Lycium barbarum efficient genetic transformation system and application

Abstract

The present application relates to the field of plant genetic transformation, and particularly relates to a high-efficiency genetic transformation system of Lycium barbarum and application. The root system tissue of aseptic seedling of Lycium barbarum is used as an explant, a visual plant gene expression vector is mediated by Agrobacterium GV3101 strain to invade, and melatonin is added in culture media such as co-culture, callus induction, adventitious bud and adventitious root differentiation and selection, which plays a key role in aspects such as plant cell differentiation regeneration, ion osmotic balance and plant hormone regulation, and significantly improves the genetic transformation efficiency of Lycium barbarum. The visual plant gene expression vector used in the present application comprises three genes CYP76AD1, DODA and GT which are codon-optimized for Solanaceae plants and a self-cleavable connecting sequence between the genes, has the advantages of naked-eye visualization and non-injury, can be used as a marker gene to track gene expression or visualize the gene transformation process, optimize the gene transformation system and improve the genetic transformation efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant tissue culture and genetic (gene) transformation, and in particular to a high-efficiency genetic transformation system of Lycium barbarum and application. The present application claims priority from CN2024108389191. BACKGROUND

[0002] Lycium barbarum L. is a plant of Solanaceae Lycium, which has become a traditional Chinese medicinal material in China. It has the effects of tonifying kidney and liver, moistening lung and eyesight, and is highly valued by Chinese and foreign medical experts and food therapy health experts. The directional cultivation of fresh food, medicinal, leaf, processing and other special new varieties using genetic transformation, gene editing and other technologies, and the acceleration of high-value production of Lycium barbarum are the key to realize the high-quality development of Lycium barbarum industry. Therefore, a stable and efficient tissue culture and genetic (gene) transformation system of Lycium barbarum is established.

[0003] At present, the reported genetic transformation system of Lycium barbarum has generally low efficiency, and the marker genes used in plant genetic transformation at the present stage are mostly limited to β-glucuronidase (β-glucuronidase, GUS) and green fluorescent protein (green fluorescent protein, GFP) genes. GUS chemical tissue staining needs to add expensive substrate X-Gluc, and it is invasive, so the material cannot be cultured after observation; GFP needs a specific light source to visualize the fluorescent signal, which is not practical under natural conditions. Therefore, selecting suitable marker genes and improving the efficiency of genetic transformation system are a major problem to be solved in Lycium barbarum genetic transformation. SUMMARY

[0004] In order to realize the high-efficiency and stable genetic transformation of Lycium barbarum, the present application establishes a technical system for obtaining Lycium barbarum genetic transformation and regeneration positive plant by co-culturing and selecting culture, which uses root tissue of Lycium barbarum as genetic transformation material (explant), and uses Agrobacterium GV3101 containing visual plant gene expression vector pNULPGE706 as infection strain.

[0005] The present application also uses a multi-gene joint method to construct a visual plant gene expression vector pNULPGE706 containing three gene fragments optimized by Solanaceae plant codon. Figure 1), namely CYP76AD1 (P450 oxygenase, cytochrome P450 monooxygenase), DODA (L-3,4-dihydroxyphenylalanine 4,5-dioxygenase, L-3,4-dihydroxyphenylalanine 4,5-dioxygenase) and GT (glucosyltransferase, glucosyltransferase) and a connecting sequence which can be self-cleaved between genes. The expression efficiency and stability of the genetic transformation system provided by the application in Lycium barbarum are effectively improved, and the three gene sequences are also codon-optimized for solanaceous plants. After the three genes are introduced into plant cells, they can catalyze tyrosine to generate betalains, which present very bright red color, have the advantages of being visible to the naked eye without chemical staining or special light source and being non-invasive, and can be used as a marker gene to track gene expression or visualize the gene transformation process, and the Lycium barbarum gene transformation system is optimized to improve the genetic transformation efficiency (such as Figures 2-5

[0006] To achieve the technical purpose of the application, in one aspect, the application provides a high-efficiency genetic transformation system of Lycium barbarum, which uses root tissue as the material for gene transformation, uses Agrobacterium GV3101 containing the visual plant gene expression vector pNULPGE706 as the infection strain, and uses co-culture medium added with melatonin, selection medium for callus induction and adventitious bud and root differentiation, and then obtains Lycium barbarum genetic transformation regenerated positive plants.

[0007] In particular, the tissue culture regeneration system using root tissue as the explant has the advantages of high callus induction and adventitious bud differentiation efficiency, short cycle, large propagation coefficient and the like.

[0008] In particular, melatonin is added in the medium for Agrobacterium infection and co-culture, callus induction, adventitious bud and root differentiation, and selection. Melatonin plays a key role in the regulation of plant cell differentiation and regeneration, ion permeation balance, and plant hormone regulation, and improves the genetic transformation efficiency of Lycium barbarum.

[0009] To achieve the technical purpose of the application, in a second aspect, the application provides a method for constructing a Lycium barbarum genetic transformation system, which comprises: placing root tissue of Lycium barbarum into infection liquid prepared from GV3101 Agrobacterium containing the pNULPGE706 visual plant gene expression vector to perform infection, to obtain infected root tissue; after co-culture of the infected root tissue, transferring to a selection medium for selection, and finally subculturing the selected positive adventitious buds to obtain Lycium barbarum genetic transformation positive plants.

[0010] ​To achieve the technical purpose of the present application, the third aspect of the present application provides a visual plant gene expression vector pNULPGE706 Figure 1 ), comprising three gene fragments of CYP76AD1 (P450 oxygenase, cytochrome P450 monooxygenase), DODA (L-3, 4-dihydroxyphenylalanine 4, 5-dioxygenase, L-3, 4-dihydroxyphenylalanine 4, 5-dioxygenase) and GT (glucosyltransferase, glucosyltransferase) optimized by Solanaceae plant codon and a connecting sequence between the genes which can be self-cleaved. The expression efficiency and stability of the genetic transformation system provided by the present application in Lycium barbarum are effectively improved. After the three genes are introduced into plant cells, they can catalyze tyrosine to generate betalains, which present very bright red color, have the advantages of being visible to the naked eye without chemical staining or special light source conditions and being non-invasive, and can be used as a marker gene to track gene expression or visualize the gene transformation process. The Lycium barbarum gene transformation system is optimized to improve the genetic transformation efficiency (as shown in Figures 2-5 ).

[0011] In particular, the GC proportion of the CYP76AD1 sequence optimized by Solanaceae plant codon is 36.35%, and the codon adaptation index (CAI) is 0.9.

[0012] Specifically, the CYP76AD1 sequence optimized by Solanaceae plant codon is as shown in SEQ ID NO. 1 (as shown in Figure 6 ).

[0013] In particular, the GC proportion of the DODA sequence optimized by Solanaceae plant codon is 39.86%, and the codon adaptation index (CAI) is 0.91.

[0014] Specifically, the DODA sequence optimized by Solanaceae plant codon is as shown in SEQ ID NO. 2 (as shown in Figure 7 ).

[0015] In particular, the GC proportion of the GT sequence optimized by Solanaceae plant codon is 38.06%, and the codon adaptation index (CAI) is 0.91.

[0016] Specifically, the GT sequence optimized by Solanaceae plant codon is as shown in SEQ ID NO. 3 (as shown in Figure 8 ).

[0017] It should be noted that the codon optimization referred to in the present application can be realized by conventional bioinformatics software, and the present application does not make special limitations.

[0018] In particular, the CYP76AD1, DODA and GT of the visualized plant gene expression vector are introduced into plant cells, and tyrosine in the plant can be converted into betalain of bright red.

[0019] To achieve the technical purpose of the present application, the fourth aspect of the present application provides a PCR identification primer for screening the visualized plant gene expression vector and genetically transformed regenerated plant, and the primer sequence is shown in SEQ ID NO. 4 and SEQ ID NO. 5:

[0020] SEQ ID NO. 4 (Sense): 5'-CCCAAGAGGAAATCAAGCAGGTC-3';

[0021] SEQ ID NO. 5 (Anti-sense): 5'-GTGTAGTTGCGGAGGATGGAGAAG-3'.

[0022] To achieve the technical purpose of the present application, the fifth aspect of the present application provides an application of the above-mentioned expression vector, which is the establishment and optimization of plant gene transformation system.

[0023] To achieve the technical purpose of the present application, the sixth aspect of the present application provides an application of plant gene transformation system, which comprises the steps of using the above-mentioned vector, the above-mentioned application, and the above-mentioned primer.

[0024] In particular, the plant in the method is Lycium barbarum.

[0025] Preferably, the plant in the method is Lycium barbarum L.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] (1) The present application establishes a complete and efficient tissue culture regeneration system taking the root system tissue of Lycium barbarum sterile seedlings as the explant, which has the advantages of high efficiency of callus induction and adventitious bud differentiation, short cycle, large propagation coefficient, etc. compared with the current tissue culture taking cotyledon, hypocotyl, leaf, petiole, stem segment as the explant, and can be applied to large-scale factory seedling raising, and solves the important problem of asexual propagation of Lycium barbarum excellent germplasm resources.

[0028] (2) The application uses a multi-gene combination method to construct a visual plant gene expression vector pNULPGE706 containing CYP76AD1, DODA and GT three gene fragments. The three gene sequences of the vector are codon optimized according to Solanaceae plants, further improving the expression efficiency and stability. After being introduced into plant cells, it can catalyze tyrosine to form betalains, showing very bright red, with the advantages of being visible to the naked eye without chemical staining or special light source and non-injury, which can be used as a marker gene to track gene expression or visualize the gene transformation process, and optimize the genetic transformation system, significantly improving the transformation efficiency. The currently used marker genes for plant genetic transformation mainly include GUS (β-glucuronidase) and GFP (green-fluorescent protein) genes. The former can be stained by histochemical method, and can be monitored during the transient expression stage after Agrobacterium infection or the entire differentiation process, but the used material cannot be cultured further; the latter can be observed by fluorescence microscope for living cells or tissues of transgenic plants, and has no damage to the used material, but requires a special visual fluorescence light source, and is often affected by the false fluorescence signal of plant cells or tissues.

[0029] (3) The application establishes a complete and efficient genetic transformation system of Lycium barbarum mediated by Agrobacterium, and adds melatonin in the medium of Agrobacterium infection and co-culture, callus induction, differentiation of adventitious buds and roots, and selection. Melatonin plays a key role in the regulation of plant cell differentiation and regeneration, ion permeation balance, and plant hormone regulation, improves the genetic transformation efficiency of Lycium barbarum, ensures the uniformity and robustness of the proliferated seedlings, effectively promotes the rooting of tissue culture seedlings, and the rooting rate reaches more than 93.33%, and the sprouting rate reaches 87.50%. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The T-DNA part of the visual plant gene expression vector pNULPGE706 constructed in the embodiments of the application;

[0031] Figure 2 The callus induction effect of the application example using the root system tissue of Lycium barbarum sterile seedlings as the explant and Agrobacterium GV3101 as the infection strain;

[0032] Figure 3 The differentiation effect of adventitious buds in the embodiments of the application;

[0033] Figure 4 The subculture effect of adventitious buds and the differentiation effect of adventitious roots in the embodiments of the application;

[0034] Figure 5The Lycium chinense genetic transformation positive plant obtained through selection and identification in the embodiments of the present application;

[0035] Figure 6 The CYP76AD1 sequence of the Solanaceae plant codon optimization of the present application is SEQ ID NO. 1;

[0036] Figure 7 The DODA sequence of the Solanaceae plant codon optimization of the present application is SEQ ID NO. 2;

[0037] Figure 8 The GT sequence of the Solanaceae plant codon optimization of the present application is SEQ ID NO. 3. DETAILED DESCRIPTION

[0038] In order to make the purpose and advantages of the present application more clear and obvious, the present application is further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. If not specifically stated, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, if not specifically stated, can be obtained from commercial channels.

[0039] Materials: Ningxia Lycium barbarum 'Zhongxuan No. 1' cultivated for many years in the laboratory was used as plant material; Agrobacterium strains heat-shocked competent cells were GV3101, LBA4404, Agl1 and EHA105; Kanamycin (Kanamycin sulfate, Kan), Carbelicilin sodium (Carbelicilin sodium, Car), Timentin (Timentin, Tim), Cefotaxime (Cefotaxime, Cef) and other antibiotics were purchased from Beijing Coolab Technology Co., Ltd.; MS (Murashige and Skoog) medium, sucrose, agar, melatonin (Melatonin, MT), 6-benzylaminopurine (6-benzylaminopurine, BAP), 1-naphthaleneacetic acid (1-naphthaleneacetic acid, NAA), Zeatin (Zeatin, ZT), Acetosyringone (Acetosyringone, Ace) and other reagents were purchased from Beijing Solabio Technology Co., Ltd.

[0040] Example 1 Construction of Lycium barbarum genetic transformation system

[0041] 1. Explant culture

[0042] The branches of Lycium barbarum 'Zhongxuan No. 1' are pruned into 5-7 cm (with 3-4 bud nodes), put into sterilized triangular bottles, 70% alcohol is added, surface sterilization for 30 seconds, and the alcohol is poured out; 1% sodium hypochlorite solution (NaClO, the original solution Cl content is 5%) is added, sterilization for 20 minutes, shake in the middle, and the disinfectant is poured out; more than 5 times of sterile water is used for washing, and the excess water is absorbed with sterilized filter paper, and the branches are cut into the sterile seedling culture medium. The sterile seedling culture medium is 1 / 2MS medium added with sucrose and agar, specifically: 2.21 g·L -1 MS + 15 g·L -1 sucrose + 7 g·L -1 agar. The culture box is at 25 DEG C, 5000 lux, 16 hours of illumination. Subculture every 4 weeks to obtain Lycium barbarum sterile seedlings containing root system.

[0043] It should be noted that the Lycium barbarum sterile seedlings can also be obtained by conventional techniques in the art, and the present application is not limited.

[0044] 2, Construction of visual plant gene expression vector

[0045] 2.1 Gene codon optimization and multi-gene connection

[0046] The Solanaceae plant codon-optimized CYP76AD1, DODA and Mirabilis jalapa GT genes are obtained, wherein the GC proportion of the CYP76AD1 sequence optimized by the Solanaceae plant codon is 36.35%, the codon adaptation index (CAI) is 0.9, and the optimized sequence is shown as SEQ ID NO. 1, and the specific sequence is shown as SEQ ID NO. 1. Figure 6 The GC proportion of the DODA sequence optimized by the Solanaceae plant codon is 39.86%, the codon adaptation index (CAI) is 0.91, and the optimized sequence is shown as SEQ ID NO. 2, and the specific sequence is shown as SEQ ID NO. 2. Figure 7 The GC proportion of the GT sequence optimized by the Solanaceae plant codon is 38.06%, the codon adaptation index (CAI) is 0.91, and the sequence is shown as SEQ ID NO. 3, and the specific sequence is shown as SEQ ID NO. 3. Figure 8 The last two genes are removed from the stop codon, and P2A (GSGATNFSLLKQAGDVEENPGP) is used for connection in turn, and then constructed into the Agrobacterium binary plasmid to obtain the visual plant gene expression vector, named pNULPGE706 (as shown in Figure 1 ).

[0047] It should be noted that any order of the connection of the optimized CYP76AD1, DODA and GT genes can achieve the technical purpose of the present application, and the present application is not limited.

[0048] It should be noted that codon optimization can be achieved using bioinformatics software commonly used in the art, which is not limited by the present application.

[0049] 2.2 Transformation of Agrobacterium competent cells

[0050] About 200 ng of pNULPGE706 was added to Agrobacterium GV3101, LBA4404, Agl1 and EHA105 heat shock competent cells, respectively, for heat shock transformation; PCR positive detection was performed on the transformed Agrobacterium single colonies, and the primer sequence was:

[0051] SEQ ID NO. 4 (Sense), 5'-CCCAAGAGGAAATCAAGCAGGTC-3';

[0052] SEQ ID NO. 5 (Anti-Sense), 5'-GTGTAGTTGCGGAGGATGGAGAAG-3'.

[0053] Positive GV3101, LBA4404, Agl1 and EHA105 monoclonal strains containing pNULPGE706 vector were screened out and stored at -80°C for standby.

[0054] 3. Genetic transformation

[0055] 3.1 Preparation of Agrobacterium infection solution

[0056] The positive Agrobacterium GV3101 containing the vector pNULPGE706 screened out in step 2.2 was placed on ice to melt, and a disposable inoculation loop was used to streak on solid LB medium and placed in a 37°C incubator for activation for 48 hours to obtain activated bacteria; a small amount of activated bacteria was scraped with an inoculation loop and added to the infection solution (as shown in Table 1), and cultured at 28°C with 180-200 rpm vibration until the logarithmic growth phase, then centrifuged at 4°C at 5500 rpm for 10 minutes to collect the bacterial cells. The concentration of the infection solution was adjusted to OD 550 = 0.25 to obtain the Agrobacterium infection solution.

[0057] Table 1 Agrobacterium infection solution

[0058]

[0059] Melatonin (MT) plays an important role in regulating plant growth and development and biotic and abiotic stress, such as melatonin is involved in leaf senescence, flowering, fruit ripening and root development and other biological processes. The present application first uses it as one of the infection liquid components of genetic transformation of wolfberry, especially uses the root system as an explant to be infected in the infection liquid added with melatonin, promotes the transformation of the target gene and integrates into the genome of the wolfberry root tissue, and the addition of melatonin can not only reduce the browning necrosis and vitrification of the explant in the process of Agrobacterium-mediated transformation, but also can enhance the activity of Agrobacterium and promote the growth of the explant, realize and improve the efficiency of Agrobacterium-mediated genetic transformation of wolfberry.

[0060] It should be noted that the concentration of Agrobacterium is an important factor affecting the efficiency of genetic transformation, and too high concentration will cause the explant to be toxic and necrotic, and too low concentration will affect the transformation efficiency, and the present application determines that the concentration OD550 of Agrobacterium is 0.25, which can realize high-efficiency transformation of the root as an explant, and the effect is remarkable.

[0061] 3.2 Explant infection

[0062] The aseptic seedling root tissue of about 45 days of subculture is selected as the explant, and the length is 0.5-1cm. It is immersed in the Agrobacterium infection liquid (as shown in Table 1) for 3 minutes, the explants are collected and placed on sterile filter paper, and then the liquid is absorbed, and then the explants are moved to the co-culture medium (as shown in Table 2), 15-20 explants are placed in each culture dish, and the culture is carried out at 22℃ in the dark for 3 days.

[0063] Table 2 Co-culture medium

[0064]

[0065] 3.3 Callus induction, adventitious bud differentiation and selection

[0066] After co-culture, the explants are transferred to the callus induction, adventitious bud differentiation and selection medium (as shown in Table 3), and the culture is carried out at 25℃ for 16 hours per day; generally, the formation of red callus can be observed after 7-10 days (as shown in Figure 2 ), and the differentiation of red adventitious buds can be observed after about 20 days (as shown in Figure 3 ); every 4 weeks, the same medium is subcultured, and during the period, the well-grown adventitious buds are moved to the test tube containing the adventitious root differentiation medium (as shown in Table 3 and Table 4) for continuous culture (as shown in Figure 4 ) and adventitious root differentiation (as shown in Figure 5from the above genetic transformation process can be seen, the present application is established in the root system of the sterile seedling of wolfberry as explant tissue culture regeneration system, the efficiency of Agrobacterium-mediated genetic transformation system of wolfberry is higher; the expression of visual gene expression vector is stable, without the help of special light source condition or chemical staining can track gene expression or visual gene transformation process, the red plant obtained by regeneration is identified by PCR, and is determined as a positive plant of successful genetic transformation. The primer sequence is:

[0067] SEQ ID NO. 4 (Sense): 5'-CCCAAGAGGAAATCAAGCAGGTC-3';

[0068] SEQ ID NO. 5 (Anti-Sense): 5'-GTGTAGTTGCGGAGGATGGAGAAG-3'.

[0069] Table 3 Callus induction, adventitious bud differentiation and selection medium

[0070]

[0071] Figure 4 For the genetic transformation of wolfberry plant seedling stage, Figure 5 For the genetic transformation of wolfberry plant seedling stage. According to Figure 4 and Figure 5 It can be observed that in different growth stages of transgenic positive plants, the tissues such as leaves, stems and roots are red, which indicates that there is a large amount of betalain accumulation in all tissues of the whole plant. The CYP76AD1, DODA and GT gene fragments in the pNULPGE706 visual plant gene expression vector constructed by the present application can be used as marker genes to track gene expression or visualize gene transformation process, which is convenient and fast, reduces cost, improves the accuracy of screening culture positive plants, and shortens the screening period.

[0072] Table 4 Adventitious root differentiation medium

[0073]

[0074] The present application adds melatonin (as shown in Tables 1-3) in the culture medium of Agrobacterium infection and co-culture, callus induction, adventitious bud differentiation and selection, which plays a key role in the regulation of plant cell differentiation and regeneration, ion permeation balance and plant hormone regulation, and improves the genetic transformation efficiency of wolfberry.

[0075] In order to screen and obtain wolfberry genetic transformation system and efficient expression, the inventors have carried out a large amount of test processing work, and the following is part of the comparison test.

[0076] Test of different explants of comparative test example 1

[0077] In theory, plant cells have totipotency, and cotyledon, embryo axis, embryo, stem segment and leaf are commonly used as explants for tissue culture and genetic transformation. However, due to different plant species, the difficulty level and transformation efficiency are different. In one comparative test of the present application, leaf, petiole, stem segment and root system are used as explants, Agrobacterium GV3101 is used as the infection strain, and other operations are performed according to the method in the example. Specifically, the same sterile seedlings cultured for about 45 days are selected, the leaves are cut into 5*5mm, and the petiole and stem segment are cut into about 0.5-1cm in length.

[0078] The culture state and genetic transformation state of each explant are observed, and the callus induction rate and adventitious bud differentiation rate of various explants are counted (as shown in Table 5).

[0079] Table 5 Callus induction rate and adventitious bud differentiation rate of various explants

[0080]

[0081] It is found from the test observation that the callus induction rates of root system and leaf explants are both above 90%, but the adventitious bud differentiation rate of leaf is only 35%, which is much lower than the 87.50% of root system. Moreover, the callus obtained by differentiation of root system is denser and in good condition, and it is easier to differentiate into a large number of adventitious buds, and the adventitious buds grow vigorously, so that the regenerated seedlings are easily obtained.

[0082] The present application first uses root system as the tissue culture and genetic transformation material (explant) of wolfberry, and the callus induction rate and adventitious bud differentiation rate are much higher than those of other explants, which achieves an unexpected effect.

[0083] Test of different infection strains of comparative test example 2

[0084] In one comparative test of the present application, root system is used as the explant, Agrobacterium GV3101, Agl1, LBA4404 and EHA105 are used as the infection strains, and other operations are performed according to the method in the example, and the callus induction rate and adventitious bud differentiation rate after infection of various strains are counted (as shown in Table 6).

[0085] Table 6 Callus induction rate and adventitious bud differentiation rate after infection of various Agrobacterium strains

[0086]

[0087] The test observation found that using Agrobacterium EHA105 as the infection strain, the explant grew bacteria immediately after infection, and no callus and adventitious bud appeared. Other strains could appear callus and adventitious bud; using Agrobacterium GV3101 strain had a high callus induction rate and adventitious bud differentiation rate, which reached 95% and 87.5%, respectively.

[0088] Test of different culture media in comparative test example 3

[0089] The present application compares the hormone ZT, BAP and NAA concentration in the culture medium (as shown in Table 7). The root system is used as the explant, Agrobacterium GV3101 is used as the infection strain, and the other methods are carried out according to the method in the example.

[0090] Table 7 Hormone concentration ratio of culture medium

[0091]

[0092] After repeated tests, it was found that the culture medium Lb8 had the best effect, the callus grew larger, the structure was compact and emerald green, and the most adventitious buds were obtained; followed by culture media Lb4, Lb5, Lb17, Lb26 and Lb27, the callus volume was small, the color state was not good, and there were a small amount of adventitious buds; the callus volume of culture media Lb13, Lb15 and Lb18 was the smallest, and almost no adventitious buds were generated.

[0093] Test of different antibiotics in comparative test example 4

[0094] The present application compares the antibiotic kanamycin concentration in the culture medium (as shown in Table 8). The root system is used as the explant, Agrobacterium GV3101 is used as the infection strain, and the other methods are carried out according to the method in the example.

[0095] Table 8 Antibiotic concentration ratio

[0096]

[0097] After repeated experiments, it was found that when the concentration of kanamycin was 6.25 mg·L -1 , the explant after infection would grow excessively, and did not play a selection role; but when the concentration was 25 mg·L -1 , the wolfberry explant would almost all die, indicating that the antibiotic concentration was too high; when the concentration was 12.5 mg·L -1 , the gene transformed wolfberry explant grew moderately, and part of the gene transformed positive plants could be successfully selected, so 12.5 mg·L -1 was set as the best screening concentration of antibiotic kanamycin.

[0098] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims include all such modifications and variations as fall within the scope of the present application.

[0099] It is apparent that those skilled in the art can make various changes and modifications to the application without departing from the spirit and scope of the application. It is therefore intended that the present application cover all such changes and modifications that are within its scope.

Claims

1. A method for efficient genetic transformation of Lycium ruthenicum Murr, comprising the following steps: constructing a visual plant gene expression vector pNULPGE706 containing a CYP76AD1 gene, a DODA gene and a GT gene; wherein the sequence of the CYP76AD1 gene is shown as SEQ ID NO.1, the sequence of the DODA gene is shown as SEQ ID NO.2, and the sequence of the GT gene is shown as SEQ ID NO.3; using the root tissue of a sterile seedling of Lycium ruthenicum Murr as an explant, and after being infected in an Agrobacterium infection solution, transferring the explant to a co-culture medium containing melatonin, and then transferring the explant to a callus induction, adventitious bud differentiation and selection medium containing melatonin after dark culture to obtain an adventitious bud; wherein the concentration of melatonin is 1 mg / L; and transferring the adventitious bud to an adventitious root differentiation medium to obtain a regenerated positive plant of Lycium ruthenicum Murr. The components of the Agrobacterium infection solution are 30 g of sucrose, 4.41 g of MS medium, 1 mg of melatonin and 20 mg of acetosyringone per liter, and the pH value is 5.

8. The visual plant gene expression vector pNULPGE706 containing the CYP76AD1 gene, the DODA gene and the GT gene is transformed into Agrobacterium GV3101, and the transformed Agrobacterium is cultured and then added to a medium containing melatonin to obtain an Agrobacterium infection solution; wherein the concentration OD of the Agrobacterium infection solution is 0.

25. 550 0.

25. The components of the co-culture medium are 30 g of sucrose, 4.41 g of MS medium, 1 mg of melatonin, 1 mg of 6-benzylaminopurine, 0.5 mg of 1-naphthaleneacetic acid, 7 g of agar and 20 mg of acetosyringone per liter, and the pH value is 5.

8. The components of the callus induction, adventitious bud differentiation and selection medium are 30 g of sucrose, 4.41 g of MS medium, 1 mg of melatonin, 1 mg of 6-benzylaminopurine, 0.5 mg of 1-naphthaleneacetic acid, 7 g of agar, 125 mg of carbenicillin, 125 mg of timentin and 12.5 mg of kanamycin per liter, and the pH value is 5.

8.

2. The efficient genetic transformation method for Ningxia wolfberry as described in claim 1, characterized in that, The components of the adventitious root differentiation medium are 15 g of sucrose, 2.21 g of MS medium, 0.5 mg of 1-naphthaleneacetic acid, 7 g of agar, 125 mg of carbenicillin and 125 mg of cephalothin per liter, and the pH value is 5.

8.

3. The efficient genetic transformation method for Ningxia wolfberry as described in claim 1, characterized in that, ​ 4. The method of claim 1, wherein the Lycium chinense is efficiently transformed.

4. The method of claim 1, wherein the Lycium chinense is efficiently transformed. ​ 5. The method of claim 1, wherein the Lycium chinense is efficiently transformed. ​ ​

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