CN113265421A - Method for establishing agrobacterium-mediated shortstem ephedra stem callus transgenic system - Google Patents
Method for establishing agrobacterium-mediated shortstem ephedra stem callus transgenic system Download PDFInfo
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Abstract
The invention discloses an establishing method of an agrobacterium-mediated ephedra brachypoda callus transgenic system, which comprises the following steps: taking the ephedra brachypoda branch as an explant, and inducing the callus to obtain callus; selecting callus, and placing the callus into agrobacterium infection solution for infection culture; co-culturing the infected callus, and then screening and culturing; performing adventitious bud screening culture and rooting culture on the resistance callus which is light yellow, full in shape and obviously expanded; and finally, judging whether the regeneration seedlings of the ephedra brachypoda are transgenic positive plants or not by a GUS (glucuronidase) staining method to obtain the ephedra brachypoda transgenic plants. The invention adopts a specific callus induction culture medium, a co-culture medium, a resistant callus screening culture medium and an adventitious bud screening culture medium to construct an agrobacterium-mediated brevicaulis callus transgenic system, so that a transgenic plant of the brevicaulis can be obtained, the time of the transformation system can be greatly shortened, and the transformation rate is improved.
Description
Technical Field
The invention relates to the technical field of forest tree transgenosis, in particular to a method for establishing an agrobacterium-mediated ephedra brachypoda callus transgenic system.
Background
Casuarina (Casuarina) is a generic name of Casuarina species, some tropical islands of australia and pacific, which can grow in extremely heavily saline soil, and is called "pioneer of world plants".
The dry shape of the casuarina equisetifolia is high and straight, the small branches are soft and droop, the casuarina equisetifolia grows rapidly, the wind resistance is strong, the salt and alkali resistance is realized, the drought and barren resistance is realized, the sea tide immersion is not feared, the plant diseases and insect pests are few, and the like. In the 50 th century of the 20 th century, coastal areas such as Taiwan, Guangdong, Hainan, Guangxi and southern Fujian in China began to be introduced and cultivated, and the three species and a part of hybrid species of Ephedra sinica Stapf, Ephedra sinica Stapf and Ephedra sinica Stapf were mainly used. At present, the casuarina equisetifolia has become a main tree species for building the windproof sand-fixation forest on the coast beach in the above areas. Besides, the planting of the casuarina equisetifolia can quickly and effectively improve coastal soil and change barren salt into good field, so that the planting of the casuarina equisetifolia is continuously popularized.
During the planting process, casuarina suffers from some problems. Firstly, casuarina equisetifolia is a thermophilic plant and has poor tolerance to low temperature. Casuarina equisetifolia introduced to Zhejiang province often suffers from severe freezing injury and is difficult to survive in early winter when the temperature is low. Although the plants are adapted by low-temperature domestication for many years, the plants can be planted in Taizhou province of Zhejiang at present. Secondly, along with the change of global climate environment and the large-area planting of casuarina equisetifolia artificial forests, the occurrence and harm of casuarina equisetifolia diseases are getting more and more serious. The bacterial wilt and the non-invasive disease decline are the main diseases of the casuarina equisetifolia, and the harm is the most serious. In addition, casuarina equisetifolia protection forest has self-allelopathic effect, and is easy to cause planting problems such as continuous planting productivity reduction, protection function decline and the like. Research also shows that the casuarina equisetifolia has strong tolerance to high salt stress, but has lower resistance to salt and alkali mixed stress. Therefore, the establishment of a rapid regeneration system and a genetic transformation system of the casuarina equisetifolia by utilizing the modern molecular biology means has very important significance for cultivating new casuarina equisetifolia varieties to enlarge the cultivation and planting area of the casuarina equisetifolia and improve the stress resistance.
In view of the value of Casuarina, Le et al (Le Q V, Bogusz D, Gherbi H, et al, Agrobacterium tumefaciens gene transfer to Casuarina glauca, a tropical nitrogen-matching tree [ J ] in 1996]Plant Science,1996,118(1):57-69) began transgenic studies of plants of the family Musca. They used Agrobacterium C58C1(GV 2260; pBIN19GUSINT) to perform genetic transformation experiments on Ephedra sinica (Casuarina glauca Sieb. ex Spr.) and established a relatively complete genetic transformation system: culturing 45d seedling epicotyl as explant, and co-culturing for 3d (adding 25 μmol. L to co-culture medium)-1Acetosyringone) of (4), callus growth screening medium contained 0.5. mu. mol. L-1NAA+2.5μmol·L-1BAP+50mg·L-1Kanamycin +250 mg. L-1And (3) the cefamycin. The target gene is detected by beta-glucuronidase analysis and detection, PCR reaction and Southern hybridization, and the transgenic technology is proved to be successful in the ephedra mauritiana and has high transformation efficiency. Since then, Le et al also conducted genetic transformation studies using the particle gun method, but it was found that the transformation efficiency was not as high as that of Agrobacterium-mediated transformation by comparison with the results of GUS staining identification of Agrobacterium transformation, and the number of transgenic plants obtained was not described therein. Subsequently, in 1997, Franche et al (Franche C, Diouf D, Le Q V, et al. genetic transformation of the actinomycin tree allocastionina Verticillata by Agrobacterium tumefaciens [ J]Plant Journal,1997,11(4):897-904) transgenic studies were carried out using Agrobacterium tumefaciens, and Ephedra verticillata (Allocaerulina Verticillata Lam) as the subject, and finally 100 transgenic plants were obtained from 23 successful independent transformation experiments. Then, they infected these transgenic plants with Frankia Allo2 strainAnd roots, wherein nitrogen-fixing nodules can grow on the roots, thereby proving that the obtained transgenic plants can be symbiotic with the Frankia actinomycetes like normal casuarina plants. The successful establishment of the method provides an effective technical basis for the research of the symbiotic mechanism of the casuarina equisetifolia-Frankia actinomycetes and the development of the research work of genetic breeding improvement. In 2003, Santi et al (Santi C, Svisoton S, Constants L, et al, breathing a reporter for gene expression students in genetic organic organisms plants of the Casuarinaceae family [ J].Plant&Soil,2003,254(1): 229-. The result shows that GFP does not affect the regeneration of casuarina equisetifolia tissues, and GFP gene can be used as a reporter gene in the process of establishing a genetic transformation system. Obertello et al (Obertello M, Santi C, Sy M O, et al. Complex of four consistent reagents for the expression of genes in the radial nitro-five transforming tree allocastarina Verticata [ J]Plant Cell Reports,2005,24(9):540-548.) the expression specificity of 4 promoter genes (CaMV 35S, e35S, e35S-4ocs and UBQ1) in Arabidopsis in roots of Ephedra verticillata, rhizobia and aerial part mature tissues was studied in 2005. Gherbi et al (Gherbi H, Nambiar-Veetil M, Zhong C, et al post-translational gene differentiating in the root system of the actinomycin tree allocastiana Verticillata [ J]Mol Plant Microbe Interact,2008,21(5): 518-. 2011 study on Hedychium arborvitae (Hedychium arborvitae) regeneration system and Agrobacterium mediated genetic transformation [ D ]]China forestry science research institute 2011.) establishes a callus in-vitro culture regeneration system and an agrobacterium tumefaciens-mediated genetic transformation system of Casuarina cunninghamiana Miq, performs genetic transformation research on exogenous genes LEA by using 3 selected clone materials, and obtains a small amount of transgenic plants. 2012 LingyongTissue culture and transgenic research (English) of Ma Huang Lian (Lin Yong Sheng, Jiang Jing, Qiao Gui Rong, etc.)].Agricultural Science&Technology,2012,40(1):1313-1314.) callus culture and transgenic system of Ephedra tenuifolia were studied, but no data were shown for obtaining whole transgenic plants.
In 2019, genome sequencing of Casuarina equisetifolia (Casuarina equisetifolia) is completed, and the Casuarina equisetifolia is expected to become a salt-tolerant model tree species of the forest trees by combining the salt stress resistance of the Casuarina equisetifolia, so that development of a transgenic system of the Casuarina equisetifolia is urgently needed for molecular research service. However, the existing transgenic system is limited to the regeneration casuarina equisetifolia, the fine casuarina equisetifolia and the coarse casuarina equisetifolia, and the method is not suitable for the short casuarina equisetifolia.
Disclosure of Invention
The invention provides an establishing method of an agrobacterium-mediated brevifolin callus transgenic system, which uses young branches of brevifolin (Casuarina equisetifolia) as research materials, realizes agrobacterium-mediated genetic transformation on the basis of a Casuarina equisetifolia callus regeneration system, and establishes a stable and rapid genetic transformation system of the brevifolin.
The specific technical scheme is as follows:
an establishment method of an agrobacterium-mediated ephedra brachycarpa callus transgenic system is characterized by comprising the following steps:
(1) taking the branches of the ephedra brachypoda as explants, cleaning and disinfecting the branches, cutting the branches into small sections, and placing the small sections on a callus induction culture medium to induce callus to obtain callus;
the callus induction culture medium comprises: 0.1mg/L NAA +0.1mg/L TDZ +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(2) selecting light yellow and bright callus, and placing the light yellow and bright callus into an agrobacterium infection solution for infection culture;
(3) after infection is finished, pouring out infection liquid, airing infected callus, putting the callus into a co-culture medium after airing, and carrying out co-culture;
the co-culture medium comprises: 0.1mg/L NAA +0.1mg/L TDZ +20mg/L As +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(4) after the co-culture is finished, cleaning and airing the callus, transferring the callus into a resistant callus screening culture medium for screening culture after airing until the callus is obviously proliferated;
the resistant callus screening culture medium comprises: 0.5mg/L of 6-BA +0.1mg/L of TDZ +5mg/L of Hyg +250mg/L of Car +1/2MS +30g/L of cane sugar +3.5g/L of plant gel, and the pH value is 5.7 +/-0.1;
(5) transferring the resistance callus which is light yellow, full in shape and obviously expanded into a resistance adventitious bud screening culture medium for adventitious bud screening culture to obtain callus containing adventitious buds;
the adventitious bud screening culture medium is 0.5 mg/L6-BA +5mg/L Hyg +250mg/L Car +1/2MS +30g/L sucrose +3.5g/L plant gel, and the pH value is 5.7 +/-0.1;
(6) transferring the callus containing the adventitious buds to a rooting culture medium for rooting culture, transferring the callus into a screening culture medium containing activated carbon for continuous growth after the length of a radicle is 2-2.5 cm, and then hardening and transplanting to obtain a transgenic seedling of the ephedra brachypoda; the rooting medium is 0.02mg/L IBA, 0.4mg/L IAA, 5mg/L Hyg, 250mg/L Car, 1/2MS, 30g/L cane sugar and 3.5g/L plant gel, and the pH value is 5.7 +/-0.1; the activated carbon culture medium is 0.5g/L activated carbon, 5mg/L Hyg, 250mg/L Car, 1/2MS, 30g/L cane sugar and 3.5g/L plant gel, and the pH value is 5.7 +/-0.1;
(7) and judging whether the transgenic ephedra brachypoda seedling is a positive plant or not to obtain the transgenic ephedra brachypoda plant.
The invention can judge the transgenic positive plants by a GUS staining method.
Further, in the step (1), the ephedra brachypoda branch takes an tender branch of the root of an ephedra seedling which grows in an artificial greenhouse for 30-90 days and is more than 1cm as an explant material; the cut small sections are 0.8-1 cm.
Further, in the step (2), the agrobacterium in the agrobacterium infection solution is GV 3101.
If the concentration of the agrobacterium is low, the infection time is short, and the amount of bacteria contacting the wound surface of the explant is small, the conversion rate is low; and if the concentration of the agrobacterium is higher, the excessive propagation of the agrobacterium is easy to damage materials, and the transformation rate can be reduced.
Further, in the step (2), the OD600nm of the agrobacterium-infected liquid is 0.6-0.8.
Further, in the step (2), the conditions of the infection culture are as follows: and (4) carrying out table infection for 2h at the temperature of 28 ℃.
Further, in the step (3), the co-cultivation conditions are as follows: culturing at 28 deg.C in dark for 2 days.
Further, in the step (4), after the co-culture is finished, the callus is cleaned by sterilized RO water containing 250mg/L carbenicillin for 5 times, and the callus is soaked for 5-10min for the last time; the screening culture conditions are as follows: subculturing once every two weeks during the screening culture, and culturing at 28 ℃ in a dark environment until callus with obvious proliferation appears, wherein part of the callus can have obvious proliferation phenomenon after about 15 days to 20 days generally. The method can finish the bacteria removal within 25-50 min, is more convenient to operate, has good effect and can not grow bacteria.
Further, in the step (5), the conditions for the adventitious bud screening culture are as follows: culturing under illumination for 45-60 days at 28 deg.C for 16h, culturing in dark for 8h, and culturing under illumination intensity of 60-80 μmol photons m-2s-1(ii) a Subcultured every two weeks.
Further, in the step (6), when the adventitious bud grows to 0.5-1cm, the adventitious bud is cut off and inserted into a rooting medium; the rooting culture conditions are as follows: culturing at 28 deg.C for 16h and culturing in dark for 8h, wherein the illumination intensity is 60-80 μmol photons m-2s-1。
Further, in the step (6), when the root length is 2cm, the seedling is transferred to a rooting medium containing activated carbon for culturing.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts a specific callus induction culture medium, a co-culture medium, a resistant callus screening culture medium and an adventitious bud screening culture medium to construct an agrobacterium-mediated brevicaulis callus transgenic system, so that a transgenic plant of the brevicaulis can be obtained, the time of the transformation system can be greatly shortened, and the transformation rate is improved.
Drawings
FIG. 1 is the procedure for constructing transgenic system of Ephedra brachypodium brevicornum callus mediated by Agrobacterium in example 1.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
Example 1
An establishment method of an agrobacterium-mediated ephedra brachypoda callus transgenic system comprises the following specific steps
(1) Taking a ephedra brachypoda (young branches with the root of more than 1cm of an ephedra seedling growing in an artificial greenhouse for 30-90 days are taken as explant materials), cleaning and sterilizing, cutting the cut ephedra brachypoda into small sections with the length of 0.8-1cm (the section is as large as possible), placing the small sections on a callus induction culture medium for inducing callus to obtain callus, wherein the callus induction rate can reach about 95%;
the callus induction culture medium is: 0.1mg/L NAA +0.1mg/L TDZ +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(2) selecting a callus with a good growth state (light yellow and transparent), and putting the callus into an agrobacterium GV3101 invasion solution with OD600 of 0.6-0.8 for infection culture; the infection culture conditions are as follows: carrying out table infection for 2h at 28 ℃; the preparation method of the agrobacterium infection solution adopts the conventional method in the field for preparation.
(3) After infection is finished, pouring out infection liquid, placing infected callus on sterile filter paper for airing, placing the aired callus into a co-culture medium, and performing co-culture;
the co-culture medium is as follows: 0.1mg/L NAA +0.1mg/L TDZ +20mg/L As +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1; a piece of sterile filter paper can be placed on the culture medium in advance to prevent large-area bacteria growth, 15-20 infected calluses can be placed in each culture medium, and the co-culture conditions are as follows: culturing at 28 deg.C in dark for 2 d;
(4) after the co-culture is finished, cleaning the callus and drying in the air, cleaning the callus by adopting sterilized RO water containing 250mg/L carbenicillin for 5 times, and finally soaking for 5-10min for the first time; after air drying, transferring the callus into a resistant callus screening culture medium for screening culture until the callus has obvious proliferation, wherein the callus proliferation rate is 90%;
the resistant callus screening culture medium comprises: 0.5mg/L of 6-BA +0.1mg/L of TDZ +5mg/L of Hyg +250mg/L of Car +1/2MS +30g/L of cane sugar +3.5g/L of plant gel, and the pH value is 5.7 +/-0.1; the conditions of the screening culture are as follows: 4-6 pieces of the callus are placed in each dish, subculture is carried out once every two weeks during the screening culture period, and the callus with obvious proliferation is cultured in dark at the temperature of 28 ℃, and part of the callus can have obvious proliferation phenomenon generally within about 15 d-20 days;
(5) transferring the resistance callus which is light yellow, full in shape and obviously expanded into a resistance adventitious bud screening culture medium for adventitious bud screening culture to obtain callus containing adventitious buds; the adventitious bud inductivity is about 55%;
the adventitious bud screening culture medium is 0.5 mg/L6-BA +5mg/L Hyg +250mg/L Car +1/2MS +30g/L sucrose +3.5g/L plant gel, and the pH value is 5.7 +/-0.1; the conditions for screening and culturing the adventitious buds are as follows: culturing under illumination for 45-60 days at 28 deg.C for 16h, culturing in dark for 8h, and culturing under illumination intensity of 60-80 μmol photons m-2s-1(ii) a Carrying out subculture once every two weeks, wherein green bud points can grow on the callus, adventitious buds can grow slowly, if the callus grows to be large, the callus can be broken into two blocks by using forceps for continuous culture, and the brown dead part needs to be peeled off in time; the adventitious bud induction rate of about 0.5cm in length can reach 55 percent after illumination culture for about 60 days.
(6) When the adventitious bud grows to 0.5-1cm, cutting the adventitious bud, inserting the cut adventitious bud into a rooting culture medium for rooting culture, hardening and transplanting the seedling to obtain the ephedra brachypoda regenerated seedling with the rooting rate of 92%;
the rooting medium is 0.02mg/L IBA, 0.4mg/L IAA, 5mg/L Hyg, 250mg/L Car, 1/2MS, 30g/L cane sugar and 3.5g/L plant gel, and the pH value is 5.7 +/-0.1; the rooting culture conditions are as follows: the culture medium is prepared in glass test tube (a slant is prepared when pouring) and tissue culture bottle, and the bud base is inserted into the culture medium by 0.1cmThe culture medium is vertically placed in a light culture box, and is subjected to light culture for 16h and dark culture for 8h at 28 ℃ every day, wherein the light intensity is 60-80 mu mol photons m-2s-1(ii) a After the roots grow for 6-10 days, the young roots grow out, and when the roots grow for 2cm, the young seedlings are transferred into a rooting culture medium containing activated carbon for culture; the rooting rate is 92% after 1 month of rooting.
(7) And judging whether the regeneration seedlings of the ephedra brachypoda are transgenic positive plants or not by a GUS (glucuronidase) staining method to obtain the ephedra brachypoda transgenic plants.
According to an experimental result, the period of the whole transgenic system is 3.5-6 months, and the time of the transformation system is greatly shortened.
Comparative example 2
In the comparative example, another antibiotic Cef is selected, and the transgenic material is screened by the same screening concentration, so that most of calluses are browned in the induction stage of the adventitious bud, and the adventitious bud cannot be induced.
The specific steps are as follows (the contents which are not described in detail in the steps (1) to (5) are the same as the steps (1) to (5) in the example 1):
(1) taking the branches of the ephedra brachypoda as explants, cleaning and disinfecting the branches, cutting the branches into small sections, and placing the small sections on a callus induction culture medium to induce callus to obtain callus;
the callus induction culture medium comprises: 0.1mg/L NAA +0.1mg/L TDZ +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(2) selecting light yellow and bright callus, and placing the light yellow and bright callus into an agrobacterium infection solution for infection culture;
(3) after infection is finished, pouring out infection liquid, airing infected callus, putting the callus into a co-culture medium after airing, and carrying out co-culture;
the co-culture medium comprises: 0.1mg/L NAA +0.1mg/L TDZ +20mg/L As +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(4) after the co-culture is finished, cleaning and airing the callus, transferring the callus into a resistant callus screening culture medium for screening culture after airing until the callus is obviously proliferated;
the resistant callus screening culture medium comprises: 0.5 mg/L6-BA +0.1mg/L TDZ +5mg/L Hyg +250mg/L Cef +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 +/-0.1;
(5) transferring the resistance callus which is light yellow, full in shape and obviously expanded into a resistance adventitious bud screening culture medium for adventitious bud screening culture to obtain callus containing adventitious buds;
the adventitious bud screening culture medium is 0.5 mg/L6-BA +5mg/L Hyg +250mg/L Cef +1/2MS +30g/L sucrose +3.5g/L plant gel, and the pH value is 5.7 +/-0.1;
however, in the adventitious bud induction stage, serious browning occurs after two subcultures of most calluses, the browning rate can reach about 70-80% through statistics, compared with Car with the same concentration, the browning rate is obviously increased, the adventitious bud induction rate is obviously reduced and is only 20-30%, the time required for the adventitious bud to reach 0.5cm is correspondingly prolonged, and the transgenic efficiency is greatly influenced.
Claims (9)
1. An establishment method of an agrobacterium-mediated ephedra brachycarpa callus transgenic system is characterized by comprising the following steps:
(1) taking the branches of the ephedra brachypoda as explants, cleaning and disinfecting the branches, cutting the branches into small sections, and placing the small sections on a callus induction culture medium to induce callus to obtain callus;
the callus induction culture medium comprises: 0.1mg/L NAA +0.1mg/L TDZ +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(2) selecting light yellow and bright callus, and placing the light yellow and bright callus into an agrobacterium infection solution for infection culture;
(3) after infection is finished, pouring out infection liquid, airing infected callus, putting the callus into a co-culture medium after airing, and carrying out co-culture;
the co-culture medium comprises: 0.1mg/L NAA +0.1mg/L TDZ +20mg/L As +1/2MS +30g/L sucrose +3.5g/L plant gel, pH 5.7 + -0.1;
(4) after the co-culture is finished, cleaning and airing the callus, transferring the callus into a resistant callus screening culture medium for screening culture after airing until the callus is obviously proliferated;
the resistant callus screening culture medium comprises: 0.5mg/L of 6-BA +0.1mg/L of TDZ +5mg/L of Hyg +250mg/L of Car +1/2MS +30g/L of cane sugar +3.5g/L of plant gel, and the pH value is 5.7 +/-0.1;
(5) transferring the resistance callus which is light yellow, full in shape and obviously expanded into a resistance adventitious bud screening culture medium for adventitious bud screening culture to obtain callus containing adventitious buds;
the adventitious bud screening culture medium is 0.5 mg/L6-BA +5mg/L Hyg +250mg/L Car +1/2MS +30g/L sucrose +3.5g/L plant gel, and the pH value is 5.7 +/-0.1;
(6) transferring the callus containing the adventitious buds to a rooting culture medium for rooting culture, transferring the callus into a screening culture medium containing activated carbon for continuous growth after the length of a radicle is 2-2.5 cm, and then hardening and transplanting to obtain a transgenic seedling of the ephedra brachypoda; the rooting medium is 0.02mg/L IBA, 0.4mg/L IAA, 5mg/L Hyg, 250mg/L Car, 1/2MS, 30g/L cane sugar and 3.5g/L plant gel, and the pH value is 5.7 +/-0.1; the activated carbon culture medium is 0.5g/L activated carbon, 5mg/L Hyg, 250mg/L Car, 1/2MS, 30g/L cane sugar and 3.5g/L plant gel, and the pH value is 5.7 +/-0.1;
(7) and judging whether the transgenic ephedra brachypoda seedling is a positive plant or not to obtain the transgenic ephedra brachypoda plant.
2. The method for establishing an agrobacterium-mediated casuarina brachycarpa callus transgene system according to claim 1, wherein in the step (1), the casuarina brachycarpa branches take tender branches of more than 1cm of roots of casuarina brachycarpa seedlings which grow in an artificial greenhouse for 30-90 days as explant materials; the cut small sections are 0.8-1 cm.
3. The method for establishing the agrobacterium-mediated ephedra brachycarpa callus transgenic system as claimed in claim 1, wherein in step (2), the agrobacterium in the agrobacterium infection solution is GV 3101.
4. The method for establishing the agrobacterium-mediated casuarina brachycarpa callus transgenic system according to claim 1, wherein in the step (2), the infection culture conditions are as follows: and (4) carrying out table infection for 2h at the temperature of 28 ℃.
5. The method for establishing an agrobacterium-mediated casuarina brachycarpa callus transgene system according to claim 1, wherein in the step (3), the co-culture conditions are as follows: culturing at 28 deg.C in dark for 2 days.
6. The method for establishing the agrobacterium-mediated breviburnum brevifolia callus transgenic system as claimed in claim 1, wherein in the step (4), after the co-culture is finished, sterilized RO water containing 250mg/L carbenicillin is adopted to clean the callus for 5 times, and the callus is soaked for 5-10min for the last time; the screening culture conditions are as follows: subculture every two weeks during the screening culture period, and culturing at 28 ℃ in a dark environment until callus with obvious proliferation exists.
7. The method for establishing the agrobacterium-mediated casuarina brachycarpa callus transgene system according to claim 1, wherein in the step (5), the conditions for the adventitious bud selection culture are as follows: culturing under illumination for 45-60 days at 28 deg.C for 16h, culturing in dark for 8h, and culturing under illumination intensity of 60-80 μmol photons m-2s-1(ii) a Subcultured every two weeks.
8. The method for constructing an Agrobacterium mediated casuarina equisetifolia callus transgene system according to claim 1, wherein in the step (6), when the adventitious bud grows to 0.5-1cm, the adventitious bud is cut off and inserted into a rooting medium; the rooting culture conditions are as follows: culturing at 28 deg.C for 16h and culturing in dark for 8h, wherein the illumination intensity is 60-80 μmol photons m-2s-1。
9. The method for constructing transgenic system of Ephedra brachypomum callus mediated by Agrobacterium according to claim 1, wherein in step (6), when the root length is 2cm, the seedling is transplanted to rooting medium containing activated carbon for culturing.
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