of the buffer culture is 12d to 16d.
According to the embodiment of the invention, cefotaxime sodium (Cef) is added into a T1 culture medium, a T2 culture medium, a T3 culture medium and a T4 culture medium, and the Cef is used for controlling the excessive growth of agrobacterium tumefaciens and avoiding serious pollution in the tissue culture process; in the embodiment of the invention, kanamycin (Kan) is added into a T2 culture medium, a T3 culture medium and a T4 culture medium for effectively screening transformed tissues, so that explants which are not successfully transformed die.
The method provided by the embodiment of the invention combines a high-efficiency regeneration system of the toona sinensis, carries out multi-aspect experiments and groping in the construction of a genetic transformation system, can realize multi-aspect genetic improvement such as stress resistance research of the toona sinensis, fills the blank that the precious tree species obtains transgenic plants in genetic transformation, and provides an effective way for variety improvement and genetic analysis of the toona sinensis. The method provided by the embodiment of the invention has the advantages that the conversion rate is about 10%, a reliable research platform is provided for the rapid propagation technology of the toona sinensis, and favorable conditions are created for the genetic engineering modification of the toona sinensis.
It is understood that the "plant expression vector" described in the embodiments of the present invention includes a selection gene and a reporter gene, and may further include a vector carrying an insect-resistant gene and a disease-resistant gene. Taking plant expression vector pCAMBIA1305.2 as an example, the screening gene is kanamycin-resistant gene, and the reporter gene is Gus gene.
Preferably, the "leaf blade" described in the embodiment of the present invention is a seedling blade of sterile seed of toona sinensis. The source of the explant is not limited by environment and season, and the material is convenient to obtain; in addition, the problems of difficult sterilization, serious browning, difficult rooting and the like of the explant are solved; the toona sinensis leaves are selected as the receptor, the advantage that the leaves contain more protoplasts and are easy to generate adventitious buds is fully utilized, a solid experimental foundation is laid for selecting the mother tree leaves as the receptor in the following process, and the purpose of ensuring the excellent properties of the mother tree can be achieved.
In one example, the T1 medium (buffer medium) contains 90mg/L to 110mg/L of cefotaxime sodium, and the culture medium for screening culture comprises an induction medium (T2 medium) from callus induced by explant wound to adventitious bud, an induction medium (T3 medium) from adventitious bud to bud, and an induction medium (T4 medium) from bud to bud root, which all contain 9mg/L to 11mg/L of kanamycin and 90mg/L to 110mg/L of cefotaxime sodium. Referring to fig. 11 and 12, the effect of different concentrations of kanamycin and cefotaxime sodium on explant induction rate, respectively.
In one example, the bacterial liquid contains 45 mmol/L-155 mmol/L acetosyringone, and the co-culture medium contains 145 mmol/L-155 mmol/L acetosyringone.
In one example, the co-culture medium is MS medium containing 2.8 mg/L-3.2 mg/L6-BA, 0.8 mg/L-1.2 mg/L KT, 0.03 mg/L-0.06 mg/L NAA, 28 g/L-32 g/L sucrose, 4.8 g/L-5.2 g/L agar and 145 mmol/L-155 mmol/L acetosyringone.
In one example, the co-cultivation conditions include: and keeping away from light, wherein the temperature is 23-27 ℃, and the time duration is 22-26 h.
In one example, the explant is subjected to ultrasonic treatment before infection, the ultrasonic treatment time is 25-35 s, and the ultrasonic treatment power is 4.5-5.5 kHz. According to the embodiment of the invention, the short-time crushing treatment is carried out on the toona sinensis explants by combining ultrasonic oscillation before the agrobacterium infection is carried out, so that the success rate of the agrobacterium infection on the explant cells is greatly increased.
In one example, the plant expression vector is pcambia1305.2 and the agrobacterium tumefaciens is agrobacterium tumefaciens EHA105.
In one example, the callus induction culture (i.e. the explant after co-culture is subjected to buffer culture) adopts a T1 culture medium, and then the adventitious bud is subjected to elongation culture by using a T3 culture medium until the bud is 2-4 cm high;
the T1 culture medium is an MS culture medium containing 2.8-3.2 mg/L6-BA, 0.8-1.2 mg/L KT, 0.03-0.06 mg/L NAA, 28-32 g/L sucrose, 4.8-5.2 g/L agar and 90-110 mg/L cefotaxime sodium;
the induction culture of the adventitious bud (namely the induction culture of healthy callus till the adventitious bud grows) adopts a T2 culture medium, wherein the T2 culture medium is an MS culture medium containing 2.8-3.2 mg/L of 6-BA, 0.8-1.2 mg/L of KT, 0.03-0.06 mg/L of NAA, 28-32 g/L of sucrose, 4.8-5.2 g/L of agar, 90-110 mg/L of cefotaxime sodium and 9-11 mg/L of kanamycin;
the bud induction culture (the adventitious bud is subjected to extension culture until the bud height is 2-4 cm) adopts a T3 culture medium, and the T3 culture medium is an MS culture medium containing 0.25-0.35 mg/L of 6-BA, 0.18-0.22 mg/L of NAA, 28-32 g/L of sucrose, 4.8-5.2 g/L of agar, 90-110 mg/L of cefotaxime sodium and 9-11 mg/L of kanamycin.
In one example, the induction culture of the root comprises subjecting the bud to root culture with T4 medium;
the induction culture of the root (i.e. the induction culture of the bud-bud rooting) adopts a T4 culture medium, and the T4 culture medium is an MS culture medium containing 0.08-0.12 mg/L NAA, 13-17 g/L sucrose, 4.8-5.2 g/L agar, 90-110 mg/L cefotaxime sodium and 9-11 mg/L kanamycin.
In one example, the conditions of the induction culture include: the temperature is 23-27 ℃, the illumination intensity is 2300 lx-2700 lx, the illumination time is 10-14 h every day, and the replacement frequency of the culture medium is changed once every 13-16 d.
Aiming at the characteristics of the toona sinensis, the embodiment of the invention ensures that a large number of transgenic plants are obtained and the transformation efficiency is improved, the embodiment of the invention improves the culture mode, firstly carries out pre-culture, co-culture and buffer culture, can improve the transformation efficiency and overcome the mass death caused by the fact that fragile explants are immediately contacted with antibiotics, and then selects 3 selective culture mediums to carry out selective screening culture on the explants infected by agrobacterium sequentially, thereby not only meeting the bacteriostatic effect of cefotaxime sodium and the screening effect of kanamycin, but also meeting the nutritional requirements of the explants at different development stages.
In the embodiment of the invention, the early-stage experiment determines that the optimal concentration for kanamycin screening is 10mg/L and the optimal concentration for cefotaxime sodium bacteriostasis is 100mg/L, a proper amount of antibiotics is added into various induction culture media, untransformed plants can be screened out and transgenic plants with good growth vigor can be obtained, and then the transgenic plant materials can be verified through PCR molecular detection, GUS dyeing and laser confocal fluorescence microscopy.
In one example, the OD of the bacteria liquid 600 0.4 to 0.8, for example 0.6.
In one example, the method further comprises the step of identifying the products of the genetic transformation.
In one example, the identification method includes one or more of PCR molecular detection, reporter gene detection and laser confocal fluorescence microscopy.
In one example, the method further comprises the step of hardening off and transplanting the identified positive plants.
Example 1
The embodiment provides an agrobacterium-mediated genetic transformation method for toona sinensis, which comprises the following operation steps:
1. obtaining and pre-culturing of sterile seedlings and explants
1.1 removing double wings of the Chinese toon seeds, soaking the Chinese toon seeds in sterile water with the initial temperature of 45 ℃ for 4 hours, and removing the shriveled seeds floating on the upper part.
1.2 the seeds soaked for 4 hours are sterilized by alcohol solution with the volume percentage concentration of 75 percent for 1min, washed by sterile water for 1 time, sterilized by sodium hypochlorite solution with the mass percentage concentration of 10 percent for 18min, washed by sterile water for 4 times, and washed by each time for 5min on a sterile super clean bench.
1.3 sowing seeds in a blank MS culture medium for culturing, wherein the seeds begin to sprout after 3d of seed culture, and aseptic Miao Zhenshe grows out after 35d, cutting leaves on a super clean bench and cutting 2-3 wounds on the leaves to serve as explants; in the step: the culture conditions are that the culture temperature is 25 +/-2 ℃, the culture is carried out in the dark until radicles grow out, and then the culture is carried out under the conditions that the illumination intensity is 2500lx and the illumination time is 12h.
1.4 inoculating the leaf into a pre-culture medium for pre-culture, wherein the paraxial surface of the leaf is tightly attached to the culture medium during inoculation (see figure 1); in the step:
the pre-culture medium is an MS culture medium added with 6-BA 3mg/L, KT 1mg/L, NAA0.05mg/L, sucrose 30g/L and agar 5 g/L;
the culture condition of the pre-culture is 25 +/-2 ℃, the dark culture is carried out, and the pre-culture time is 3d.
2. Explant agrobacterium infection and co-culture treatment
2.1 the explant obtained in the step 1 is firstly put into sterile water and treated in an ultrasonic oscillator for 30s, and the ultrasonic power is 5kHz, so that the purpose of breaking cells is achieved.
2.2 infecting the explant subjected to ultrasonic treatment by using an agrobacterium tumefaciens bacterial liquid carrying a plant expression vector, wherein the plant expression vector contains a screening marker gene and a reporter gene, the explant is placed in a shaking table to be continuously shaken during the infection period, the explant is taken out after being shaken for 20min, the explant is washed by sterile water for 3 times, 3min is carried out each time, the explant is continuously shaken during the period, then the bacterial liquid on the surface of a leaf is sucked by sterile filter paper, and the bacterial liquid is placed in a seed inoculation disc to be dried; the method comprises the following steps:
the plant expression vector is a plasmid pCAMBIA1305.2 containing a screening gene, carries a screening gene of Kanamycin resistance (Kanamycin), and carries a GUS reporter gene containing an intron;
the agrobacterium tumefaciens bacterial liquid carrying the plant expression vector is prepared according to the following steps:
taking out the agrobacterium tumefaciens EHA105 competent cells which are preserved by freezing, putting the agrobacterium tumefaciens EHA105 competent cells into a precooled electric shock cup, and carrying out electric shock on the agrobacterium tumefaciens EHA105 competent cells and the plasmids; adding 1mL of LB culture medium into an electric shock cup, transferring the electric shock cup into a centrifuge tube, and culturing for 1.5h at 28 ℃ and 150 rpm; uniformly coating 100 mu L of the bacterial liquid on LB +50mg/L kanamycin +50mg/L rifampicin solid culture medium, and carrying out inverted culture at 29 ℃ for 2d until bacterial colonies grow out; selecting a single colony for PCR detection, and determining a positive colony; the identified Agrobacterium was added to LB liquid medium containing 50mg/L kanamycin and 50mg/L chloramphenicol, incubated at 28 ℃ overnight at 150rpm, shaken for 13h, added with AS 50mmol/L until shaken to OD 600 Carried on =0.6Agrobacterium tumefaciens bacterial liquid of the plant expression vector;
the infection is that the processed explant is soaked in sterile water and processed in an ultrasonic oscillator for 30s, then the explant is transferred to an infection liquid (namely, an agrobacterium tumefaciens liquid carrying a plant expression vector), and the infection liquid is placed in a shaking table for 20min at 28 ℃ and 120 rpm.
2.3 inoculating to a co-culture medium paved with a layer of sterile filter paper for co-culture, wherein the paraxial surface of the leaf is tightly attached to the culture medium during inoculation (see figure 2); the method comprises the following steps:
the co-culture medium is an MS culture medium added with 6-BA 3mg/L, KT 1mg/L, NAA0.05mg/L, sucrose 30g/L, agar 5g/L and AS 150 mmol/L;
the culture condition of the co-culture is 25 +/-2 ℃, the culture is carried out in the dark, and the co-culture time is 1d.
3. Acquisition of Positive seedlings
3.1 transferring the explants after co-culture to a culture medium 1 (T1) for buffer callus induction culture, wherein the culture medium 1 (T1) is an MS culture medium added with 6-BA 3mg/L + KT 1mg/L + NAA0.05mg/L + sucrose 30g/L + agar 5g/L + cefotaxime sodium 100 mg/L; buffer callus induction culture 14d (see fig. 3), excise the callus portion that appeared browned; in the step:
the buffer callus induction culture is mainly based on the fact that the toona sinensis leaves are very sensitive to kanamycin, and in order to avoid the situation that a large number of deaths occur in the early development stage, the agrobacterium-mediated transformation frequency can be obviously improved by giving a buffer culture period to the explants.
3.2 transfer of healthy callus obtained in step 3.1 to adventitious bud induction medium 2 (T2) supplemented with 6-BA 3mg/L + KT 1mg/L + NAA0.05mg/L + sucrose 30g/L + agar 5g/L +100mg/L cefotaxime sodium +10mg/L kanamycin for adventitious bud induction, see FIG. 4 (culture 30 d) and FIG. 5 (culture 45 d).
3.3 transferring the adventitious buds induced by the step 3.2 into an adventitious bud elongation culture medium (T3) for adventitious bud elongation, wherein the adventitious bud elongation culture medium (T3) is an MS culture medium added with 6-BA0.3mg/L, NAA 0.2mg/L, sucrose 30g/L, agar 5g/L, cefotaxime sodium 100mg/L and kanamycin antibiotic 10mg/L until robust buds transgenic for toona sinensis are obtained (see figure 6, culture for 60 d).
In steps 3.1, 3.2 and 3.3, the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, the illumination intensity of the culture is 2500lx, the illumination time is 12h every day, and the culture medium is replaced every 15d, so that the components of the culture medium are not lost, and the mass propagation of agrobacterium is prevented; in the process of callus and adventitious bud induction culture, the leaf explant is placed in a mode that the paraxial surface is tightly attached to a culture medium for callus induction.
4. Rooting culture of transgenic plant
When the strong bud of the red Chinese toon transgene extends to 4cm, cutting an adventitious bud, and inoculating the bud into a rooting culture medium (T4) of MS + NAA 0.1mg/L + sucrose 15g/L + agar 5g/L +100mg/L cefotaxime sodium +10mg/L kanamycin for induced rooting culture; in the step: the induced rooting culture is to cut 4cm buds and cut necrotic callus from root base, wherein the pH value of (T4) in the rooting culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, the illumination intensity is 2500lx, and the illumination time is 12h. Positive shoots obtained after rooting culture for 90 days are shown in FIG. 7.
5. Transgenic plant detection
Carrying out PCR molecular detection (figure 8), GUS staining (figure 9) and a laser confocal fluorescence microscope (figure 10) by using a screening marker gene and a reporter gene on a plant expression vector, and detecting whether the toona sinensis transgenic bud or transgenic plant obtained in the step 3 is a positive transgenic material; the method comprises the following steps:
the PCR molecular detection primer comprises:
GUS-F(SEQ ID No.1)：5′-GTC GCG CAA GAC TGT AAC CA-3′，
GUS-R(SEQ ID No.2)：5′-CGG CGA AAT TCC ATA CCT G-3′；
the PCR procedure was 94 ℃ for 2min,35 cycles (94 ℃ for 30s,58 ℃ for 30s,72 ℃ for 30 s), 72 ℃ for extension for 5min, and 4 ℃ for storage.
6. Exercising and transplanting of transgenic plants
And (3) opening a cover of the plant identified as the positive transgenic material for adaptation, taking the plant out of a culture bottle, cleaning a culture medium on roots, soaking the plant in sterile water for 1h, transplanting the plant into a sterilized matrix with the mass ratio of peat soil to vermiculite being 2:1, covering a plastic bag on a nutrition cup for moisturizing, moving the plastic bag away after 3d, and watering according to growth requirements to obtain the successfully transplanted transgenic plant.
Results of this example: the genetic transformation rate was 15.56%.
Example 2
The embodiment provides an agrobacterium-mediated genetic transformation method for toona sinensis, which comprises the following operation steps:
1. obtaining and pre-culturing of sterile seedlings and explants
1.1 removing double wings of the Chinese toon seeds, soaking the Chinese toon seeds in sterile water with the initial temperature of 45 ℃ for 3 hours, and removing the shriveled seeds floating on the upper part.
1.2 the seeds soaked for 3 hours are sterilized by alcohol solution with the volume percentage concentration of 75 percent for 1min, washed by sterile water for 1 time, sterilized by sodium hypochlorite solution with the mass percentage concentration of 10 percent for 15min, washed by sterile water for 3 times, and washed for 5min each time on a sterile super clean bench.
1.3 sowing seeds in a blank MS culture medium for culturing, wherein the seeds begin to sprout after 3 days of seed culture, and aseptic Miao Zhenshe grows out after 30 days, cutting leaves on a super clean bench and cutting 2-3 wounds on the leaves to serve as explants; the method comprises the following steps: the culture conditions are that the culture temperature is 25 +/-2 ℃, the culture is carried out in the dark until radicles grow out, and then the culture is carried out under the conditions that the illumination intensity is 2500lx and the illumination time is 12h.
1.4 inoculating the leaves into a pre-culture medium for pre-culture, wherein the paraxial surfaces of the leaves are tightly attached to the culture medium during inoculation; the method comprises the following steps:
the pre-culture medium is an MS culture medium added with 2.8mg/L of 6-BA, 0.8mg/L of KT, 0.03mg/L of NAA, 28g/L of sucrose and 4.8g/L of agar;
the pre-culture is carried out at the temperature of 25 +/-2 ℃ in the dark for 1d.
2. Explant agrobacterium infection and co-culture treatment
2.1 the explant obtained in the step 1 is firstly put into sterile water and treated in an ultrasonic oscillator for 25s, and the ultrasonic power is 4.5kHz, so that the purpose of breaking cells is achieved.
2.2 infecting the explant subjected to ultrasonic treatment by using an agrobacterium tumefaciens bacterial liquid carrying a plant expression vector, wherein the plant expression vector contains a screening marker gene and a reporter gene, the explant is placed in a shaking table to be continuously shaken during the infection period, the explant is taken out after being shaken for 20min, the explant is washed by sterile water for 3 times, 3min is carried out each time, the explant is continuously shaken during the period, then the bacterial liquid on the surface of a leaf is sucked by sterile filter paper, and the bacterial liquid is placed in a seed inoculation disc to be dried; the method comprises the following steps:
the plant expression vector is a plasmid pCAMBIA1305.2 containing a screening gene, carries a screening gene of Kanamycin resistance (Kanamycin), and carries a GUS reporter gene containing an intron;
the agrobacterium tumefaciens bacterial liquid carrying the plant expression vector is prepared according to the following steps:
taking out the agrobacterium tumefaciens EHA105 competent cells which are preserved by freezing, putting the agrobacterium tumefaciens EHA105 competent cells into a precooled electric shock cup, and carrying out electric shock on the agrobacterium tumefaciens EHA105 competent cells and the plasmids; adding 1mL of LB culture medium into an electric shock cup, transferring the electric shock cup into a centrifuge tube, and culturing for 1h at 28 ℃ and 150 rpm; uniformly coating 100 mu L of the bacterial liquid on LB +50mg/L kanamycin +50mg/L rifampicin solid culture medium, and carrying out inverted culture at 28 ℃ for 2d until bacterial colonies grow out; selecting a single colony for PCR detection, and determining a positive colony; the identified Agrobacterium was added to LB liquid medium containing 50mg/L kanamycin and 50mg/L chloramphenicol, incubated at 28 ℃ overnight at 150rpm, shaken for 12h, added with AS 45mmol/L until shaken to OD 600 =0.6 agrobacterium tumefaciens liquid carrying plant expression vector;
the infection is that the processed explant is soaked in sterile water and processed in an ultrasonic oscillator for 25s, then the explant is transferred to an infection liquid (namely, an agrobacterium tumefaciens liquid carrying a plant expression vector), and the infection liquid is placed in a shaking table for 20min at 28 ℃ and 120 rpm.
2.3 inoculating to a co-culture medium paved with a layer of sterile filter paper for co-culture, wherein the blade paraxial surface is tightly attached to the culture medium during inoculation; the method comprises the following steps:
the co-culture medium is an MS culture medium added with 6-BA2.8mg/L, KT 0.8mg/L, NAA0.03mg/L, sucrose 28g/L, agar 4.8g/L and AS 145 mmol/L;
the co-culture is carried out at the temperature of 25 +/-2 ℃ in the dark, and the co-culture time is 22h.
3. Obtaining positive seedling
3.1 transferring the co-cultured explant to an adventitious bud induction culture medium 1 (T1) for buffer callus induction culture, wherein the adventitious bud induction culture medium 1 (T1) is an MS culture medium added with 6-BA2.8mg/L + KT 0.8mg/L + NAA0.03mg/L + sucrose 28g/L + agar 4.8g/L + cefotaxime sodium 90 mg/L; buffering callus induction culture until the explant grows out callus, and cutting the callus part with browning; in the step:
the buffer callus induction culture is mainly based on the fact that the toona sinensis leaves are very sensitive to kanamycin, and in order to avoid the situation that a large number of deaths occur in the early development stage, the agrobacterium-mediated transformation frequency can be obviously improved by giving a buffer culture period to the explants.
3.2 transfer of healthy callus obtained in step 3.1 to adventitious bud induction medium 2 (T2) supplemented with 6-BA2.8mg/L + KT 0.8mg/L + NAA0.03mg/L + sucrose 28g/L + agar 4.8g/L +90mg/L cefotaxime sodium +9mg/L kanamycin for adventitious bud induction.
3.3 transferring the adventitious bud induced in the step 3.2 into an adventitious bud elongation culture medium (T3) for adventitious bud elongation, wherein the adventitious bud elongation culture medium (T3) is an MS culture medium added with 6-BA0.25mg/L + NAA 0.18mg/L + sucrose 28g/L + agar 4.8g/L +90mg/L cefotaxime sodium +9mg/L kanamycin antibiotic until obtaining a robust adventitious bud of the toona sinensis transgene.
In the steps 3.1, 3.2 and 3.3, the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, the illumination intensity of the culture is 2300lx, the illumination time is 10 hours every day, and the culture medium is replaced every 13 days, so that the components of the culture medium are not lost, and the mass propagation of agrobacterium is prevented; in the process of callus and adventitious bud induction culture, the leaf explant is placed in a mode that the paraxial surface is tightly attached to a culture medium for callus induction.
4. Rooting culture of transgenic plant
When the robust adventitious bud of the toona sinensis transgene extends to 3cm, cutting the adventitious bud, and inoculating the bud into a rooting culture medium (T4) of MS + NAA 0.08mg/L + sucrose 13g/L + agar 4.5g/L +90mg/L cefotaxime sodium +9mg/L kanamycin antibiotic to induce rooting culture; the method comprises the following steps:
the induced rooting culture is to cut 3cm adventitious buds and cut off the callus of the roots, wherein the pH value of (T4) in a rooting culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, the illumination intensity is 2300lx, and the illumination time is 10h.
5. Transgenic plant detection
Carrying out PCR molecular detection, GUS dyeing and a laser co-focusing fluorescence microscope by using a screening marker gene and a reporter gene on a plant expression vector, and detecting whether the transgenic bud or the transgenic plant of the toona sinensis obtained in the step 3 is a positive transgenic material; the method comprises the following steps:
the PCR molecular detection primer comprises:
GUS-F(SEQ ID No.1)：5′-GTC GCG CAA GAC TGT AAC CA -3′，
GUS-R(SEQ ID No.2)：5′-CGG CGA AAT TCC ATA CCT G-3′；
the PCR procedure was 94 ℃ for 2min,35 cycles (94 ℃ for 30s,58 ℃ for 30s,72 ℃ for 30 s), 72 ℃ for extension for 5min, and 4 ℃ for storage.
6. Hardening and transplanting of transgenic plants
And (3) opening a cover of the plant identified as the positive transgenic material for adaptation, taking the plant out of a culture bottle, cleaning a culture medium on roots, soaking the plant in sterile water for 1h, transplanting the plant into a sterilized matrix with the mass ratio of peat soil to vermiculite being 2:1, covering a plastic bag on a nutrition cup for moisturizing, moving the plastic bag away after 3d, and watering according to growth requirements to obtain the successfully transplanted transgenic plant.
Results of this example: the genetic transformation rate was 13.33%.
Example 3
The embodiment provides an agrobacterium-mediated genetic transformation method for toona sinensis, which comprises the following operation steps:
1. obtaining and pre-culturing of sterile plantlets and explants
1.1 removing double wings of the Chinese toon seeds, soaking the Chinese toon seeds in sterile water with the initial temperature of 45 ℃ for 5 hours, and removing the shriveled seeds floating on the upper part.
1.2 the seeds soaked for 5 hours are sterilized by alcohol solution with the volume percentage concentration of 75 percent for 1min, washed by sterile water for 1 time, sterilized by sodium hypochlorite solution with the mass percentage concentration of 10 percent for 20min and washed by sterile water for 5 times, and the seeds are washed for 5min each time on a sterile super clean bench.
1.3 sowing seeds in a blank MS culture medium for culturing, wherein the seeds begin to sprout after 3 days of seed culture, and after 40 days, sterile Miao Zhenshe grows out, and cutting leaves as explants; the method comprises the following steps:
culturing at 25 + -2 deg.C in dark until radicle grows out, and culturing under the conditions of illumination intensity of 2500lx and illumination time of 12h.
1.4 inoculating the leaves into a pre-culture medium for pre-culture, wherein the paraxial surfaces of the leaves are tightly attached to the culture medium during inoculation; the method comprises the following steps:
the pre-culture medium is an MS culture medium added with 6-BA3.2mg/L, KT 1.2mg/L, NAA0.06mg/L, sucrose 32g/L and agar 5.2 g/L;
the culture condition of the pre-culture is 25 +/-2 ℃, the dark culture is carried out, and the pre-culture time is 3d.2. Explant agrobacterium infection and co-culture treatment
2.1 the explant obtained in the step A is firstly put into sterile water and treated for 35s in an ultrasonic oscillator, and the ultrasonic power is 5.5kHz, so that the purpose of breaking cells is achieved.
2.2 infecting the explant subjected to ultrasonic treatment by using an agrobacterium tumefaciens bacterial liquid carrying a plant expression vector, wherein the plant expression vector contains a screening marker gene, the explant is placed in a shaking table to be continuously shaken during the infection period, the explant is taken out after being shaken for 20min, the explant is washed by sterile water for 3 times, the explant is continuously shaken every time for 3min, then the bacterial liquid on the surface of a leaf is sucked by sterile filter paper, and the bacterial liquid is placed in a seed inoculation table to be dried; in the step:
the plant expression vector is a plasmid pCAMBIA1305.2 containing a screening gene, carries a screening gene of Kanamycin resistance (Kanamycin), and carries a GUS reporter gene containing an intron;
the agrobacterium tumefaciens bacterial liquid carrying the plant expression vector is prepared according to the following steps:
taking out the agrobacterium tumefaciens EHA105 competent cells which are frozen and stored, putting the agrobacterium tumefaciens EHA105 competent cells into a precooled electric shock cup, and electrically shocking the agrobacterium tumefaciens EHA105 competent cells with plasmids; adding 1mL of LB culture medium into an electric shock cup, transferring the electric shock cup into a centrifuge tube, and culturing for 2h at 28 ℃ and 150 rpm; uniformly coating 100 mu L of the bacterial liquid on LB +50mg/L kanamycin +50mg/L rifampicin solid culture medium, and carrying out inverted culture at 30 ℃ for 2d until bacterial colonies grow out; selecting a single colony for PCR detection, and determining a positive colony; the identified Agrobacterium was added to LB liquid medium containing 50mg/L kanamycin and 50mg/L chloramphenicol, cultured overnight at 28 ℃ at 150rpm, shaken for 15 hours, added with AS 155mmol/L until shaken to OD 600 =0.8 agrobacterium tumefaciens liquid carrying plant expression vector;
the infection is that the processed explant is soaked in sterile water and processed in an ultrasonic oscillator for 35s, then the explant is transferred to an infection liquid (namely, an agrobacterium tumefaciens liquid carrying a plant expression vector), and the infection liquid is placed in a shaking table for 20min at 28 ℃ and 120 rpm.
2.3 inoculating to a co-culture medium paved with a layer of sterile filter paper for co-culture, wherein the paraxial surface of the leaf is tightly attached to the culture medium during inoculation; the method comprises the following steps:
the co-culture medium is an MS culture medium added with 6-BA3.2mg/L + KT 1.2mg/L + NAA0.06mg/L + sucrose 32g/L + agar 5.2g/L + AS 155 mmol/L;
the co-culture is carried out at the temperature of 25 +/-2 ℃ in the dark, and the co-culture time is 26h.
3. Acquisition of Positive seedlings
3.1 transferring the co-cultured explant to an adventitious bud induction culture medium 1 (T1) for buffer callus induction culture, wherein the adventitious bud induction culture medium 1 (T1) is an MS culture medium added with 6-BA3.2mg/L + KT 1.2mg/L + NAA0.06mg/L + sucrose 32g/L + agar 5.2g/L + cefotaxime sodium 110 mg/L; performing buffer callus induction culture for 14d, and cutting the browned callus part; the method comprises the following steps:
the buffer callus induction culture is mainly based on the fact that toona sinensis leaves are very sensitive to kanamycin, and in order to avoid the situation that a large number of deaths occur in the early development stage, the agrobacterium-mediated transformation frequency can be obviously improved by giving a buffer culture period to the explants.
3.2 transfer of healthy callus obtained in step 3.1 to adventitious bud induction medium 2 (T2) supplemented with 6-BA3.2mg/L + KT 1.2mg/L + NAA0.06mg/L + sucrose 32g/L + agar 5.2g/L +110mg/L cefotaxime sodium +11mg/L kanamycin antibiotic for adventitious bud induction, see FIG. 3 (culture 30 d) and FIG. 4 (culture 45 d).
3.3 transferring the adventitious bud induced in the step 3.2 into an adventitious bud elongation culture medium (T3) for adventitious bud elongation, wherein the adventitious bud elongation culture medium (T3) is an MS culture medium added with 6-BA0.35mg/L + NAA 0.22mg/L + sucrose 32g/L + agar 5.2g/L +110mg/L cefotaxime sodium +11mg/L kanamycin antibiotic until obtaining the transgenic robust bud of the toona sinensis (see figure 5, culture for 60 d).
In steps 3.1, 3.2 and 3.3, the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, the illumination intensity of the culture is 2700lx, the illumination time is 14h every day, and the culture medium is replaced every 16d, so that the components of the culture medium are not lost, and the mass propagation of agrobacterium is prevented; in the process of callus and adventitious bud induction culture, the leaf explant is placed in a mode that the paraxial surface is tightly attached to a culture medium for callus induction.
4. Rooting culture of transgenic plant
When the robust adventitious bud of the toona sinensis transgene extends to 5cm, cutting the adventitious bud, and inoculating the bud into a rooting culture medium (T4) of MS + NAA 0.12mg/L + sucrose 17g/L + agar 5.2g/L +110mg/L cefotaxime sodium +11mg/L kanamycin antibiotic to induce rooting culture; the method comprises the following steps:
the induced rooting culture is to cut 5cm adventitious buds and cut off the callus of the root, wherein the pH value of (T4) in the rooting culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, the illumination intensity is 2700lx, and the illumination time is 14h.
5. Transgenic plant detection
Carrying out PCR molecular detection, GUS dyeing and a laser co-focusing fluorescence microscope by using a screening marker gene and a reporter gene on a plant expression vector, and detecting whether the transgenic bud or the transgenic plant of the toona sinensis obtained in the step 3 is a positive transgenic material; the method comprises the following steps:
the PCR molecular detection primer comprises:
GUS-F(SEQ ID No.1)：5′-GTC GCG CAA GAC TGT AAC CA-3′，
GUS-R(SEQ ID No.2)：5′-CGG CGA AAT TCC ATA CCT G-3′；
the PCR procedure was 94 ℃ for 2min,35 cycles (94 ℃ for 30s,58 ℃ for 30s,72 ℃ for 30 s), 72 ℃ for extension for 5min, and 4 ℃ for storage.
6. Hardening and transplanting of transgenic plants
And (3) uncovering the plant identified as the positive transgenic material for adaptation, then taking out the plant from a culture bottle, cleaning the culture medium on the root, soaking in sterile water for 1h, then transplanting into a sterilized matrix with the mass ratio of peat soil to vermiculite of 2:1, covering a plastic bag on a nutrition cup for moisturizing, moving away the plastic bag after 3d, and watering according to growth requirements to obtain the successfully transplanted transgenic plant.
Results of this example: the genetic transformation rate was 11.11%.
Example 4
The present embodiment is a modification of embodiment 1, and the main changes with respect to embodiment 1 are: the AS concentration in the bacterial liquid was 20mmol/L. Results of this example: the genetic transformation rate was 6.67%.
Example 5
The present embodiment is a modification of embodiment 1, and the main changes with respect to embodiment 1 are: the concentration of AS in the co-cultivation medium was 200mmol/L. Results of this example: the genetic transformation rate was 7.67%.
Example 6
The present embodiment is a modification of embodiment 1, and the main changes to embodiment 1 are as follows: the duration of the co-cultivation was 48h. Results of this example: the genetic transformation rate was 7.07%.
Example 7
The present embodiment is a modification of embodiment 1, and the main changes with respect to embodiment 1 are: the duration of the ultrasonic treatment was 20 seconds. Results of this example: the genetic transformation rate was 7.16%.
Example 8
The present embodiment is a modification of embodiment 1, and the main changes with respect to embodiment 1 are:
(1) The step of induction culture of the adventitious bud comprises the following steps:
inducing and culturing the explants subjected to the co-culture by using a T1 culture medium until callus grows out, inducing and culturing the healthy callus by using a T2 culture medium until adventitious buds grow out, and inducing and culturing the adventitious buds by using a T3 culture medium;
the T1 culture medium is an MS culture medium containing 3.0mg/L of 6-BA, 0.2mg/L of KT, 0.5mg/L of NAA, 30g/L of sucrose, 5g/L of agar and 100mg/L of cefotaxime sodium;
the T2 culture medium is an MS culture medium containing 3.0mg/L of 6-BA, 0.2mg/L of KT, 0.5mg/L of NAA, 30g/L of sucrose, 5.0g/L of agar, 100mg/L of cefotaxime sodium and 10mg/L of kanamycin;
the T3 culture medium is an MS culture medium containing 0.3mg/L of 6-BA, 0.5mg/L of NAA, 30g/L of sucrose, 5.0g/L of agar, 100mg/L of cefotaxime sodium and 10mg/L of kanamycin.
(2) The root induction culture comprises the following steps:
performing induction culture on the adventitious bud by using a T4 culture medium;
the T4 culture medium is an MS culture medium containing 0.5mg/L NAA, 15g/L sucrose, 5g/L agar, 100mg/L cefotaxime sodium and 10mg/L kanamycin.
Results of this example: the genetic transformation rate was 6.94%.
Example 9
The present embodiment is a modification of embodiment 1, and the main changes with respect to embodiment 1 are:
the T1 culture medium is an MS culture medium added with 6-BA 3mg/L, KT 1mg/L, NAA0.05mg/L, sucrose 30g/L, agar 5g/L and cefotaxime sodium 200 mg/L;
the T2 culture medium is an MS culture medium added with 6-BA 3mg/L, KT 1mg/L, NAA0.05mg/L, sucrose 30g/L, agar 5g/L, cefotaxime sodium 200mg/L and kanamycin antibiotic 5 mg/L;
the T3 culture medium is an MS culture medium added with 6-BA0.3mg/L + NAA 0.2mg/L + sucrose 30g/L + agar 5g/L +200mg/L cefotaxime sodium +5mg/L kanamycin antibiotic;
the T4 culture medium is an MS culture medium added with NAA 0.1mg/L, sucrose 15g/L, agar 5g/L, cefotaxime sodium 200mg/L and kanamycin antibiotic 5 mg/L.
Results of this example: the genetic transformation rate was 6.21%.
Comparative example 1
The comparative example is that of example 1, and the main difference relative to example 1 is that in "2. Agrobacterium explant infection and cocultivation treatment", only the culture solution contains 150mmol/L of AS, and the cocultivation medium does not contain AS, i.e., the cocultivation medium is MS medium containing 6-BA 3mg/L + KT 1mg/L + NAA0.05mg/L + sucrose 30g/L + agar 5 g/L. The rest is the same as example 1.
Results of this example: the genetic transformation rate was 3.34%.
Comparative example 2
The comparative example is that of example 1, and the main difference relative to example 1 is that "2. In the explant agrobacterium infection and co-culture treatment", the bacterial liquid does not contain AS, and only AS is added to the co-culture medium, namely the co-culture medium is an MS medium added with 6-BA 3mg/L + KT 1mg/L + naa0.05mg/L + sucrose 30g/L + agar 5g/L + AS 50 mmol/L.
Results of this example: the genetic transformation rate was 2.21%.
The inventive examples relate to the following preferred tests: the following preferred tests were performed with respect to example 1 with one variable being changed, and the data in the table below were performed in triplicate, including mean, standard deviation (which can be used as a criterion for reproducibility), and significance of difference in duncan analysis (which can be used to determine whether there is a significant difference in the different gradients of the variable).
(1) The sensitivity of explants to different concentrations of cana was compared and the results are shown in the following table and in fig. 11:
TABLE 1 sensitivity of explants to different concentrations of kanamycin
(2) The bacteriostatic effect of different concentrations of cefotaxime sodium and the sensitivity of explants to different concentrations of cefotaxime sodium are compared, and the results are shown in the following table and fig. 12:
table 2, bacteriostatic effect of different concentrations of cefotaxime sodium and explant sensitivity to different concentrations of cefotaxime sodium
(3) The effect of pre-culture on genetic transformation was studied and the results are shown in the following table:
TABLE 3 Effect of Pre-culture on genetic transformation
(4) Bacteria liquid OD 600 The impact of genetic transformation was optimized and the results are shown in the following table:
TABLE 4
(5) The effect of infection patterns on genetic transformation was studied and the results are shown in the following table:
TABLE 5
(6) The effect of AS on genetic transformation was optimized and the results are shown in the following table:
TABLE 6
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Sequence listing
<110> southern China university of agriculture
<120> agrobacterium-mediated genetic transformation method for toona sinensis
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Claims (7)
1. An agrobacterium-mediated genetic transformation method of toona sinensis, which is characterized by comprising the following steps:
taking leaves of the toona sinensis as explants, and infecting the explants by using a bacterial solution of agrobacterium tumefaciens carrying a plant expression vector after pre-culturing the explants; the pre-culture duration is 1 d-3 d, the pre-culture medium is an MS culture medium containing 2.8 mg/L-3.2 mg/L6-BA, 0.8 mg/L-1.2 mg/L KT, 0.03 mg/L-0.06 mg/L NAA, 28 g/L-32 g/L sucrose and 4.8 g/L-5.2 g/L agar, and the pre-culture conditions comprise: keeping out of light at the temperature of 23-27 ℃; the plant expression vector is pCAMBIA1305.2; the agrobacterium tumefaciens is agrobacterium tumefaciens EHA105; the OD600 of the bacterial liquid is 0.4-0.8; the bacterial liquid contains 45 mmol/L-155 mmol/L acetosyringone, the explant is subjected to ultrasonic treatment before infection, the ultrasonic treatment time is 25 s-35 s, and the ultrasonic treatment power is 4.5 kHz-5.5 kHz;
inoculating the infected explants to a co-culture medium for co-culture; the co-culture medium is an MS culture medium containing 2.8 mg/L-3.2 mg/L6-BA, 0.8 mg/L-1.2 mg/L KT, 0.03 mg/L-0.06 mg/L NAA, 28 g/L-32 g/L sucrose, 4.8 g/L-5.2 g/L agar and 145 mmol/L-155 mmol/L acetosyringone, and the co-culture conditions comprise: keeping out of light, wherein the temperature is 23-27 ℃, and the time duration is 22-26 h;
subjecting the co-cultured explants to an induction culture comprising: induction culture of callus, induction culture of adventitious buds, induction culture of buds and induction culture of roots; carrying out induction culture on the explants subjected to co-culture, wherein the induction culture of the callus adopts a T1 culture medium, and the T1 culture medium is an MS culture medium containing 2.8-3.2 mg/L of 6-BA, 0.8-1.2 mg/L of KT, 0.03 mg-0.06 mg/L of NAA, 28-32 g/L of sucrose, 4.8-5.2 g/L of agar and 90-110 mg/L of cefotaxime sodium; the induction culture of the adventitious bud adopts a T2 culture medium, wherein the T2 culture medium is an MS culture medium containing 2.8-3.2 mg/L6-BA, 0.8-1.2 mg/L KT, 0.03 mg/L-0.06 mg/L NAA, 28-32 g/L sucrose, 4.8-5.2 g/L agar, 90-110 mg/L cefotaxime sodium and 9-11 mg/L kanamycin; the induction culture of the bud adopts a T3 culture medium, wherein the T3 culture medium is an MS culture medium containing 0.25-0.35 mg/L of 6-BA, 0.18-0.22 mg/L of NAA, 28-32 g/L of cane sugar, 4.8-5.2 g/L of agar, 90-110 mg/L of cefotaxime sodium and 9-11 mg/L of kanamycin; the root is induced and cultured by a T4 culture medium, wherein the T4 culture medium is an MS culture medium containing 0.08-0.12 mg/L NAA, 13-17 g/L sucrose, 4.8-5.2 g/L agar, 90-110 mg/L cefotaxime sodium and 9-11 mg/L kanamycin.
2. The agrobacterium-mediated toona sinensis genetic transformation method according to claim 1, wherein the bacterial liquid contains 50mmol/L acetosyringone, and the co-culture medium contains 150mmol/L acetosyringone.
3. The agrobacterium-mediated genetic transformation method of toona sinensis according to claim 2, wherein the co-culture medium is an MS medium containing 3 mg/L6-BA, 1mg/L KT, 0.05mg/L NAA, 30g/L sucrose, 5g/L agar and 150mmol/L acetosyringone.
4. The agrobacterium-mediated genetic transformation method of toona sinensis according to claim 1, wherein the co-cultivation conditions comprise: protected from light at 25 ℃ for 24h.
5. The agrobacterium-mediated toona sinensis genetic transformation method according to claim 1, wherein the induction culture of the callus uses a T1 medium, and the T1 medium is an MS medium containing 3mg/L of 6-BA, 1mg/L of KT, 0.05mg/L of NAA, 30g/L of sucrose, 5g/L of agar and 100mg/L of cefotaxime sodium;
the induction culture of the adventitious bud adopts a T2 culture medium, wherein the T2 culture medium is an MS culture medium containing 3mg/L of 6-BA, 1mg/L of KT, 0.05mg/L of NAA, 30g/L of sucrose, 5g/L of agar, 100mg/L of cefotaxime sodium and 10mg/L of kanamycin;
the induction culture of the bud adopts a T3 culture medium, and the T3 culture medium is an MS culture medium containing 0.3 mg/L6-BA, 0.2mg/L NAA, 30g/L sucrose, 5g/L agar, 100mg/L cefotaxime sodium and 10mg/L kanamycin;
the induction culture of the root adopts a T4 culture medium, and the T4 culture medium is an MS culture medium containing 0.1mg/L NAA, 15g/L sucrose, 5g/L agar, 100mg/L cefotaxime sodium and 10mg/L kanamycin;
the conditions of the induction culture include: the temperature is 23-27 ℃, the illumination intensity is 2300 lx-2700 lx, the illumination time is 10-14 h every day, and the replacement frequency of the culture medium is changed once every 13-16 d.
6. The agrobacterium-mediated genetic transformation method of toona sinensis according to any of claims 1 to 5, further comprising the step of identifying the product resulting from genetic transformation; the identification step comprises one or more than two combination of PCR molecular detection, reporter gene detection and laser confocal fluorescence microscopy.
7. An Agrobacterium mediated genetic transformation method of Toona sinensis as claimed in any one of claims 1 to 5 further comprising the step of acclimatizing and transplanting the identified positive plants.
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