CN112553248B - Establishment method and genetic transformation method of Miscanthus stramineus genetic transformation system - Google Patents
Establishment method and genetic transformation method of Miscanthus stramineus genetic transformation system Download PDFInfo
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Abstract
The invention discloses a method for establishing a genetic transformation system of Miscanthus stramineus and a genetic transformation method, belonging to the technical field of plant bioengineering. The method for establishing the old mango wheat genetic transformation system tests and counts the callus induction capability of five explants, and determines the callus induced by young ears as an optimal transformation receptor; performing mediated transformation on young ears and young ear calluses of the Miscanthus stramineus by using a particle gun method, respectively selecting embryonic calluses of 25d and 35d for inducing the young ears of the Miscanthus stramineus as target calluses, and performing two pretreatment methods on the target calluses before bombardment: hypertonic treatment, filter paper drying, and subculture on the bombarded callus to perform recovery culture based on dark culture conditions. Finally, the genetic transformation method of the Sichuan grass No.2 old mango wheat mediated by the gene gun with the young ear callus of 25d as the transformation receptor and the filter paper drying pretreatment for 2 hours is determined. The method can obtain the old mango wheat seedlings with the regeneration efficiency of 63% and the old mango wheat positive calluses with the transformation efficiency of about 40%.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, particularly relates to a plant genetic transformation method, and particularly relates to an establishment method and a genetic transformation method of a Miscanthus floridulus genetic transformation system.
Background
Old miscanthus (Elymus sibiricus) also known as lolium sagittatum, siberian Elymus, belongs to the wheat family of Gramineae, belongs to the genus perennial pasture of Elymus, and is a dominant species and a population building species in meadow steppe and meadow communities. The Miscanthus perenneus has the advantages of good tillering, strong regeneration capacity, early green turning, rich leaf quantity, high crude protein content and good palatability. Meanwhile, the Amur aweto has the characteristics of drought tolerance, cold resistance, moisture resistance, soil impoverishment resistance, salt tolerance and the like, and has an important effect in the improvement of deteriorated grasslands and the establishment of the cultivation grasslands. In particular, sichuan grass No.2 old mango wheat (Elymus sibiricus L.cv. 'Chuancao No. 2') is a new high-quality old mango wheat variety which is bred by taking the local old mango in the Aban as an original material by Sichuan province grassland scientific research institute for ten years, and has the characteristics of strong stress resistance, high grass yield, long utilization period, high forage grass quality and the like, so that the new high-quality old mango wheat variety is the main cultivated grass variety for constructing a high-quality forage grass base in the pastoral area in the northwest of Sichuan province.
In recent years, with the development of molecular biology technology, functional gene research of plants becomes a hotspot, which lays a foundation for molecular breeding, especially the generation of CRISPR/Cas9 technology, greatly accelerates the molecular breeding process of crops, but due to the limitation of genetic transformation system, the current plant functional gene research and molecular breeding are limited to some large crops and model plants, such as tobacco (Nicotiana tabacum lin.), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum l.), and the like. However, gramineous forage grass crops develop slowly in this respect, so that the development of a matched genetic transformation system for serving the research and breeding work of important crops is urgently needed.
At present, relatively few research reports about the establishment of the genetic transformation system of Miscanthus floridulus. Mainly focuses on the establishment of a tissue culture and rapid propagation system, only Li Daxu and the like preliminarily establish an agrobacterium-mediated szechuan lovage 2 # old mango genetic transformation system by taking the callus induced by mature seeds as a transformation receptor, the transformation efficiency is 11 percent, but the requirement of genetic improvement of old mango is difficult to meet. In addition, due to the complex genetic background and uneven quality of seeds, the conventional genetic transformation system of the Miscanthus floridulus still has the problems of unstable system, poor repeatability, difficulty in realizing batch transformation and the like. In addition, compared with an agrobacterium-mediated genetic transformation system, the genetic transformation system mediated by the gene gun is not limited by species and explant types, is simple to operate and strong in controllability, and cannot introduce microbial genes except a target sequence into plant cells, so that the efficiency and safety of the system are high. With the continuous development and improvement of the gene gun transformation technology, the problem of gene multi-copy tandem connection caused by gene gun transformation can be effectively overcome by controlling the plasmid concentration. Therefore, the gene gun-mediated genetic transformation system is important in plants, especially gramineae which are difficult to be genetically transformed, for example, in wheat which is mainly genetically transformed by the gene gun method and the agrobacterium-mediated method, the use of the gene gun method accounts for 68.8%, however, the technology has not been tried in Miscanthus giganteus which is difficult to be genetically transformed so far.
Disclosure of Invention
The invention aims to provide a method for establishing a gene gun-mediated Laomangmai genetic transformation system and a genetic transformation method. By utilizing the method, the old mango wheat seedlings with the regeneration efficiency of about 63% and the old mango wheat positive callus with the transformation efficiency of about 40% can be obtained.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme, which specifically comprises the following contents:
a method for establishing a genetic transformation system of Miscanthus stramineus comprises the following steps:
1) Determination of transformation receptors: testing and counting the callus induction capability of five explants of mature seeds, roots, hypocotyls, stems and young ears, and then testing and screening the differentiation and regeneration capability of the five explants induced callus, wherein the result shows that only the young ear callus can be differentiated and regenerated; finally determining the young ear induced callus as the optimal transformation receptor;
2) Agrobacterium-mediated transformation of young ears of Miscanthus stramineus and young ear callus: 4 different infection modes and different vectors are utilized to carry out agrobacterium-mediated transformation on young ears and young ear calluses of the Miscanthus strawberries, the transformation is not successful, and then a gene gun transformation method is adopted to carry out the mediation;
3) Transformation of E.coli DH 5. Alpha. Competent cells: converting Escherichia coli DH5 alpha competent cells by PANIC6A, carrying out PCR identification on hygromycin (hph) resistance genes, preserving positive colonies, sucking about 50 mu L of positive bacteria, culturing in 50mL of LB liquid culture medium containing kanamycin at 37 ℃ for 6h, extracting plasmids, and placing at-20 ℃ for later use;
4) Selecting an embryogenic target callus: respectively selecting young ears of Miscanthus straminea to induce embryonic callus with compact structure of about 25 days and 35 days, and uniform granular shape, placing in two sterile culture dishes, kneading, and respectively mixing uniformly for use;
5) Pretreatment of target callus before bombardment: two pretreatment modes are as follows: (1) hypertonic treatment: placing the callus on a hypertonic culture medium for culturing for 6h; (2) drying filter paper: placing the callus on 2 layers of sterile filter paper, drying for 2h, placing the filter paper on the surface of a callus induction culture medium for micro-soaking before using, and sealing with a Pirifilm film;
6) Preparation work before bombardment and bombardment: (1) washing microcarrier gold powder with the concentration of 60 mg/mL; (2) DNA molecule embedding microcarrier: 2.5mol/L CaCl 2 (calcium chloride) and 0.1mol/L C 7 H 22 CL 3 N 3 (spermidine) as precipitant, DNA solution concentration is 1 mug/μ L; (3) Selecting the target callus pretreated in the step 3), placing the target callus in the range of 2.5cm of the central diameter of a hypertonic culture medium, and starting to prepare for bombardment; (4) starting bombardment; bombarding 6 mu L of the embedded microcarrier each time, wherein the bombardment pressure is 1100psi, the bombardment distance is 9cm, and each dish of material is bombarded once;
7) Placing the bombarded callus in the step 6) in dark culture at 26 ℃ for 12h, then translating the callus to a subculture medium for recovery culture, keeping the contact surface of the callus and the original culture medium unchanged in the translation process, continuously carrying out RFP fluorescence tracking observation and statistics under a stereoscope at fixed time and fixed point for 10d in the recovery culture period, and finally establishing a genetic transformation method of the Guest-mediated Siberian haya 2 with 25d young ear callus as a transformation receptor and filter paper drying pretreatment for 2h.
The method for genetic transformation of Miscanthus formosanus mediated by a gene gun established by the method comprises the following specific steps:
1) Taking callus induced by young ears of old wheat and miscanthus sinensis as a transformation receptor, mediating by adopting a gene gun transformation method, transforming escherichia coli DH5 alpha competent cells, extracting positive plasmids, and placing at-20 ℃ for later use;
2) Selecting compact-structure granular embryonic callus with consistent state induced by young ear of Miscanthus stramineus, placing in two sterile culture dishes, kneading with forceps, and mixing well for use
3) Pretreatment of target callus before bombardment: placing the callus in a culture dish containing 2 layers of sterile filter paper, drying for 2h, placing the filter paper on the surface of a callus induction culture medium for micro-soaking before using, and sealing the culture dish by a Pirifilm film;
4) Preparation work before bombardment and bombardment: (1) washing microcarrier gold powder with the concentration of 60 mg/mL; (2) DNA molecule embedding microcarrier: 2.5mol/L CaCl 2 (calcium chloride) and 0.1mol/L C 7 H 22 CL 3 N 3 (spermidine) as precipitant, DNA solution concentration is 1 mug/μ L; (3) Selecting the target callus pretreated in the step 3), placing the target callus in the range of 2.5cm of the central diameter of a hypertonic culture medium, and starting to prepare for bombardment; (4) starting bombardment; bombarding the embedded microcarrier by 6 mu L each time, wherein the bombardment pressure is 1100psi, the bombardment distance is 9cm, and each dish of material is bombarded once;
5) Placing the bombarded callus in the step 4) in dark culture at 26 ℃ for 12h, then translating to a subculture medium for recovery culture, and performing RFP fluorescence tracking observation and statistics under a stereoscope at a fixed point for 10d continuously during the recovery culture.
Further, the young ear callus of the old wheat and mango in the step 1) is induced for 30 d.
Further, the contact surface of the callus and the culture medium is kept unchanged during the translation process of the step 5).
Compared with the prior art, the invention has the beneficial effects that:
1. according to the method, the induction and differentiation capacities of the calli of different explants are tested and screened, young ears are screened to serve as an ideal explant material, and the callus induced by the young ears of the Miscanthus strawberries is used for establishing a high-efficiency tissue culture regeneration system, so that the regeneration system can be stably and efficiently differentiated and regenerated, and the regeneration efficiency reaches 63%.
2. The method transforms the Amur aweto by the gene gun method for the first time, optimizes and improves the gene gun target callus pretreatment mode, adopts filter paper drying treatment, belongs to the first report at present, and is original. Compared with the conventional method (treating the target callus by using a hypertonic culture medium), the filter paper drying treatment has the advantages of simplicity, convenience, time saving and the like.
3. The genetic transformation system mediated by the gene gun is not limited by species and explant types, is simple to operate and strong in controllability, and microbial genes except for a target sequence cannot be introduced into plant cells, so that the efficiency and the safety of the system are high.
According to the invention, the primula sikkmensis mascot positive callus with the transformation efficiency reaching 40% is finally obtained by improving the pretreatment method of the target callus before the gene gun bombardment. Compared with the traditional pretreatment method, the final conversion efficiency is improved by 20 percent. Finally establishing an identification foundation for a stable and efficient genetic transformation system of the old mango wheat, and simultaneously laying a solid foundation for the gene function research and genetic improvement of the old mango wheat in the later period.
5. Compared with the conventional pretreatment method, the method shortens the time required in the whole transformation process and simultaneously obviously improves the genetic transformation efficiency of the Miscanthus floridulus. Has important practical and guiding significance.
Description of the drawings:
FIG. 1 five explants induced callus status (30 d callus):
(A) - (E) represent in turn the calli induced from the seeds (mature embryos), roots, hypocotyls, stems and young shoots, respectively. Bars =50 μm.
FIG. 2 five explant callus induction capacities: time for callus induction of different explants of Sichuan grass No.2 Amur blumea and callus induction rate
FIG. 3 differentiation and regeneration:
(A) - (F) a flow chart of a tissue culture regeneration system established by the young ear callus of the Sichuan grass No.2 old mango wheat; (A) inoculating young spikes of Miscanthus stramineus to a callus induction culture medium; (B) taking a picture after the young ears of the Sichuan grass No.2 old mango wheat are induced to callus for 35d; (C) Selecting the callus with compact and hard structure in the graph B and placing the callus in a subculture medium 15d; (D) placing the callus of the graph C in a differentiation medium for about 35D; (E) the red in-frame callus differentiation micrograph in Panel D; (F) in the diagram D, the differentiated seedling is placed in a rooting culture medium for about 30 days; (G) - (K) shows a process diagram of establishing a regeneration system by inducing the callus by the seeds of the Sichuan grass No.2 Amur barley, and the results show that the callus can not be differentiated and regenerated, and the periods shown by (G) - (K) correspond to the periods (A) - (E) one by one; the regeneration system is not successfully established for roots, stems and hypocotyls, so the result is not shown; (L) the callus differentiation efficiency of the young ears of the Sichuan grass No.2 old mango wheat changes along with time; (D) Bars =2cm in (F), (G) and (J), the remaining bars =1cm.
FIG. 4 rooting of young ear seedlings. bar =1cm.
FIG. 5 vector diagram.
The T-DNA segments of the vectors PANIC6A (A), PANIC6D (B) and PANIC6E (C).
FIG. 6 results of Agrobacterium infection.
(A) - (D) Chuan 2 old mango wheat young ear induced callus; (A) - (B) pictures before and after Agrobacterium infection Sichuan 2 Laomangmai young ear induced callus gus staining; (C) - (D) bright field and dark field pictures of callus RFP fluorescence induced by infecting young ears of Miscanthus stramineus with agrobacterium; (E) - (H) young ears of Sichuan 2 old mango wheat; (E) - (F) performing gus dyeing on the Sichuan 2 old mango wheat young ear directly infected by the agrobacterium; (G) - (H) directly infecting Sichuan 2 Amur awn wheat young ear with Agrobacterium under the RFP bright field and dark field pictures. Bars =50 μm.
FIG. 7 RFP red fluorescence after gun transformation of young ear calli. Among them BDFH has red fluorescence:
(A) - (B) 25d young ear callus (not bombarded); (C) - (D) carrying out conventional hyperosmotic treatment on the young ear callus; (E) - (F) drying 25d young ear callus filter paper for 2h; and (G) - (H) 35d of young ear callus filter paper is dried for 2H. Bars =20 μm.
FIG. 8 is a graph showing the percentage of fluorescent callus as a function of time after particle gun bombardment in two different pretreatment modes.
The specific implementation mode is as follows:
the process steps for obtaining the positive callus through the gene gun mediated young ear callus of the Miscanthus floridulus will be described below. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Materials, reagents and the like used in the following examples are commercially available from companies unless otherwise specified.
In the experiment of the invention, all solvents of the basic MS culture medium are water; solutes and concentrations (see table 1):
TABLE 1
The formula of the culture medium is as follows:
basic culture medium: MS culture medium, 30g/L of cane sugar, 7.5-7.8g/L of Agar and 5.9-6.0 of pH value; note: the culture medium was autoclaved at 121 deg.C for 15min before use.
Induction medium: MS culture medium +2,4-D (5 mg/L) +6-BA (0.15 mg/L);
subculture medium: MS culture medium +2,4-D (3 mg/L) +6-BA (0.15 mg/L);
differentiation medium: MS culture medium +6-BA (0.5 mg/L) + KT (2 mg/L);
rooting culture medium: 1/2MS culture medium;
hypertonic culture medium: MS medium +72g/L mannitol.
Note: the Chinese explanation of the English abbreviation of the invention is as follows:
2,4-D:2,4-Dichlorophenoxyacetic acid
6-BA: 6-benzylaminopurine
KT kinetin
Example 1: the specific method and the steps for determining the young ear callus as a better transformation receptor comprise the following steps:
(1) Five explants induced callus: selecting seeds of Sichuan grass No.2 Laomangmai with full young grains, complete structure and consistent state, and peeling off glumes. 100mg/L gibberellin (GA 3) is soaked for 24h, the mixture is washed by sterile water for 3 times, 2 drops of Tween-20 are added, the mixture is washed by the sterile water until no foam is generated, the mixture is soaked by 75% ethanol for 50s, the mixture is washed by the sterile water for 3 times, the content of 5% NaClO is added, the mixture is soaked for 8min, the mixture is washed by the sterile water for 5 times and then is dried on sterilized filter paper, 30 grains/dish of dried old mango wheat seeds are inoculated on a callus induction culture medium, and dark culture is carried out at 26 ℃. Meanwhile, partial seeds are put in a rooting culture medium, after about 7 days, the fine roots (cut into small sections of about 3-4 mm) and hypocotyls are cut and inoculated on a callus induction culture medium, and dark culture is carried out at 26 ℃. A part of Sichuan grass No.2 old mango seeds are sown in soil (3 th ten days) of a base of a team (Qingdao biological energy and energy crop molecular breeding team of Process research institute), the upper half of the old mango seeds are collected when the seeds reach the booting stage (6 th middle days), after outer-layer leaves are removed, the seeds are sterilized by 75% ethanol for 8min under aseptic conditions, then the seeds are aired on filter paper, young ears are carefully stripped by using a pair of tweezers, the young ears are cut into small sections of 2-3mm and inoculated on a callus induction culture medium for dark culture at 26 ℃, parts with stem nodes of young plants are selected alternatively, the middle of the stem nodes are cut into small sections of 1.5-2mm, and one end with the cut is contacted with the callus induction culture medium for dark culture at 26 ℃. The different explants were observed to induce callus status (FIG. 1). The observation result shows that the young ear (figure 1E) induces the relatively best callus state, and the callus is compact in structure, has obvious boundary and is granular embryogenic callus. The callus induced by the seeds (figure 1A), roots (figure 1B), hypocotyls (figure 1C) and stems (figure 1D) is water-soaked, spongy and loose in structure.
(2) Statistics of callus induction capacity of different explants: the time and the callus induction rate (figure 2) for the five explants of mature seeds, roots, hypocotyls, stems and young ears to be placed in the callus induction culture medium to start dedifferentiation and form the callus are counted, and the result shows that the young ears have the shortest recovery time and about 23 days. The young ear callus induction rate is the highest and can reach 90%.
(3) Differentiation and regeneration capacity tests of calli induced by different explants are carried out: after the callus is induced by the different explants for 30 days, transferring the callus into a subculture medium, subculturing for 1 time every 20 days, placing the callus in a differentiation medium after a granular yellowish embryogenic callus with an obvious boundary and a compact structure is grown, and counting the differentiation rate (figure 3). The results show that the green spots and the seedlings can not be differentiated from the callus induced by the other four explants except the young spike, the differentiation rate of the young spike callus is increased along with the time within a certain time range, and the differentiation efficiency of the young spike callus reaches about 63% of the peak value after 50 days. The differentiated plantlets were then put on rooting medium (FIG. 4).
Example 2: agrobacterium transformed young ear callus
(1) Preparing an infection working solution: the heat shock method converts PANIC6A/6D/6E (figure 5) into Agrobacterium tumefaciens EHA105 (Qingdao biological energy source of Chinese academy of sciences)And provided by energy and crop molecule breeding team of institute of Process research), selecting single clone after about 48h of plate coating, inoculating the single clone into 600 muL LB culture solution added with 50mg/L kanamycin and 50mg/L rifampicin, shaking at 28 ℃,200rpm for about 14h, after detection is positive, taking 200 muL of bacterial liquid, placing the bacterial liquid into 50mL LB culture solution added with 50mg/L kanamycin and 50mg/L rifampicin at 28 ℃, shaking at 200rpm to OD 600 Adding 100 μmol/L Acetosyringone (AS) when the content is 0.4, and shaking for 2 hr to OD 600 When =0.6, the cells were collected by centrifugation at 3500rpm for 15min. Resuspending the bacterial suspension with infection solution (Agar-free subculture medium) to final concentration OD 600 =0.3, acetosyringone (AS) concentration 100 μmol/L initial infestation.
(2) Preparing infection receptor materials: selecting young ear callus of Sichuan grass No.2 old mango wheat (about 30 days for culture) with compact structure and relatively consistent state and part of fresh young ear.
(3) Callus infection: putting the young ear callus prepared in advance into the prepared infection working solution, wherein the specific 4 infection schemes are shown in table 2.
(4) Co-culturing: after the four infection schemes are finished, respectively slightly shaking and incubating the callus fragments and the agrobacterium tumefaciens for 10min, sucking dry bacteria liquid by sterile filter paper after incubation, and airing the callus. The calli were placed on sterile filter paper wetted with the infection solution (the number of callus pieces on each filter approximately equals 20 original whole calli) and dark-cultured at 23 ℃ for 2-3d.
(5) And (3) measuring the conversion efficiency: red fluorescence tracking observation and photographing are carried out under a body type microscope (SZX 16, japan Olibbus company) and the ratio of fluorescence callus and the ratio of Gus staining are counted, and the experimental result shows that under 4 different infection schemes (table 2), young ears and young ear calluses of Miscanthus floridulus are infected by agrobacterium, and the Gus staining result and the RFP fluorescence observation result are negative. The results show that neither infection of young ears with Agrobacterium nor infection of young ear calli was successful.
Fluorescent callus ratio = (number of calli with red fluorescence/number of inoculated calli) × 100%
Gus staining ratio = (number of calluses with blue-green/number of inoculated stained calluses) × 100%
TABLE 2
Note: "+" indicates "execute"; "-" indicates "not executed"
Example 3: gene gun transformed young ear callus
(1) Preparing a callus receptor: young ears of Sichuan grass No.2 Miscanthus straminea induce callus of 25 days and 35 days, respectively select embryonic callus with compact structure and consistent state, place in two sterile culture dishes, and knead with forceps, and respectively mix them uniformly for later use.
(2) Two pretreatment modes are as follows: 1) And (3) hypertonic treatment: placing the callus on a hypertonic culture medium for culturing for 6h; 2) Drying by using filter paper: the calli were placed on 2 layers of sterile filter paper for drying treatment for 2h, the filter paper was placed on the surface of the callus induction medium before being used for micro-soaking, and the culture dish was sealed with a Pirifilm membrane.
(3) And (3) transforming escherichia coli DH5 alpha competent cells by a freeze-thawing method and extracting plasmids: thawing Escherichia coli DH5 alpha competent cells on ice, adding 600ng plasmid PANIC6A containing RFP (Escherichia coli DH5 alpha and plasmid are both provided by PANI researchers in Qingdao bioenergy and Process research institute energy and crop molecular breeding team of China academy of sciences), standing on ice for 30min, then thermally shocking at 42 ℃ for 90s, then icing for 3min, adding 600 mu L of LB without antibiotics, culturing at 37 ℃ and 200rpm, and shaking for 1h. Centrifuging at 4500rpm for 2min to concentrate bacterial liquid, dissolving thallus with 100 μ L LB, and applying the dissolved thallus to a container containing 50mg/L -1 Kanamycin was cultured on solid LB medium at 37 ℃ for 12h. After the single clone grows out, the single clone is selected to be cultured in 600 mu L LB liquid culture medium containing kanamycin at 37 ℃ for 3h, PCR is carried out to identify the hygromycin (hph) resistance gene, and a positive colony is preserved. Sucking about 50 μ L of positive thallus, culturing in 50mL LB liquid culture medium containing kanamycin at 37 deg.C for 5-6h, extracting plasmid with kit, and keeping at-20 deg.C.
(4) Preparation work before bombardment and bombardment: bombarding by a gene gun of PDS-1000He type of Bio-Rad company, and the basic steps of the bombardment are as follows: 1) Washing the microcarrier (gold powder) (concentration of microcarrier is 60 mg/mL); 2) DNA molecule embedding micro-carrierBody (2.5 mol/L CaCl) 2 (calcium chloride) and 0.1mol/L C 7 H 22 CL 3 N 3 (spermidine) as precipitant, DNA solution concentration is 1 mug/μ L); 3) Selecting the pretreated target callus in the step (2), placing the pretreated target callus in a hypertonic culture medium with the central diameter of 2.5cm, and beginning to prepare for bombardment; 4) Starting bombardment; each bombardment of 6. Mu.L of embedded microcarriers (500. Mu.g microcarriers and 0.83. Mu.g DNA) was performed at 1100psi, at a distance of 9cm, and once per dish of material.
(5) And (4) placing the bombarded callus tissues in the step (4) for dark culture at 26 ℃ for 12h, and then translating the bombarded callus tissues to a subculture medium for recovery culture (keeping the contact surface of the callus and the original medium unchanged during translation). During the recovery culture period, RFP fluorescence tracking observation and statistics are carried out under a stereoscope at fixed time and fixed points for 10 days continuously (figure 7, figure 8), and the experimental result shows that under the condition that other conditions are consistent, the total trend of the fluorescence callus ratio of the 2 pretreatment modes is increased and then reduced in a certain time period (1-10 days) after bombardment and finally tends to be stable. The filter paper drying treatment of the 25d young ear callus is higher than the fluorescence callus rate of the conventional hypertonic treatment. And finally, when the stability is reached, the fluorescence callus rate under the filter paper drying treatment is up to 40%, and the fluorescence callus rate under the conventional hypertonic treatment is up to 20%. The fluorescent callus rate of the young ear callus is higher at 25d than at 35d under the filter paper drying treatment.
Claims (2)
1. A method for establishing a genetic transformation system of Miscanthus stramineus is characterized by comprising the following steps:
1) Determination of transformation receptors: testing and counting the callus induction capability of five explants of mature seeds, roots, hypocotyls, stems and young ears, and then testing and screening the differentiation and regeneration capability of the five explants induced callus, wherein the result shows that only the young ear callus can be differentiated and regenerated; finally determining the young ear induced callus as the optimal transformation receptor;
2) Agrobacterium-mediated transformation of young ears of Miscanthus stramineus and young ear callus: 4 different infection modes and different vectors are utilized to carry out agrobacterium-mediated transformation on young ears and young ear calluses of the Miscanthus strawberries, the transformation is not successful, and then a gene gun transformation method is adopted to carry out the mediation;
3) Transformation of E.coli DH 5. Alpha. Competent cells: converting Escherichia coli DH5 alpha competent cells by PANIC6A, carrying out PCR identification on hygromycin resistance genes, storing positive colonies, sucking about 50 mu L of positive bacteria, culturing in 50mL of LB liquid culture medium containing kanamycin at 37 ℃ for 6h, extracting plasmids, and placing at-20 ℃ for later use;
4) Selecting an embryogenic target callus: respectively selecting young ears of Miscanthus straminea to induce embryonic callus with compact structure of about 25 days and 35 days, and uniform granular shape, placing in two sterile culture dishes, kneading, and respectively mixing uniformly for use;
5) Pretreatment of target callus before bombardment: two pretreatment modes are as follows: (1) hypertonic treatment: placing the callus on a hypertonic culture medium for culturing for 6h; (2) drying filter paper: placing the callus on 2 layers of sterile filter paper, drying for 2h, placing the filter paper on the surface of a callus induction culture medium for micro soaking before using, and sealing the culture dish by a Pirifilm film;
6) Preparation work before bombardment and bombardment: (1) washing microcarrier gold powder with the concentration of 60 mg/mL; (2) embedding a DNA molecule into a microcarrier: 2.5mol/L CaCl 2 And 0.1mol/L C 7 H 22 CL 3 N 3 As a precipitating agent, the concentration of the DNA solution is 1 mug/muL; (3) Selecting the target callus pretreated in the step 5), placing the target callus in the range of 2.5cm of the central diameter of a hypertonic culture medium, and starting to prepare for bombardment; (4) starting bombardment; bombarding 6 mu L of the embedded microcarrier each time, wherein the bombardment pressure is 1100psi, the bombardment distance is 9cm, and each dish of material is bombarded once;
7) Placing the bombarded callus in the step 6) at 26 ℃ for dark culture for 12h, then translating the callus to a subculture medium for recovery culture, keeping the contact surface of the callus and an original culture medium unchanged in the translation process, continuously carrying out RFP fluorescence tracking observation and statistics under a stereoscope at a fixed point for 10d at regular time during the recovery culture period, and finally establishing a genetic transformation method of the gene gun mediated Amberrypt by using 25d scion callus as a transformation receptor for filter paper drying pretreatment for 2h;
the young ear callus of the old mango wheat in the step 2) is induced for 30 d.
2. The method according to claim 1, wherein the callus contact surface with the culture medium is kept constant during the translation of step 7).
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