A genetic transformation method of Longxiangtaro

Abstract

The invention discloses a genetic transformation method of taro, and belongs to the field of biotechnology. The genetic transformation method comprises the following steps: using the apical bud or axillary bud of taro as an explant to induce callus tissue to obtain a seedling; cutting off the stem segment or leaf of the seedling as an explant, and pre-culturing on a pre-culture medium; transferring to an Agrobacterium infection solution containing AS components for incubation to obtain an infected explant; transferring to a co-culture medium for co-culture; washing the explant obtained by the co-culture, transferring to a resistance dedifferentiation medium for subculture, and obtaining a resistant callus; placing the resistant callus on a recovery medium for subculture, and transferring to a differentiation medium containing activated carbon for culture to obtain a clustered bud; the clustered bud is rooted, hardened, transplanted and PCR identified to obtain a positive transgenic taro seedling. The genetic transformation method of the invention lays a foundation for establishing a stable and efficient taro genetic transformation system.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a genetic transformation method of taro. Background Art

[0002] Taro (Colocasia esculenta (L.) Schott), commonly known as taro, is a monocotyledonous plant of the Araceae family. Its utilization history can be traced back to the Solomon Island 28,000 years ago. The main edible part of taro is the underground corm, which is the staple food and vegetable of more than 500 million people in Asia-Pacific, Africa and the Americas. my country has a taro cultivation history of more than 2,000 years and is the world's second largest taro producer and largest exporter. Taizhou, as an important taro production area in China, has a cultivation history of more than 800 years and is famous at home and abroad for its "Four Golden Flowers". One of them, Longxiang Taro, is produced in Taizhou's characteristic Duotian area. It is a rare variety of Que taro in Jiangsu Province, with an annual planting area of ​​about 20,000 mu.

[0003] With the development of the economy, the food consumption structure of the Chinese people has undergone new changes, gradually shifting from eating enough to pursuing a health-oriented consumption model that is more nutritious, healthier and safer. In line with this, my country's agriculture has also entered the 3.0 era, which is mainly centered on nutrition. This requires more attention to the improvement of nutritional quality and the enhancement of the nutritional and health benefits of taro. Variety improvement is the best means to improve the nutritional quality of taro. However, taro rarely blooms under natural conditions, which limits the sexual hybridization of taro. Relying solely on a single breeding method, it is impossible to effectively improve the nutritional quality of taro. With the rapid development of crop genomics research, the use of molecular breeding technology to improve taro is obviously a more effective alternative method to improve the nutritional quality of taro.

[0004] Due to the large genome and high heterozygosity, the research on taro at the molecular level is relatively backward. In the past two decades, the genetic research of taro has mainly focused on the development of molecular markers for diversity analysis and genetic positioning. Compared with other crops, the research on taro transgenic technology has been slow, lacking an efficient and stable genetic transformation system. So far, there has been no successful case of using transgenic technology to improve taro varieties in China, and there are not many successful cases in the world. The existing method of preparing transgenic taro by Agrobacterium-mediated method still has the shortcomings of narrow variety application range, long transformation cycle, and low transformation rate (1-3%). In addition, the existing method mainly uses embryonic callus as the receptor of genetic transformation. Callus tissue is usually composed of multiple cells and is easy to produce chimeras. In addition, the clones of regenerated plants obtained by callus tissue have large variations and poor stability of foreign gene integration.

[0005] Plant genetic modification technology is an emerging biotechnology in the 21st century. By transferring foreign genes into plants, the genetic characteristics of plants can be improved or enhanced. Common transformation methods include protoplast fusion, pollen tube channel method, gene gun method, Agrobacterium-mediated method, etc. Among them, the Agrobacterium-mediated method has strong feasibility, simple operation, low cost, good repeatability and other characteristics. Transgenic technology provides a feasible basis for improving the nutritional quality of taro and cultivating resistant varieties. Therefore, it is of great significance to establish a set of efficient genetic transformation system for taro. Summary of the invention

[0006] The purpose of the present invention is to provide a genetic transformation method of taro to solve the problems existing in the above-mentioned prior art. The genetic transformation method of the present invention has a transformation rate of up to 10.1%, which lays a foundation for establishing a stable and efficient taro genetic transformation system.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] Technical solution 1: A genetic transformation method for taro, comprising the following steps: using the apical bud or axillary bud of taro as an explant to induce callus tissue to obtain seedlings; cutting off the stem segment or leaf of the seedling as an explant, and pre-culturing on a pre-culture medium; transferring to an Agrobacterium infection solution containing AS components for incubation to obtain infected explants; using sterile filter paper to absorb the bacterial solution remaining on the surface of the explant, and transferring to a co-culture medium for co-culture for 3-4 days; washing the explant obtained by the co-culture with sterile water containing timentin, removing the moisture on the surface of the explant, and transferring to a resistance dedifferentiation medium containing glutamine for subculture to obtain resistant callus; placing the resistant callus on a recovery medium with a decreasing timentin concentration for subculture, and transferring to a differentiation medium containing activated carbon for culture to obtain clustered buds; after the clustered buds are rooted, hardened, transplanted and PCR identified, positive transgenic taro seedlings are obtained, and finally transplanted.

[0009] Furthermore, the callus induction is carried out on a callus induction medium, and the components of the callus induction medium are: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 6-BA+1mg﹒L -1 NAA.

[0010] Furthermore, the composition of the pre-culture medium is: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L-1 6-BA+0.5mg﹒L -1 NAA.

[0011] Furthermore, the preparation method of the Agrobacterium infection solution is: inoculating a single colony of Agrobacterium into a YEB medium and culturing until the OD 600 The value reaches 0.6-0.8; the Agrobacterium activated overnight is inoculated into the YEB medium at a ratio of 1:100-1:50 and cultured until the logarithmic growth phase; after centrifugation and discarding the supernatant, it is added to the infection solution to resuspend the bacteria, and the OD of the resuspended solution is adjusted. 600 The value is 0.1-0.3.

[0012] Furthermore, the composition of the infection solution is: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose + 2mg﹒L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+100μmol﹒L -1 AS; the composition of the co-culture medium is: 4.4g﹒L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+20μmol﹒ L -1 A.S.

[0013] Furthermore, the composition of the resistance dedifferentiation medium is: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+200mg﹒L -1 Glutamine +7.5mg﹒L -1 Hygromycin + 300mg﹒L -1 Timentin.

[0014] Furthermore, during the subculture on the resistance dedifferentiation medium, if Agrobacterium overgrows, 500 mg﹒ L -1 Rinse the explants with sterile water containing Timentin for 3-5 minutes.

[0015] Furthermore, the method of subculturing the resistant callus on a recovery medium with decreasing concentration of timentin is as follows: subculturing once every 14-16 days, for a total of 3 times, during which the concentration of timentin is reduced by 50 mg﹒ L each time the subculture is performed. -1 .

[0016] Furthermore, the components of the recovery medium for the first subculture are: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 1 mg L -1 2,4-D+2mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+250mg﹒L -1 Timentin + 0.5g﹒ L -1 Activated carbon.

[0017] Furthermore, the composition of the differentiation medium is 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2.5 mg L -1 TDZ+150mg﹒L -1 Timentin + 0.5g﹒ L -1 Activated carbon.

[0018] The present invention discloses the following technical effects:

[0019] The explants of the present invention select terminal buds and axillary buds. Compared with other explants, the terminal buds and axillary buds can induce callus tissues and adventitious buds and adventitious roots, thereby shortening the time for obtaining sterile seedlings. Compared with the taro genetic transformation method reported in the prior art, the time for obtaining callus and adventitious buds is greatly shortened. The method of the prior art is to cut the bud tip and induce buds in a liquid culture medium first, then cut the buds for secondary induction, induce callus tissue, and then differentiate into adventitious buds through callus tissue. The whole process takes 8-10 months, while the present invention takes 2-3 months.

[0020] Taro is a monocotyledonous plant with poor sensitivity to Agrobacterium. Adding an appropriate amount of acetosyringone (AS) to the Agrobacterium infection solution can improve the transformation efficiency. However, it is difficult to inhibit the excessive growth of Agrobacterium in the later stage when a high concentration of bacterial solution is used to infect callus. Therefore, the present invention adopts the addition of 100 μmol﹒ L -1 Resuspend the bacteria in AS infection solution, adjust the OD value of the resuspended solution to 0.1-0.3, and use the resuspended solution to infect the callus.

[0021] The step of using sterile filter paper to absorb the bacterial solution remaining on the surface of the explant is very critical for avoiding excessive growth of Agrobacterium and obtaining good transformation efficiency.

[0022] The present invention takes the generation of resistant callus as a node, stops adding the screening agent in the later callus proliferation and differentiation process, increases the conversion rate, and shortens the conversion time. The present invention adds glutamine to the resistant callus dedifferentiation medium, effectively accelerating the generation of resistant callus. The resistant callus is more seriously browned during the proliferation and differentiation process, and grows slowly. The present invention gradually reduces the addition of antibiotics during the proliferation and differentiation of callus tissue, and adds 0.05% of activated carbon, which has a good effect on preventing callus tissue from browning, and is conducive to the induction and proliferation of adventitious buds.

[0023] The invention has the advantages of simple operation, strong repeatability, good adaptability, low cost, etc., and has obvious advantages over the gene gun method and the polyethylene glycol method, and is of great significance to the theoretical research of taro genetic engineering and the optimization of germplasm resources. The genetic transformation method of the invention has been successfully applied to the dragon fragrant taro, and the transformation rate can reach 10.1%, which lays a foundation for establishing a stable and efficient taro genetic transformation system. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Selecting the induction conditions of the terminal buds and axillary buds of the explants of the present invention;

[0026] Figure 2 is the effect of AS concentration on the production of resistant callus; where resistant callus rate (%) = number of resistant callus blocks / total number of infected callus blocks × 100;

[0027] Figure 3 The hygromycin sensitivity of the recipient tissue;

[0028] Figure 4 The results of resistant callus screening using hygromycin as the screening agent; A is the second round of screening (adding 7.5 mg﹒L -1 Hygromycin), B is the first round of screening (add 7.5 mg﹒L -1 Hygromycin);

[0029] Figure 5 The figure is a screening comparison of different Agrobacterium inhibitors, wherein A is a culture medium with cefotaxime added, and B is a culture medium with timentin added;

[0030] Figure 6 The antibacterial effect of Timentin and the sensitivity of wound healing to Timentin;

[0031] Figure 7 The Agrobacterium contamination rate when the explants were washed with sterile water containing different concentrations of Timentin;

[0032] Figure 8 The following are photos of the growth of each link in the genetic transformation process in Example 1;

[0033] Fig. 9 The results of PCR detection of HPT gene in the regenerated plants of Longxiang Taro in Example 1, wherein the first on the left is the Marker, the second on the left is the positive control, the third on the left is the negative control, and the rest are regenerated plants. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] 1. Culture parameter screening

[0041] (1) The explants of the present invention are terminal buds and axillary buds. Compared with other explants, terminal buds and axillary buds can induce callus tissue and adventitious buds and adventitious roots (see Figure 1 ), shortening the time to obtain sterile seedlings. Compared with the taro genetic transformation method reported in the prior art, the time to obtain callus and adventitious buds is greatly shortened. The method of the prior art is to cut the bud tip and induce buds in a liquid culture medium first, then cut the bud for secondary induction, induce callus tissue, and then differentiate the callus tissue into adventitious buds. The whole process takes 8-10 months, while the present invention takes 2-3 months.

[0042] (2) Monocotyledonous plants cannot produce enough phenolic substances during the transformation process, and it is difficult to activate the infection ability of the vir gene. The addition of acetosyringone (AS) to the Agrobacterium infection solution can effectively increase the production rate of resistant callus, thereby improving the transformation efficiency. When the addition amount is 100 μmol﹒ L -1 When the resistant callus rate can be increased to 26.8%, Figure 2 .

[0043] (3) Hygromycin was used as a taro selection agent (see Figure 4 ), previous studies have found that kanamycin has a low selective stress on taro, and its effect on the screening of taro resistant callus is not obvious, and many of the callus after screening cannot differentiate again, so kanamycin is not suitable for use as a screening agent for taro. Compared with it, hygromycin has a more efficient screening effect. The present invention selects hygromycin as a screening agent for taro, and the hygromycin sensitivity of the receptor tissue is shown in Figure 3 The results showed that the concentration of hygromycin reached 7.5 mg﹒ L -1 The browning rate of callus tissue increased greatly, exceeding 90%, reaching the screening concentration. In comparison, the leaves had a stronger tolerance, reaching a concentration of 10 mg﹒ L -1 When the browning rate exceeds 90%. Since high mass concentration of antibiotics can kill cells quickly, and dead cells have a strong inhibitory effect on the growth of surrounding cells, which is not conducive to the growth of transformed cells, the screening mass concentration should generally be slightly lower than the total lethal concentration. Based on this, the present invention determines that the screening concentration of hygromycin for taro resistant callus is 7.5 mg﹒ L -1 , taking the production of resistant callus as the node, stopping the addition of screening agent in the later callus proliferation and differentiation process, reducing the effect of hygromycin on callus growth and differentiation, improving transformation efficiency and shortening transformation time.

[0044] (4) The present invention uses timentin as an Agrobacterium inhibitor to better inhibit the growth of Agrobacterium. The present invention adds 250 mg﹒ L -1 Ceftriaxone was used as an inhibitor of Agrobacterium. As a result, the color of the culture medium began to turn red around 10 days after culture, the growth of callus tissue was inhibited, and the browning rate of callus tissue was accelerated. However, the effect of Timentin on callus tissue was not obvious. Therefore, Timentin was selected as an inhibitor of Agrobacterium in the present invention (see Figure 5 ), the antibacterial effect of Timentin and the sensitivity of wound healing to Timentin are shown in Figure 6 Timentin can effectively inhibit the proliferation of Agrobacterium without inhibiting the normal growth and differentiation of callus tissue. Its concentration is 150 mg﹒ L -1 Therefore, the concentration of Timentin was determined to be 300 mg﹒ L in the dedifferentiation stage of resistant callus. -1 In the later stage, the concentration of Timentin can be gradually reduced to 150 mg﹒ L -1 .

[0045] (5) The step of using sterile filter paper to absorb the residual bacterial solution on the surface of the explant is very critical for avoiding excessive growth of Agrobacterium and obtaining good transformation efficiency. By using sterile filter paper to absorb the residual bacterial solution on the surface of the explant, the inhibition rate of Agrobacterium can reach 82.1%.

[0046] (6) The present invention adds glutamine to the resistant callus dedifferentiation medium, which can shorten the time for callus production from the original 40-50 days to about 30 days, effectively accelerating the production of resistant callus.

[0047] (7) The present invention gradually reduces the addition of antibiotics and increases activated carbon during the proliferation and differentiation of callus tissue, which has a good effect on preventing browning of callus tissue and is beneficial to the induction and proliferation of adventitious buds.

[0048] (8) If Agrobacterium overgrows during subculture on the resistance dedifferentiation medium, 500 mg﹒ L -1 The explants were rinsed with sterile water containing Timentin for 3-5 minutes. The Agrobacterium contamination rate was shown in Figure 7 .

[0049] 2. Experimental Materials

[0050] Test materials: Taro was Xinghua Longxiang taro, which was commercially available.

[0051] 2. Competent strains and vector plasmids

[0052] Agrobacterium EHA105 competent cells (Shanghai Weidi Biotechnology Co., Ltd.); pHB expression vector of CeMYB: pHB-MYB plasmid.

[0053] 3. Culture medium formula

[0054] Callus induction (M1) medium formula: 4.4 g L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 6-BA+1mg﹒L -1 NAA, pH = 5.8;

[0055] Pre-culture (MP) medium formula: 4.4 g L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA, pH = 5.8;

[0056] Co-culture (MC) medium formula: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+20μmol﹒ L -1 AS, pH = 5.8;

[0057] Screening (CSM) medium formula: 4.4 g L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+200mg﹒L -1 Glutamine +7.5mg﹒L -1 Hygromycin + 300mg﹒L -1 Timentin, pH = 5.8;

[0058] Recovery (CHM) medium formula: 4.4 g L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 1 mg L -1 2,4-D+2mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+150-300mg﹒L -1Timentin + 0.5g﹒ L -1 Activated carbon, pH = 6.0;

[0059] Differentiation (M2) medium: 4.4 g L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2.5 mg L -1 TDZ+150mg﹒L -1 Timentin + 0.5g﹒ L -1 Activated carbon, pH = 6.0;

[0060] Rooting (M3) medium formula: 2.2 g L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 0.1 mg L -1 6-BA+0.5mg﹒L -1 NAA+150mg﹒L -1 Timentin + 0.2g﹒ L -1 Activated carbon, PH=6.0.

[0061] 4. Infection solution formula

[0062] 4.4g﹒L -1 MS medium + 30g﹒L -1 Sucrose + 2mg﹒L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+100μmol﹒L -1 AS, PH=5.8.

[0063] 5. Nutritional soil formula (volume ratio)

[0064] Vermiculite: perlite: peat soil = 1:1:1.

[0065] 3. Experimental Methods

[0066] 1. Preparation of sterile seedlings

[0067] (1) Select a taro of suitable size, free of disease spots and rot, and with buds. Peel off the outermost 2-3 layers of skin of the top bud and cut off a 1 cm 3 Place the bud tips of different sizes in a bottle and wash them 3-4 times with running water;

[0068] (2) Under sterile conditions, sterilize the bud tip in 75% alcohol for 30 seconds, rinse it with sterile water, sterilize it with 5% sodium hypochlorite for 10 minutes, and rinse it with sterile water 3-4 times;

[0069] (3) Use sterile filter paper to absorb the moisture on the surface of the bud tip, inoculate it onto callus induction medium (M1), and culture it at 25°C, 16 h / d, and 3000 lx light conditions for 60-90 days to obtain sterile taro seedlings.

[0070] 2. Pre-cultivation

[0071] (1) Under sterile conditions, fully expanded leaves of the taro seedlings were cut into 5 mm × 5 mm pieces, and then the explants were inoculated on the pre-culture medium (MP) for culture;

[0072] (2) The culture conditions are as follows: light intensity of 3000 lx, temperature of 25°C, and alternating light for 16 h and dark for 8 h for 3-4 days.

[0073] 3. Transformation of Agrobacterium competent cells

[0074] (1) Take the competent cells out of -80°C, partially thaw them in the palm of your hand or at room temperature for a while, and insert them into ice when they are in an ice-water mixture state;

[0075] (2) Take 100 μL of competent medium and add 1 μg of pHB-MYB plasmid DNA. Gently stir the bottom of the tube to mix well. Place on ice for 10 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0076] (3) Add 900 μL of YEB liquid medium without antibiotics and culture at 28°C with shaking for 2-3 hours;

[0077] (4) Collect the bacteria by centrifugation at 6000 rpm for 1 min, add 100 μL YEB liquid culture medium, gently blow to resuspend the bacteria, and spread on the plate containing 50 mg﹒ L -1 Kanamycin and 20 mg﹒ L -1 On the YEB plate containing rifampicin, place it upside down in a 28°C incubator and incubate it in the dark for 2-3 days;

[0078] (5) Pick a single colony, extract the plasmid, perform restriction digestion, and perform electrophoresis examination;

[0079] (6) Take the culture medium of the transformed strain and place it in a 1.5 ml centrifuge tube, add 15% sterile glycerol, and store it at -80°C for long-term storage.

[0080] 4. Preparation of Agrobacterium infection solution

[0081] (1) Use an inoculating loop to pick up a small amount of glycerol bacteria and place it in a 50 mg L -1 Streak on YEB solid medium containing kanamycin and culture at 28°C for 2 days;

[0082] (2) Pick a single colony of freshly grown Agrobacterium and inoculate it in 3-5 mL of 50 mg﹒ L -1 Incubate in YEB medium containing kanamycin at 28°C with shaking at 180 rpm overnight until OD 600 The value reaches 0.6-0.8;

[0083] (3) Inoculate the activated Agrobacterium overnight into 50 ml YEB liquid medium at a ratio of 1:100 and culture at 28°C with shaking at 180 rpm until OD 600 value = 0.6-0.8;

[0084] (4) Centrifuge the Agrobacterium culture at 5000 rpm for 10 min, discard the supernatant, add 5 mL of infection solution to resuspend the cells, and measure the OD of the resuspended solution. 600 Value, use infection solution to adjust the OD value to 0.1-0.3.

[0085] 5. Co-cultivation

[0086] (1) Transfer the pre-cultured explants to the Agrobacterium infection solution and incubate at 25° C. with shaking for 30 min to obtain infected explants;

[0087] (2) Transfer the explants onto sterile filter paper and use the sterile filter paper to absorb the bacterial solution remaining on the surface of the explants. Transfer the explants into co-culture medium (MC) and culture them at 25°C in the dark for 3-4 days.

[0088] 6. Screening of resistant callus

[0089] (1) Under sterile conditions, use a solution containing 500 mg L -1 The explants obtained by co-cultivation were washed with sterile water containing Timentin for 5-10 minutes, and the water on the surface of the explants was absorbed dry with sterile filter paper.

[0090] (2) The explants were transferred to a resistance dedifferentiation medium (CSM) and cultured once every 14-16 days under the conditions of 25°C, 16 h / d, and 3000 lx light intensity for 45-60 days to obtain resistant callus tissue.

[0091] 7. Differentiation culture

[0092] (1) The resistant callus was cultured on recovery medium (CHM) with decreasing concentrations of timentin at 25°C, 16 h / d, and 3000 lx light. The culture was subcultured once every 14-16 days for a total of 3 times. The concentrations of timentin were 250 mg﹒ L -1 , 200mg﹒L -1 、150mg﹒L -1 ;

[0093] (2) The explants with callus tissue and adventitious buds were transferred to differentiation medium (M2) and cultured at 25°C, 16 h / d, and 3000 lx light conditions for 40-60 days to obtain secondary buds.

[0094] 8. Rooting culture

[0095] When the sprouts grew to between 3 and 5 cm, they were separated from the base and transferred to a rooting medium (M3) for rooting culture at 25°C, 16 h / d, and 3000 lx light. The resistant regenerated seedlings of Longxiang taro were obtained after culturing for 30 to 40 days.

[0096] 9. Acclimation and transplanting

[0097] When the roots grow to about 3-4 cm, remove the bottle cap, harden the seedlings for 3-5 days, and transplant the seedlings to the nutrient soil on the seedbed and culture them for 30-40 days.

[0098] 10. PCR detection of regenerated plants

[0099] The DNA of leaves of regenerated plants was extracted and PCR detection was performed using untransformed taro plants as negative control and pHB-MYB plasmid as positive control.

[0100] HPT gene primers:

[0101] F: 5'-GGTCGCCGGAGGCTATGGATGC-3' (SEQ ID NO. 1);

[0102] R: 5'-GCTTCTGCGGGCGATTTGTGT-3' (SEQ ID NO. 2).

[0103] The reaction system for PCR detection: 2μl 10×PCR Buffer, 2μl 2mM dNTPs, 2μl DNA, 1μl DMSO, 0.8μl 10pmol﹒ μl -1 Upstream and downstream primers and 0.5U kod DNA polymerase (KOD-401, TOYOBO), add water to make up to 20μl.

[0104] PCR amplification program: pre-denaturation at 94°C for 4 min; denaturation at 98°C for 10 s, annealing at 50-64°C for 30 s, extension at 68°C for 30 s, 40 cycles; insulation at 68°C for 8 min.

[0105] PCR electrophoresis detection: PCR products were detected by 1.2% agarose gel electrophoresis.

[0106] IV. Experimental Results

[0107] A total of 15 regenerated plants of Longxiang Taro were obtained, and PCR detection showed that (see Fig. 9 ), 13 regenerated plants and the positive control all had the target band and the same size, and the negative control did not detect the target band. The experiment proved that the genetic transformation method of this example can obtain 13 transgenic positive plants of Longxiang taro. The growth photos of each link in the genetic transformation process of this example are shown in Figure 8 The genetic transformation results are shown in Table 1.

[0108] Table 1 Genetic transformation results of Example 1 Longxiangtaro

[0109]

[0110] Note: Transformation rate = number of positive plants / number of explants × 100%; Positive rate = number of positive plants / number of regenerated plants × 100%.

[0111] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A genetic transformation method for taro, It is characterized in that The following steps are involved: The apical buds or axillary buds of taro are used as explants to induce callus tissue to obtain seedlings; stem segments or leaves of the seedlings are cut off as explants and pre-cultured on a pre-culture medium; the explants are transferred to an Agrobacterium infection solution containing AS components for incubation to obtain infected explants; the bacterial solution remaining on the surface of the explants is dried with sterile filter paper, and the explants are transferred to a co-culture medium for co-culture for 3-4 days; the explants obtained by the co-culture are washed with sterile water containing timentin, the surface moisture of the explants is removed, and the explants are transferred to a resistance dedifferentiation medium containing glutamine for subculture to obtain resistant callus tissue; the resistant callus tissue is placed on a recovery medium with a decreasing timentin concentration for subculture, and the callus tissue is transferred to a differentiation medium containing activated carbon for culture to obtain clustered buds; the clustered buds are rooted, hardened, transplanted and PCR identified to obtain positive transgenic taro seedlings, and finally transplanted; The callus induction is carried out on a callus induction medium, and the components of the callus induction medium are: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 6-BA+1mg﹒L -1 NAA; The components of the pre-culture medium are: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA; The preparation method of the Agrobacterium infection solution is as follows: a single colony of Agrobacterium is inoculated into a YEB medium and cultured until the OD 600 The value reaches 0.6-0.8; the Agrobacterium activated overnight is inoculated into the YEB medium at a ratio of 1:100-1:50 and cultured until the logarithmic growth phase; after centrifugation and discarding the supernatant, it is added to the infection solution to resuspend the bacteria, and the OD of the resuspended solution is adjusted. 600 Value to 0.1-0.3; The composition of the infection solution is: 4.4g﹒L -1 MS medium + 30g﹒L -1 Sucrose + 2mg﹒L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+100μmol﹒L -1 AS; the composition of the co-culture medium is: 4.4g﹒L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+20μmol﹒ L -1 AS; The components of the resistance dedifferentiation medium are: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L - 1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+200mg﹒L -1 Glutamine +7.5mg﹒L -1 Hygromycin + 300mg﹒ L -1 Timentin; The method of placing the resistant callus on the recovery medium with decreasing concentration of timentin for subculture is: subculture once every 14-16 days, and subculture 3 times in total, during which the concentration of timentin is reduced by 50 mg﹒ L each time the subculture is performed. -1 ; The components of the recovery medium for the first subculture are: 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 1 mg L -1 2,4-D+2mg﹒L -1 6-BA+0.5mg﹒L -1 NAA+250mg﹒L -1 Timentin + 0.5g﹒ L -1 Activated carbon; The composition of the differentiation medium is 4.4 g﹒ L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2.5 mg L -1 TDZ+150mg﹒L -1 Timentin + 0.5g﹒ L -1 Activated carbon.

2. The genetic transformation method according to claim 1, It is characterized in that During the culture on the resistance dedifferentiation medium, if Agrobacterium overgrows, it can be replaced with a medium containing 500 mg﹒ L -1 Rinse the explants with sterile water containing Timentin for 3-5 min.

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