CN113604498B - Method for inducing hairy roots of stylosanthes guianensis - Google Patents
Method for inducing hairy roots of stylosanthes guianensis Download PDFInfo
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Abstract
The invention discloses a method for inducing hairy roots of colpitis grass, which comprises the steps of agrobacterium rhizogenes selection and culture, colpitis grass explant acquisition, colpitis grass explant infection, colpitis grass hairy root induction, hairy root degerming culture and propagation, and the hairy root induction rate reaches 58.3%. The method for inducing hairy roots of the columna of the invention provides important technical support for molecular biology research of the columna, and has a great application prospect.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for inducing hairy roots of stylosanthes guianensis.
Background
The coltsfoot (Stylosanthes guianensis) is perennial leguminous forage grass mainly distributed in tropical and subtropical areas, has higher yield and better quality, and is mainly used for animal feed, woodland greening and the like. The coltsfoot is an excellent pioneer crop for improving acid soil.
In nature, agrobacterium rhizogenes (Agrobacterium rhizogenes) can stably integrate rol genes inducing rooting into a host plant genome through T-DNA of Ri plasmid to generate hairy roots, and simultaneously integrate exogenous genes carried in Ti plasmid into the plant genome together, so as to realize cotransformation. The use of Agrobacterium rhizogenes to obtain transgenic plant hairy roots has been achieved in a variety of plants. For example, hairy roots obtained from plants such as kidney bean (Phaseolus vulgaris), soybean (Glycine max), astragalus (Astragalus membranaceus), catharanthus roseus (Catharanthus roseus) are widely used for obtaining secondary metabolites of root system and researching gene functions.
At present, the coltsfoot transgenic material is mainly obtained by a whole plant transformation method. In 1987, manners et al succeeded in introducing NTP II gene into P.stylosa and obtaining whole transgenic P.stylosa by the method of Agrobacterium tumefaciens infection for the first time (Transformation of Stylosanthes spp.using Agrobacterium tumefaciens [ J ]. Plant Cell Reports, 1987). In addition, manners used agrobacterium rhizogenes to infect stems and leaves of coltsfoot to obtain 23% complete transgenic material. Sarria et al transferred the binary vector epGV1040 into P.stylosa with Agrobacterium tumefaciens and found that the foreign gene was integrated in a single copy into the genome of the transgenic Plant (Agrobacterium-mediated transformation of Stylosanthes guianensis and production of transgenic plants [ J ]. Plant Science, 1994). In addition to methods using Agrobacterium mediation to obtain transgenic materials, quechini et al used the gene gun method to introduce Be2S1 gene transformation into P.stylosa, creating a genetic transformation system for the gene gun method (Microparticle bombardment of Stylosanthes guianensis: transformation parameters and expression of a methionine-rich 2Salbumin gene [ J ]. Plant Cell, tissue and Organ Culture, 2006). In recent years, genetic transformation systems for optimizing the transformation of the coltsfoot have been further established and optimized in China, duan Ruijun and the like, the transformation systems of the coltsfoot are optimized, and the transgenic coltsfoot containing genes of bar, hpt, npt II and the like is successfully obtained, and researches also find that the hypocotyl callus induction rate and the differentiation rate of the coltsfoot seedling are higher than those of other parts, and the coltsfoot seedling hypocotyl callus induction rate are ideal explant material parts (the research of improving the cold resistance of the coltsfoot by the transgenosis [ D ]. The university of agricultural university of south China's major academic paper, 2012). In 2012, fu Shaoping et al used the plant of the genus 5 as a material, and used agrobacterium infection to successfully introduce allene oxide cyclase gene (AOC) into the plant of the genus, to obtain the whole transformed material (Fu Shaoping, guo Jianchun, li Ruimei et al. AOC-ipt transcriptional fusion gene transformed heat-ground plant of the genus 5 [ J ]. Tropical crop theory, 2012). In addition, further application and improvement of the whole plant transformation system of the stylosanthes guianensis have been reported.
However, the transgenic material obtained by the whole plant transformation method has the defects of complex transgenic process, low transgenic efficiency, overlong transformation time and the like, and is not beneficial to research on the functions of the stylosanthes guianensis genes. Therefore, the invention of the method for inducing the hairy roots of the coltsfoot has important significance for researching the gene function of the coltsfoot, but the induction of the hairy roots of the coltsfoot has not been reported at present.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings in the prior art and provide a method for inducing hairy roots of stylosanthes guianensis.
The above object of the present invention is achieved by the following technical solutions:
a method for inducing hairy roots of stylosanthes guianensis comprises the following steps:
s1, agrobacterium rhizogenes is selected and cultured: activating and culturing agrobacterium rhizogenes strain MSU440 into agrobacterium rhizogenes bacterial liquid for later use;
s2, obtaining a coltsfoot explant: cutting cotyledonary node and/or cotyledon of germinated seedling of column flower and grass as explant;
s3, infection of the coltsfoot explants: co-culturing the post flower and grass explant in the step S2 with the agrobacterium rhizogenes bacterial liquid in the step S1;
s4, inducing hairy roots of the stylosanthes guianensis: co-culturing the post flower and grass explant infected in the step S3 in sterile water for 4-6 days, transferring to sterile filter paper infiltrated by co-culture solution for co-culturing for 12-15 days, and preliminarily obtaining hairy roots at the incision;
s5, performing degerming culture and propagation on hairy roots: after hairy roots grow to more than 2cm, flushing hairy root transformants with sterile water containing antibiotics for three times, transferring to an MS culture medium containing antibiotics, subculturing for several times until agrobacterium rhizogenes is completely killed, and finally transferring to a 1/2MS culture medium to reproduce hairy roots.
Preferably, the Agrobacterium rhizogenes bacterial liquid in the step S1 is prepared by absorbing 200-300 mu L of the activated Agrobacterium rhizogenes bacterial strain MSU440 seed bacterial liquid and inoculating the agrobacterium rhizogenes bacterial liquid into 5-10 mL of YEP liquid culture medium, and carrying out shaking culture at 26-28 ℃ at 170-190 r/min until the OD is 0.5-0.6.
Preferably, the germination in step S2 is: sterilizing the post flower seeds after dormancy removal, and placing the post flower seeds in a germination culture medium for culture germination; the culture temperature is 22-24 ℃, and the illumination intensity is 110-130 mu mol.m -2 ·S -1 The illumination time is 15-17 hours per day, and the relative humidity is 55-65%.
Further preferably, the germination medium is a 1×ms medium.
Preferably, the post flower plant explant is post flower seedling cotyledon with 1mm hypocotyl after 2 days germination.
Preferably, the co-culture solution in step S4 comprises the following components: 0.4125 g.L -1 NH 4 NO 3 、0.48g·L -1 KNO 3 、0.11g·L -1 CaCl 2 ·2H 2 O、0.0925g·L -1 MgSO 4 ·7H 2 O、0.0425g·L -1 KH 2 PO 4 、0.2075mg·L -1 KI、1.54mg L -1 H 3 BO 3 、5.56mg·L -1 MnSO 4 ·4H 2 O、2.65mg·L -1 ZnSO 4 .7H 2 O、0.0625mg·L - 1 Na 2 MoO 4 ·2H 2 O、0.00625mg·L -1 CuSO 4 ·5H 2 O、0.00625mg·L -1 CoCl 2 ·6H 2 O、9.325mg·L -1 Fe-EDTA、25mg·L -1 Inositol, 0.5 mg.L -1 Glycine; 0.5% sucrose. That is, 25 mg.L was added to 1/4 XS medium -1 Inositol, 0.5 mg.L -1 Glycine and 0.5% sucrose.
Preferably, the step S4 is cultivated in sterile water at 24-26 ℃ with the illumination intensity of 39000-39100 lx, the illumination time of 15-17 hours per day and the relative humidity of 45-55 percent for co-cultivation of 4-6; the temperature of the culture of the stylosanthes guianensis explant and the co-culture solution is 22-24 ℃, the illumination intensity is 39000-39100 lx, and the co-culture is carried out under illumination for 12-15 days.
Preferably, in step S5, the antibiotic in the sterile water containing the antibiotic and the MS culture medium containing the antibiotic is carbenicillin (Carb), and the concentration is 4-6 g/L.
According to the invention, the stylosanthes guianensis seeds are selected from different varieties of germplasm resources according to different requirements, and preferably, the stylosanthes guianensis seeds selected by the invention are RY2.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for inducing hairy roots of colpitis grass, which comprises the steps of selecting and culturing agrobacterium rhizogenes, obtaining a colpitis grass explant, infecting the colpitis grass explant, inducing the hairy roots of the colpitis grass, culturing and expanding the hairy roots, wherein the induction rate of the hairy roots reaches 58.3%, and providing important technical support for molecular biological research of the colpitis grass.
Drawings
FIG. 1 is a schematic diagram showing the induction process of hairy roots of the coltsfoot grass according to the present invention. A. Obtaining a cylindrical flower grass explant; B. culturing agrobacterium rhizogenes; C. agrobacterium rhizogenes infects the seedlings of the stylosanthes guianensis; D. co-culturing the post flower grass explant and agrobacterium rhizogenes in sterile water; E. inducing the roots of the columniform grass; F. the morphology of hairy roots of the columna. The scale is 2cm.
FIG. 2 shows the results of transgenic hairy root assay.
FIG. 3 shows the results of an experiment of the induction results of Agrobacterium tumefaciens selected from Ar1193 and ArA 4. A is the condition of Ar1193 agrobacterium rhizogenes to induce the growth of hairy roots of the stylosanthes guianensis; b is the condition of selecting ArA4 agrobacterium rhizogenes to induce the growth of hairy roots of the stylosanthes guianensis; c is a detection result of Ar1193 agrobacterium rhizogenes induced hairy roots of the stylosanthes guianensis; d is the detection result of the hairy roots of the agrobacteria induced cylindrical flowers by selecting ArA4 rooting.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The stylosanthes species is RY2, provided by the Proc. Natl. Acad. Sci. On Tropical agriculture, hainan.
Example 1
1. Method of
1. Culture medium configuration
(1) Germination medium (1×ms medium) consisted of: macroelements: 1.65 g.L -1 NH 4 NO 3 、1.9g·L - 1 KNO 3 、0.44g·L -1 CaCl 2 ·2H 2 O、0.37g·L -1 MgSO 4 ·7H 2 O、0.17g·L -1 KH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Trace elements: 0.83 mg.L -1 KI、6.2mg L -1 H 3 BO 3 、22.3mg·L -1 MnSO 4 ·4H 2 O、10.6mg·L -1 ZnSO 4 .7H 2 O、0.25mg·L -1 Na 2 MoO 4 ·2H 2 O、0.025mg·L -1 CuSO 4 ·5H 2 O、0.025mg·L -1 CoCl 2 ·6H 2 O、37.3mg·L -1 Fe-EDTA;1% sucrose; 0.3% vegetable gum. Sterilizing the germination medium at 121 ℃ for 20 minutes; after cooling to 60 ℃, the medium was poured into square petri dishes of 13 x 13cm diameter.
(2) The agrobacterium rhizogenes medium configuration steps are as follows: the YEP medium consisted of: 10 g.L -1 Tryptone, 10 g.L -1 Yeast extract, 5 g.L -1 NaCl，15g·L -1 Agar (liquid medium does not need to be added). Sterilizing in a sterilizing pot at 121deg.C for 20 min, cooling to 60deg.C, and pouring into round glass bacteria culture dish for solidification. The liquid YEP medium was stored at 4℃until use.
(3) The co-culture solution induced by the hairy roots of the columna comprises the following components: 0.4125 g.L -1 NH 4 NO 3 、0.48g·L -1 KNO 3 、0.11g·L -1 CaCl 2 ·2H 2 O、0.0925g·L -1 MgSO 4 ·7H 2 O、0.0425g·L -1 KH 2 PO 4 、0.2075mg·L -1 KI、1.54mg L -1 H 3 BO 3 、5.56mg·L -1 MnSO 4 ·4H 2 O、2.65mg·L -1 ZnSO 4 .7H 2 O、0.0625mg·L - 1 Na 2 MoO 4 ·2H 2 O、0.00625mg·L -1 CuSO 4 ·5H 2 O、0.00625mg·L -1 CoCl 2 ·6H 2 O、9.325mg·L -1 Fe-EDTA、25mg·L -1 Inositol, 0.5 mg.L -1 Glycine; 0.5% sucrose. Sterilizing the culture solution at 121 ℃ for 20 minutes; and cooling for standby. Filter paper was placed in a sterile circular petri dish with a diameter of 11cm, and the culture broth was poured into the petri dish.
(4) The composition of the degerming culture medium is as follows: macroelement 1.65g.L -1 NH 4 NO 3 、1.9g·L -1 KNO 3 、0.44g·L - 1 CaCl 2 ·2H 2 O、0.37g·L -1 MgSO 4 ·7H 2 O、0.17g·L -1 KH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Trace elements: 0.83 mg.L -1 KI、6.2mg L -1 H 3 BO 3 、22.3mg·L -1 MnSO 4 ·4H 2 O、10.6mg·L -1 ZnSO 4 .7H 2 O、0.25mg·L -1 Na 2 MoO 4 ·2H 2 O、0.025mg·L -1 CuSO 4 ·5H 2 O、0.025mg·L -1 CoCl 2 ·6H 2 O、37.3mg·L -1 Fe-EDTA;1% sucrose; 0.3% vegetable gum, 5g/LCarb
2. The method for inducing hairy roots of the stylosanthes guianensis comprises the following steps, wherein the operation steps are as shown in figure 1:
(1) Sterilizing and accelerating germination of the coltsfoot seeds: rubbing off the exocarp of the columna seed, putting the columna seed into a 1.5mL centrifuge tube to a volume of 0.5mL, adding 1mL sterile water, standing at 98 ℃ for 3 minutes in an air bath to break dormancy, sucking sterile water by a liquid transfer device, adding 75% alcohol to soak the seed, intermittently vibrating the seed to thoroughly disinfect the seed during the period, and removing the alcohol after 1 minute; cleaning with sterile water for 3 times, removing sterile water, adding 10% (v/v) sodium hypochlorite, intermittently shaking for 5min, removing sodium hypochlorite, adding sterile water, washing, and repeating the sterile water washingAnd step 3, until the sodium hypochlorite is thoroughly washed. And then carefully sowing sterilized plant seeds on the surface of the germination culture medium by using a sterile toothpick, covering a cover, sealing the edges by using a breathable medical adhesive tape, and transferring to an incubator for illumination culture until the plant seeds germinate. Conditions in the incubator were: the temperature in the day and night is 23 ℃, and the illumination intensity is 120 mu mol.m -2 ·S -1 The light time per day was 16 hours and the relative humidity was 60%.
(2) Selection and cultivation of agrobacterium rhizogenes: the Agrobacterium rhizogenes strain is MSU440 (supplied by Shanghai Biotechnology Inc.) harboring a pTF101s binary vector plasmid. A small amount of MSU440 bacterial liquid is picked by a sterile toothpick and streaked in a solid YEP culture medium. After the monoclonal colony grows out, picking a single colony from the YEP solid plate, inoculating the single colony into 0.5mL of YEP liquid culture medium, and carrying out shaking culture on the single colony at the constant temperature of 28 ℃ on a shaking table at the rotating speed of 180r/min overnight to obtain seed bacterial liquid. 200 mu L of the seed bacterial liquid is sucked up and inoculated into 5mL of YEP liquid culture medium, and the seed bacterial liquid is cultured on a constant temperature shaking table at 28 ℃ at a rotating speed of 180r/min until the OD is 0.5-0.6.
(3) Infection of the seedlings of the coltsfoot: taking out the seedlings of the columna after germination for two days from the culture medium by using sterile forceps, placing the seedlings into a sterilized culture dish, dipping agrobacterium rhizogenes bacterial liquid by using a No. 11 sterile scalpel, and cutting off hypocotyls of the columna 1mm below cotyledonary nodes of the columna. Pouring a proper amount of agrobacterium rhizogenes bacterial liquid into a culture dish, cutting two cotyledons in the bacterial liquid, removing buds at the same time, and soaking the cut cotyledons in the YEP bacterial liquid for 30 minutes to finish the infection process.
(4) Induction of hairy roots of columniform grass: placing sterilized filter paper into a circular culture dish with the diameter of 11cm, adding sterilized water to wet the filter paper to saturate the filter paper, transferring the post flower and grass explants infected for 30 minutes into the culture dish, covering a cover, sealing the edge of the culture dish with a non-breathable preservative film, and transferring the culture dish into an illumination incubator for co-culture for 5 days; conditions in the incubator were: the temperature in the daytime and the night is 25 ℃, the illumination intensity is 39060lx, the illumination time per day is 16 hours, and the relative humidity is 50%. And transferring the co-cultured cylindrical flower and grass explant into a culture dish containing co-culture solution, covering a cover, sealing the edge with a non-breathable preservative film, transferring to an illumination incubator at the temperature of 23 ℃ and the illumination intensity of 39000-39100 lx, and co-culturing for about 14 days to obtain cylindrical flower and grass hairy roots with the length of about 2cm.
(5) Degerming and expanding propagation of hairy roots of columniform grass: after hairy roots grow to more than 2cm, the hairy root transformants are washed three times with sterile water containing antibiotics (5 g/L Carb), transferred to a degerming medium containing 5g/L Carb, subcultured 1 time every 7 days until agrobacterium is completely killed, and finally transferred to a 1/2×MS medium for breeding hairy roots.
3. Transgenic hairy root detection: approximately 0.05g of the subcultured hairy roots were placed in a 2mL centrifuge tube, and the sample DNA was extracted by CTAB method, followed by detection of herbicide reporter gene by 2X Rapid Master Mix. The detection primers are as follows: bar-F CAACCACTACATCGAGACAAGCA, bar-R TCATCAGATCTCGGTGACGGG; the 20. Mu.L PCR amplification system contained: 10. Mu.L of Rapid Master Mix, 0.5. Mu.L of forward primer, 0.5. Mu.L of reverse primer, 1. Mu.L of DNA, 8. Mu.L of ddH 2 O. The reaction procedure: pre-denaturation at 98℃for 5min,35 cycles of reaction (denaturation at 98℃for 30s, annealing at 59℃for 30s, extension at 72℃for 1 min), maintenance at 72℃for 10min, and storage of amplified product at 16℃after amplification.
2. Results
The total of 24 hairy roots of 10 explants are selected for detection, and the result is shown in figure 2, and 14 hairy roots are detected to have bands in the electrophoresis result, and are positive hairy roots, so that the positive rate of the hairy roots is 58.3%.
Comparative example
The Agrobacterium rhizogenes Ar1193 and ArA4 were selected, and other culture conditions were the same as in example 1, and the induction conditions of hairy roots of the columna were as shown in FIGS. 3A-B, with induction rates of Agrobacterium rhizogenes Ar1193 and ArA4 of 23.4% and 28.5%, respectively; through DNA detection, ar1193 Agrobacterium tumefaciens is selected, the positive rate of hairy roots is 0%, arA4 Agrobacterium tumefaciens is selected, the positive rate of hairy roots is 8.3%, and the result of Ar1193 Agrobacterium tumefaciens induction is shown in FIG. 3D.
Claims (1)
1. The method for inducing hairy roots of stylosanthes guianensis is characterized by comprising the following steps of:
s1, agrobacterium rhizogenes is selected and cultured: activating and culturing agrobacterium rhizogenes strain MSU440 into agrobacterium rhizogenes bacterial liquid for later use;
s2, obtaining a coltsfoot explant: cutting germinated seedling cotyledons of the column flower and grass as explants;
s3, infection of the coltsfoot explants: co-culturing the post flower and grass explant in the step S2 with the agrobacterium rhizogenes bacterial liquid in the step S1;
s4, inducing hairy roots of the stylosanthes guianensis: co-culturing the post flower and grass explant infected in the step S3 in sterile water for 4-6 days, transferring to sterile filter paper infiltrated by co-culture solution for co-culturing for 12-15 days, and preliminarily obtaining hairy roots at the incision;
s5, performing degerming culture and propagation on hairy roots: after hairy roots grow to more than 2cm, flushing hairy root transformants with sterile water containing antibiotics for three times, transferring to an MS culture medium containing antibiotics, subculturing for several times until agrobacterium rhizogenes is completely killed, and finally transferring to a 1/2MS culture medium for breeding hairy roots;
the agrobacterium rhizogenes bacterial liquid in the step S1 is prepared by absorbing 200-300 mu L from activated agrobacterium rhizogenes bacterial strain MSU440 seed bacterial liquid, inoculating the agrobacterium rhizogenes bacterial liquid into 5-10 mL of YEP liquid culture medium, and carrying out shake culture at 26-28 ℃ at 170-190 r/min until OD is 0.5-0.6;
s2, germinating the columna seedling cotyledons with the hypocotyl of 1mm for 2 days as columna explants;
the germination in the step S2 is as follows: sterilizing the post flower seeds after dormancy removal, and placing the post flower seeds in a germination culture medium for culture germination; the culture temperature is 22-24 ℃, and the illumination intensity is 110-130 mu mol.m -2 ·S -1 The illumination time is 15-17 hours per day, and the relative humidity is 55-65%;
the co-culture solution in the step S4 comprises the following components: 0.4125 g.L -1 NH 4 NO 3 、0.48 g·L -1 KNO 3 、0.11 g·L -1 CaCl 2 ·2H 2 O、0.0925 g·L -1 MgSO 4 ·7H 2 O、0.0425 g·L -1 KH 2 PO 4 、0.2075 mg·L -1 KI、1.54 mg L -1 H 3 BO 3 、5.56 mg·L -1 MnSO 4 ·4H 2 O、2.65 mg·L -1 ZnSO 4 .7H 2 O、0.0625 mg·L -1 Na 2 MoO 4 ·2H 2 O、0.00625 mg·L -1 CuSO 4 ·5H 2 O、0.00625 mg·L -1 CoCl 2 ·6H 2 O、9.325 mg·L -1 Fe-EDTA、25 mg·L -1 Inositol, 0.5 mg.L -1 Glycine; 0.5% sucrose;
step S4, culturing in sterile water at 24-26 ℃ with the illumination intensity of 39000-39100 lx, the illumination time of 15-17 hours per day, and co-culturing at the relative humidity of 45-55% for 4-6; the temperature of the culture of the stylosanthes guianensis explant and the co-culture solution is 22-24 ℃, the illumination intensity is 39000-39100 lx, and the illumination co-culture is carried out for 12-15 days;
the antibiotic in the sterile water containing the antibiotic and the MS culture medium containing the antibiotic in the step S5 is carbenicillin, and the concentration is 4-6 g/L;
the variety of the stylosanthes guianensis is RY2.
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