CN115418372A - Non-tissue-culture-dependent agrobacterium rhizogenes-mediated genetic transformation method for Phoebe bournei - Google Patents
Non-tissue-culture-dependent agrobacterium rhizogenes-mediated genetic transformation method for Phoebe bournei Download PDFInfo
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Abstract
The invention discloses a tissue culture independent agrobacterium rhizogenes mediated phoebe bournei genetic transformation method, which comprises the following steps: the method comprises the steps of (1) treating phoebe bournei seeds, (2) germinating phoebe bournei seeds and culturing seedlings, (3) preparing infection liquid, (4) infecting, (5) co-culturing, (6) inducing and culturing hairy roots, (7) detecting transgenic roots and obtaining of a compound transgenic strain. The invention establishes a stable and efficient genetic transformation system of the phoebe bournei, obtains a compound type transgenic line containing transgenic roots, has high transformation efficiency, successfully breaks through the technical obstacle of difficult transformation of the phoebe bournei, and provides technical support for obtaining transgenic compound plants and complete transgenic plants which can be used for breeding resources, and researching the gene function, secondary metabolites, transgenic compound plants and complete transgenic plants of the phoebe bournei.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a tissue culture independent agrobacterium rhizogenes mediated genetic transformation method for a phoebe bournei.
Background
The phoebe bournei is a second-grade rare and gradually-dangerous species in China and belongs to an important precious timber tree species in China. The material is tough, is an excellent wood raw material for superior buildings and furniture, has tall, big and straight tree bodies and luxuriant branches and leaves, and can be used as landscaping species. Due to the change of climatic environment and the factors of man-made felling, the existing resources of the phoebe bournei are few, and are distributed sporadically only in the west of the Yangtze river, the Fujian, the Zhejiang river, the Guangdong, the Guangxi, the Hunan, the Hubei and the Guizhou.
With the national emphasis on the cultivation and popularization of precious timber tree species, the variety improvement of the nanmu plants becomes more and more important. The basis of variety improvement is the genetic basic research related to important characters of nanmu. At present, genome sequencing of the phoebe bournei has been completed, and as a unique precious tree species used in our country, research on the phoebe bournei also starts to take the level of molecular biology. However, there is no report on the establishment of a genetic transformation system. Greatly limits the genetic basic research related to important characters of the nanmu. Therefore, the establishment of a stable transformation system for the phoebe bournei has become a very urgent problem for genetic research and breeding of phoebe bournei.
Agrobacterium rhizogenes is a group of gram-negative bacteria with a wide host range, belongs to the genus Agrobacterium of the family Rhizobiaceae, and contains Ri plasmid capable of infecting numerous plants and inducing the plants to produce highly branched adventitious roots, also known as hairy roots. Wherein the T-DNA on the Ri-plasmid is capable of insertion, integration and expression in the plant genome, thereby inducing the formation of transgenic hairy roots in the plant. Hairy roots generated by agrobacterium rhizogenes induction have the advantages of high growth speed, stable physiological and biochemical properties, stable hereditary properties and the like, and the hairy roots after genetic transformation can be used for researching plant gene functions, secondary metabolism and the like, and even can be used for plant regeneration on the basis of hairy roots to obtain complete transgenic plants and cultivate and create new plant germplasm.
At present, the agrobacterium rhizogenes-mediated plant transgenosis is mainly realized by using a sterile seedling for tissue culture, and leaves or stem segments of the sterile seedling are used as explants to be co-cultured with bacterial liquid to obtain transgenic plants, so that the process is complicated, and more time and energy are needed in the process of plant tissue culture. The infection method which is not tissue culture dependent is simpler and more convenient to operate, and the infection method of the hairy roots induced by the agrobacterium rhizogenes which is not tissue culture dependent can be roughly divided into two methods: a bacterial liquid infection method and a bacterial liquid injection method.
The agrobacterium rhizogenes-mediated transgenosis is applied to herbaceous plants more often, and the application of woody plants is difficult due to the complex genetic background of the woody plants. Secondly, woody plants generate more secondary metabolites which are easy to brown, thus resulting in low transformation efficiency. Furthermore, the transformation method, the specific technical conditions of infection and the suboptimal culture of seedlings are the reasons for the low transformation rate. Such as: the conversion rate of the jujube is only 9.4 percent [1] The conversion rate of the tea trees is 23.96 percent [2] The conversion rate of the betula luminifera leaves is 36.4 percent [3] The conversion rate of the rubber stem section is 36.6 percent [4] . The above transgenic strains of woody plants rely on tissue culture techniques. Tissue culture independent transformation is: the conversion rate of infecting apocarya by a girdling method is 45.2 percent [5] . The transformation rate of infecting pigeon pea by injection method is about 39 percent [6] . The non-tissue-culture-dependent infection mode has a slightly higher conversion rate than that of the tissue culture mode, but the conversion rate of the above systems does not exceed 50%, and the conversion efficiency is lower.
According to the invention, a set of agrobacterium rhizogenes mediated genetic transformation system with high transformation efficiency and stable heredity for agrobacterium rhizogenes independent of tissue culture is established by screening and optimizing agrobacterium rhizogenes strains, seedling age, infection bacteria liquid concentration, seedling culture conditions and culture conditions after transformation, and a transgenic composite plant with normal growth and stable heredity is obtained, namely, hairy roots induced by roots are transgenic organs, and overground organs such as stem leaves and the like are non-transgenic organs. And the complex and time-consuming procedures of seedling transplanting, seedling hardening and the like which are required in a tissue culture system and have important influence on the growth of the plants are not needed for the growth of the composite plants. After the transformed plants are infected and co-cultured, the transformed plants can be managed according to the growth management mode of normal plants. The investment of time and resource cost is greatly reduced. The scientific research and production prospect of the phoebe bournei hairy root is facilitated. The establishment of the transgenic system is not only beneficial to the deep research of key gene functions related to the development and nutrient absorption of the phoebe bournei root, but also beneficial to the auxiliary breeding design and genetic improvement of the phoebe bournei, and provides a new way for the research of metabolites and pathways of the phoebe bournei based on the complete metabolic pathway of the hairy root.
[1] Hao Zheng. Research on agrobacterium-mediated genetic transformation of jujube trees [ D ]. University of agriculture, north river, 2012.
[2] Lin Cairong, zhang Dongmin, zhang Wenjing, et al.3 Agrobacterium affects the induction of hairy roots of tea plant [ J ]. North-West plant proceedings, 2021, 41 (3): 509-516.
[3] Liu Xueyu, du Xiaoxue, chen Saiyuan, et al. Agrobacterium rhizogenes mediated high frequency induction system and genetic transformation of hairy roots of betula luminifera [ J ]. Report on agrobiotechnology, 2021, 29 (3): 495-505.
[4] Yao Qingshou, wu Yuyong, allergine. Agrobacteria mediated establishment of genetic transformation system of rubber trees [ J ]. Anhui agricscience, 2012, 40 (28): 13729-13730, 13789.
[5] Xie Xiaoting, huang Qiaoyu, wen Anchao, et al, non-tissue-culture-dependent Agrobacterium rhizogenes-mediated Carya illinoensis transformation system construction [ J ]. Fruit tree academy, 2022,39 (01): 131-140.
[6]Meng Dong,et.al.Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants[J].Plant biotechnology journal,2019,17(9):1804-1813.
Disclosure of Invention
The invention aims to overcome the technical bottleneck that the genetic transformation of the phoebe bournei is difficult, provides a method for the genetic transformation of the phoebe bournei mediated by agrobacterium rhizogenes independent of tissue culture dependence, obtains a compound transgenic plant, provides an effective platform for the molecular biological research of the phoebe bournei, can simultaneously create a transgenic compound plant meaningful for production by utilizing the method, and is used for promoting the breeding work of the phoebe bournei. In addition, the invention greatly improves the transformation efficiency of the agrobacterium rhizogenes by optimizing the transformation process, seedling management and other condition methods, and the transformation efficiency reaches more than 70 percent and is higher than that of most genetic transformation systems.
The invention provides a method for establishing a genetic transformation system of a phoebe bournei by using stems of phoebe bournei seedling as receptors and inducing transgenic hairy roots through agrobacterium rhizogenes infection. The method comprises the following steps:
(1) Pretreating the phoebe bournei seeds:
after harvesting mature Phoebe bournei fruits, removing peel, cleaning seeds, airing the seeds in the sun until no moisture is attached to the surfaces of the seeds, and then storing the seeds in sand at a low temperature;
(2) Sowing and seedling raising:
sowing the seeds subjected to low-temperature sand storage treatment in wet sand to germinate and grow to 1-3 leaves, so as to obtain the required Phoebe bournei seedlings; wherein the temperature of the seedling stage of Phoebe bournei is 26 deg.C/22 deg.C (day/night), the illumination is 16 h/dark 8h, and the illumination intensity is 30-50 μmol. M -2 ·s -1 Covering with preservative film to preserve moisture, the humidity is 70-85%;
(3) Infection:
infecting the phoebe bournei seedling with agrobacterium rhizogenes, transplanting the seedling into a fully water-absorbing matrix after infecting, and covering the infected part with the matrix; wherein the concentration OD of the infected bacterial liquid 600 0.4-1.2, the agrobacterium rhizogenes strain is K599, C58C1 or MSU440;
(4) Co-cultivation
Co-culturing the infected Phoebe bournei seedlings for 1-3 days under the dark condition; co-culture conditions are 26 ℃/22 ℃ (day/night), and a moisturizing transparent plastic cover is covered for whole-process moisturizing, wherein the humidity is 70% -85%;
(5) And (3) carrying out induction culture on hairy roots to obtain a positive phoebe bournei plant which is successfully transformed:
carrying out induction culture of hairy roots on the co-cultured Phoebe bournei seedlings, wherein the conditions of the induction culture are as follows: the temperature is 26 deg.C/22 deg.C (day/night), the illumination time is 16 h/dark 8h, and the illumination intensity is 30-50 μmol · m -2 ·s -1 (ii) a Moisturizing the whole induction period, and opening an air hole of the cover after culturing for one week; when the induction of the hairy roots is started, irrigating the roots once a week for three weeks, wherein the roots of the seedlings are promoted by adding NAA into 1/4Hoagland nutrient solution to ensure that the final concentration of the NAA is 0.05-0.1mg/L, and taking the nutrient solution fully absorbed by the matrix as a standard, and no excess water is left in a seedling tray;
(6) And (5) culturing a compound transgenic plant.
Preferably, the sand storage treatment mode in the step (1) is as follows: uniformly mixing the seeds with clean and wet river sand with the humidity of 40-60% according to the weight ratio of 1:4-1:5, putting the mixture into a gauze bag, putting the gauze bag into a foam box with a cover, covering the cover, sealing the foam box with a preservative film, and refrigerating the mixture in a refrigerator at 4 ℃. And opening the foam box every two weeks to check the humidity of the river sand, spraying a proper amount of water by a spraying pot to keep the humidity consistent with the initial humidity of the clean and wet river sand.
Preferably, the wet sand in the step (2) is river sand with the humidity of 60-70%, and the sowing depth is 0.5-1.5cm, preferably 1cm.
Preferably, the seedling for infestation in step (2) is grown to spread 2-3 leaves.
Preferably, the infection mode in the step (3) is injection or soaking and air suction after acupuncture, and injection is preferred.
Preferably, the site of infection in step (3) is 0-1cm, preferably 0.5-1cm, of stem above the seed. Injection sites 3-5.
Preferably, the concentration OD of the infected bacterial liquid in the step (3) 600 Is 0.4-1.2, preferably OD 600 Is 0.8-1.2.
Preferably, the Agrobacterium rhizogenes strain in step (3) is K599.
Preferably, the matrix in the step (3) is peat soil, perlite and vermiculite, and the volume ratio of the peat soil to the perlite is 2:1:1, wherein the grain diameter of peat soil is 1-10mm, the grain diameter of perlite is 3-6mm, and the grain diameter of vermiculite is 1-3mm.
Preferably, the step (6) is to transfer the positive plants containing the transgenic roots to 1/4Hoagland nutrient solution for water culture or continue culturing in a substrate, wherein the culture conditions are as follows: 70-85% of humidity, 16 h/8 h of illumination/darkness and 26 ℃/22 ℃ (day/night). After the transgenic root system is fully developed, the non-transgenic root of the plant can be removed, and the compound transgenic plant is obtained.
The invention has the beneficial effects that:
1) The root system induction efficiency can be improved, the growth speed of the hairy roots can be improved, and the growth of seedlings and the improvement of transformation efficiency can be promoted only by adopting weak light illumination with proper intensity in the whole process of the hairy root induction culture stage.
2) According to the invention, trace NAA is added into the root-promoting nutrient solution in the induction culture stage of the hairy roots, so that the induction of the hairy roots is promoted and the conversion efficiency of the hairy roots is improved.
3) The invention provides the cultivation method which adopts high humidity cultivation in the whole processes of seedling cultivation, co-cultivation, hairy root induction cultivation and the like, balances transpiration level, avoids the phenomenon of leaf margin scorching and promotes the growth of overground parts and roots of plants.
The invention establishes a stable and efficient genetic transformation system of the phoebe bournei, obtains a compound transgenic line containing transgenic roots, has high transformation efficiency, successfully breaks through the technical obstacle of difficult transformation of the phoebe bournei, and provides technical support for obtaining transgenic compound plants and complete transgenic plants which can be used for breeding resources and researching the gene function, secondary metabolites, transgenic compound plants, complete transgenic plants and the like of the phoebe bournei.
Drawings
FIG. 1 vector diagram.
FIG. 2 transgenic composite Phoebe Fujiannanensis plants; wherein: A. plants under natural light conditions, plants under b.gfp protein fluorescence excitation conditions.
FIG. 3 stereomicroscope fluorescence detection of transgenic roots; wherein: A. roots under natural light, roots under fluorescent excitation of b.gfp protein. GFP is the transgenic root and CK is the root that is not genetically transformed.
FIG. 4 results of GFP production amplification of untransformed root and transgenic roots; wherein: 1 and 2 are samples of non-genetically transformed root DNA, and 3 and 4 are samples of transgenic root DNA.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples, which are provided for illustration only, but not for limiting the scope of the present invention.
Example (b): non-tissue-culture-dependent agrobacterium rhizogenes-mediated genetic transformation method for Phoebe bournei
1 materials and methods
1.1 seed treatment and Germination
1.1.1 Phoebe bournei seed treatment
Removing peel of mature Phoebe bournei fruit, cleaning, and sun drying until no moisture adheres to the surface. Mixing the seeds with clean and wet river sand (about 40% humidity, the mixture is held by hand to form a ball and is scattered by hand) uniformly according to the weight ratio of 1:5, putting the mixture into a gauze bag, putting the gauze bag into a foam box with a cover, covering the cover, sealing the foam box with a preservative film, and refrigerating the mixture in a refrigerator at 4 ℃. The foam box is opened every two weeks to check the humidity of the river sand, and the watering can sprays a proper amount of water to preserve moisture, so that the humidity is kept as consistent as possible to the humidity of the river sand during the initial sand storage.
1.1.2 Germination and seedling management of Phoebe bournei seeds
Taking a seedling raising pot with the diameter of more than 10cm and the depth of more than 15 cm, paving wet river sand with the thickness of 8 cm and the humidity of 70 percent in the pot, and scattering seeds stored in the sand with the density of about grains/10 cm 2 Covering with fine sand of about 1cm, spraying water to moisten, covering with preservative film above the pot for moistening, placing in dark place for germination, and spraying water 1 time every two days for moistening. After the seeds germinate, the seedling raising container is placed in a light intensity of 50 mu mol.m -2 ·s -1 Under the weak light, the illumination is 16 h/dark 8h, the temperature is 26 ℃/22 ℃ (day/night), the preservation film is kept to be covered and the moisture is preserved in the culture process, and a watering can is used for spraying water for 1 time every two days, so that the moisture and humidity in the seed germination and seedling growth processes are ensured.
1.2 Agrobacterium rhizogenes transformation of Phoebe Fujian
1.2.1 preparation of the invaded dye solution
The bacterial solution of Agrobacterium rhizogenes K599 transferred to the pCAMBIA1300-35sGFP vector of FIG. 1 was taken out from a-80 ℃ refrigerator, streaked and activated on a solid LB medium supplemented with 50mg/L streptomycin and 50mg/L kanamycin, and inverted cultured at 28 ℃ for 2-3 days. Selecting a single clone, performing shake culture at 28 deg.C and 200rpm for 14h in 10ml of liquid LB medium containing 50mg/L streptomycin and 50mg/L kanamycin, sucking 200. Mu.L of the single clone, adding the single clone into 50ml of liquid LB medium containing 50mg/L streptomycin and 50mg/L kanamycin, performing shake culture at 28 deg.C and 200rpm until the bacterial liquid concentration OD 600 =0.8, centrifuge cultured broth at 4722g at RT for 10min, pour off supernatant, add isovolumetric resuspension (10 mM MES-KOH, pH =5.2, 10mM MgCl) 2 100 μ M AS) was prepared AS an invaded liquid.
1.2.2 infection
Selecting Phoebe bournei seedlings with 1-3 leaves developed as infection materials, carefully digging out, extracting infection liquid by using a 5ml syringe, injecting the infection liquid to stems 0-1cm above the seeds of the seedlings, injecting 5 parts per plant, and inserting a needle head of the syringe into the stems but not penetrating the stems during injection. Immediately after injection and infection, transplanting the seedlings into a fully water-absorbing matrix (the matrix is peat soil, perlite and vermiculite, and is mixed according to a volume ratio of 2.
1.2.3 Co-cultivation
The infected seedlings planted in the substrate are covered with a moisture-preserving transparent plastic cover of a seedling tray and are cultured for two days under the conditions of temperature of 26 ℃/22 ℃ (day/night), humidity of 70% and darkness.
1.3 Induction and culture of hairy roots
And (3) carrying out induction culture on hairy roots of the co-cultured seedlings, wherein the conditions of the induction culture are as follows: the temperature is 26 ℃/22 ℃ (day/night), the illumination is 16 h/dark 8h, and the illumination intensity is 50 mu mol.m -2 ·s -1 . And in the induction period, the whole course is subjected to weak light, the seedling raising plate is covered by a moisturizing transparent plastic cover for moisturizing, and after one week of culture, the air holes of the cover are opened. When the induction of the hairy roots is started, the root-promoting nutrient solution is irrigated once every week for three weeks. The root-promoting nutrient solution is 1/4Hoagland nutrient solution, 0.1mg/L NAA is added, the nutrient solution is fully absorbed by a substrate as a standard, and no excessive water is left in a seedling culture plate.
1.4 detection of transformed hairy root
After culturing for 45 days, detecting the hairy root transformation condition by adopting a portable fluorescent protein excitation light source (LUYOR-3415), and counting the plants successfully inducing the hairy roots and the successfully transformed plants. And further carrying out DNA molecular level detection, extracting DNA of the transgenic root and the untransformed root, carrying out PCR verification by using a primer containing a GFP fragment, carrying out agarose gel electrophoresis analysis on a PCR product, and determining the plant as a positive plant if the plant has a strip and the size is correct.
FIG. 3 bulk microscope fluorescence detection of transgenic roots; wherein: A. roots under natural light, roots under the fluorescence excitation condition of B.GFP protein; in the figure, GFP is a transgenic root, and CK is a root which is not genetically transformed. Transgenic roots were able to see a bright green fluorescent signal under GFP fluorescence excitation, whereas CK had no green fluorescent signal under GFP fluorescence excitation.
FIG. 4 results of GFP production amplification of untransformed root and transgenic roots; wherein: 1 and 2 are samples of non-genetically transformed root DNA, and 3 and 4 are samples of transgenic root DNA. DNA samples from transgenic roots were amplified and electrophoresed to give bands of the correct size, whereas non-genetically transformed root DNA samples did not.
1.5 Complex transgenic plant culture
Transferring the positive plants containing the transgenic roots to 1/4Hoagland nutrient solution for water culture or continuing culture in a matrix. The culture conditions are as follows: humidity 70%, light 16 h/dark 8h, temperature 26 ℃/22 ℃ (day/night). After the transgenic root system is fully developed, the non-transgenic root of the plant can be removed, and the compound transgenic plant is obtained.
FIG. 2 transgenic composite Phoebe Fujiannanensis plants; wherein: A. plants under natural light conditions, plants under b.gfp protein fluorescence excitation conditions. The hairy root above the seeds is the transgenic root of the composite plant, and the transgenic root can see bright green fluorescent signals under the fluorescent excitation light of GFP protein.
2 results of the experiment
The specific technical effects of the invention are as follows:
the induction rate is 87.5 percent, and the conversion rate is 70.6 percent. Wherein, the inductivity is = (the number of hairy root plants/the total number of infected plants) × 100%; transformation ratio = (number of positive plants/total number of infected plants) × 100%.
2.1 results of mediating Phoebe bournei transformation by different infection methods
Pricking a stem 5 part 0-1cm above the seedling seeds by using a syringe or a needle, then completely soaking the infected part into the staining solution, vacuumizing for 5 minutes, and taking out paper to suck the redundant staining solution after the completion. The remaining steps are as described in material and method 1. The injection method uses a 5ml syringe to aspirate the invader solution and inject 5 points at the stem 0-1cm above the seed. The results show that the induction rate and the conversion rate of the injection infection are higher than those of the soaking and air exhaust infection after the acupuncture.
TABLE 1 Fujian transformation results with different infection methods
2.2 optimization of the types of strains, the concentration of bacterial liquid and the age of seedlings
The strain species, bacterial liquid concentration, seedling age were optimized using a three-factor three-level orthogonal test (table 2, table 3), with other conditions as described in material and method 1.
The induction and conversion for each treatment group are shown in Table 4. In each treatment, the induction rate is 87.5 percent at most, the treatment condition is K599 strain, the concentration of the bacterial liquid is 0.8, and the seedling age is in a three-leaf stage. The highest transformation efficiency is 70.6 percent, the treatment condition is K599 bacterial strain, the concentration of bacterial liquid is 1.2, and the seedling age is two-leaf period.
The induction rate and the transformation rate of each treatment are subjected to range analysis, and the influence of each factor on the induction rate and the transformation rate of the phoebe bournei from large to small can be known from the range value (table 4) as follows: species of the Strain>Age of seedling>Concentration of bacterial liquid. According to the results of the range analysis of the orthogonal test, the optimal treatment conditions with high induction rate are as follows: MSU440 strain, bacterial liquid concentration OD 600 =0.8, seedling age is two-leaf period; the optimal treatment conditions for high conversion are: k599 Strain, bacterial liquid concentration OD 600 =1.2, the seedling age is the trefoil stage. Therefore, the transformed seedling age of the phoebe bournei can be set to be 2-3 leaf stage.
TABLE 2 three-factor three-level table
TABLE 3 three-factor three-level orthogonal experimental design table
TABLE 4 three-factor three-level orthogonal test results and range analysis
Note: k is the mean value of the induction rate or the conversion rate of the three factors under three levels; r is the worst.
2.3 optimization of hairy root Induction culture conditions
The management conditions of the seedlings are optimized according to the illumination intensity and humidity in the seedling culture conditions and whether the root-promoting nutrient solution is irrigated during the induction period of the hairy roots. Wherein, when the hairy root induction is started, the treatment group for irrigating the root-promoting nutrient solution irrigates the root-promoting nutrient solution once every week for three weeks. Other conditions were as described in materials and methods 1. Through the screening of the culture conditions, the transformation rate after optimization is greatly improved compared with that before optimization (tables 5, 6 and 7), which shows that the culture conditions of low light, higher humidity keeping and irrigation of the root promoting nutrient solution are essential for the high induction rate and the transformation rate of a genetic transformation system of the phoebe bournei.
TABLE 5 conversion results of Phoebe bournei treated with different light
TABLE 6 Fujian nan transformation results treated at different humidities
TABLE 7 Phoebe bournei transformation results treated with different nutrient solutions
The invention establishes a genetic transformation system of the phoebe bournei by utilizing agrobacterium rhizogenes mediation, obtains a compound phoebe bournei transgenic plant line containing transgenic roots, breaks through the technical obstacle that the phoebe bournei is difficult to transform, and provides technical support for the research of the functions of important genes of the phoebe bournei and the root system resistance breeding and the like. And also provides reference for establishing genetic transformation systems of other nanmu plants close to the genetic relationship with the phoebe bournei.
Claims (9)
1. A method for the tissue culture independent agrobacterium rhizogenes mediated genetic transformation of a Phoebe bournei is characterized by comprising the following steps:
(1) Pretreating the phoebe bournei seeds:
after harvesting mature Phoebe bournei fruits, removing peel, cleaning seeds, airing the seeds in the sun until no moisture is attached to the surfaces of the seeds, and then storing the seeds in sand at a low temperature;
(2) Sowing and seedling raising:
sowing the seeds subjected to low-temperature sand storage treatment in wet sand to germinate and grow to 1-3 leaves, so as to obtain the required Phoebe bournei seedlings; wherein the Phoebe bournei adopts the temperature of 26 ℃/22 ℃ (day/night) after germination in the seedling stage of Phoebe bournei, the illumination is 16 h/dark 8h, and the illumination intensity is 30-50 mu mol.m -2 ·s -1 Covering with preservative film to preserve moisture, the humidity is 70-85%;
(3) Infection:
infecting the Phoebe bournei seedling with agrobacterium rhizogenes, transplanting the seedling into a fully water-absorbing matrix after infection, and covering the infected part with the matrix; wherein the concentration OD of the infected bacterial liquid 600 0.4-1.2, the agrobacterium rhizogenes strain is K599, C58C1 or MSU440;
(4) Co-cultivation
Co-culturing the infected Phoebe bournei seedlings for 1-3 days under the dark condition; co-culture conditions are 26 ℃/22 ℃ (day/night), and a moisturizing transparent plastic cover is covered for whole-process moisturizing, wherein the humidity is 70% -85%;
(5) And (3) carrying out induction culture on hairy roots to obtain a positive phoebe bournei plant which is successfully transformed:
carrying out induction culture of hairy roots on the co-cultured Phoebe bournei seedlings, wherein the conditions of the induction culture are as follows: the temperature is 26 deg.C/22 deg.C (day/night), the illumination time is 16 h/dark 8h, and the illumination intensity is 30-50 μmol · m -2 ·s -1 (ii) a The whole process of the induction period is moisturized, and the humidity is 70-85%; after culturing for one week, opening the vent hole of the cover; when the induction of the hairy roots is started, irrigating the hairy roots once every week for three weeks, wherein the root-promoting nutrient solution isAdding NAA into 1/4Hoagland nutrient solution to make the final concentration of NAA be 0.05-0.1mg/L;
(6) And (5) culturing a compound transgenic plant.
2. The method according to claim 1, wherein the sand storage treatment in step (1) is: uniformly mixing the seeds with clean and wet river sand with the humidity of 40-60% according to the weight ratio of 1:4-1:5, putting the mixture into a gauze bag, putting the gauze bag into a foam box with a cover, covering the cover, sealing the foam box with a preservative film, and refrigerating the mixture in a refrigerator at 4 ℃; and opening the foam box every two weeks to check the humidity of the river sand, spraying a proper amount of water by the spraying pot to keep the humidity consistent with the initial humidity of the clean and wet river sand.
3. The method as set forth in claim 1, wherein the wet sand in the step (2) is river sand having a humidity of 60% to 70% and is sown at a depth of 0.5 to 1.5cm.
4. The method according to claim 3, wherein the seeding depth in the step (2) is 1cm.
5. The method according to claim 1, wherein the seedling used for infestation in step (2) is grown to spread 2 to 3 leaves.
6. The method according to claim 1, wherein the infection in the step (3) is performed by injecting or immersing and evacuating after needling; the infected part is 0-1cm above the seed stem, and the injection position is 3-5.
7. The method according to claim 1 or 6, wherein the concentration OD of the infecting bacterium solution in the step (3) 600 0.8-1.2, and K599 is the agrobacterium rhizogenes strain.
8. The method according to claim 1, wherein the matrix in step (3) is peat soil, perlite, vermiculite, in a volume ratio of 2:1:1, wherein the grain diameter of the peat soil is 1-10mm, the grain diameter of the perlite is 3-6mm, and the grain diameter of the vermiculite is 1-3mm.
9. The method according to claim 1, wherein the step (6) is specifically to transfer the positive plants containing transgenic roots to 1/4Hoagland nutrient solution for hydroponic culture or to continue culture in a medium, and the culture conditions are as follows: humidity is 70% -85%, illumination is 16 h/dark 8h, and temperature is 26 ℃/22 ℃ (day/night); after the transgenic root system is fully developed, the non-transgenic root of the plant can be removed, and the compound transgenic plant is obtained.
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| CN106857251A (en) * | 2017-01-22 | 2017-06-20 | 浙江农林大学 | A kind of Phoebe bournei somatic embryo and adventitious bud inducing method |
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| CN111454986A (en) * | 2020-04-13 | 2020-07-28 | 南京农业大学 | Genetic transformation method for pears |
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| CN106857251A (en) * | 2017-01-22 | 2017-06-20 | 浙江农林大学 | A kind of Phoebe bournei somatic embryo and adventitious bud inducing method |
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