Establishment method of orchid cinnabar genetic transformation system

Abstract

The invention discloses a method for constructing a genetic transformation system of cymbidium kanran, which belongs to the technical field of plant genetic engineering, and comprises the following steps of: carrying out monoclonal propagation on rhizomes after germination of cymbidium kanran seeds to obtain vegetative propagules with stable characters, taking the vegetative propagules as transformation receptors, taking GFP (Green Fluorescent Protein) as marker genes, and constructing the genetic transformation system of cymbidium kanran. The method comprises the following steps: adding agrobacterium EHA105 into a YEB liquid culture medium containing 50 mg / L of kanamycin and 25 mg / L of rifampicin as a dip dyeing solution; placing the injured transformation receptor in dip dyeing for transformation, and then carrying out regeneration culture. The invention relates to a method for carrying out genetic transformation on cymbidium kanran by utilizing the innovative receptor and the screening culture medium, and the method can realize efficient and stable genetic transformation without genotype dependence and can solve the problem that the genetic transformation of cymbidium kanran varieties is difficult to carry out. According to the invention, an efficient genetic transformation and regeneration system of cymbidium kanran is established for the first time, and a foundation is laid for verification of related functional genes of cymbidium kanran and further development of biological research of cymbidium transgenic breeding and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to a construction method of a Cymbidium kanran genetic transformation system. BACKGROUND

[0002] Cymbidium kanran (Cymbidium sinense As one of the important national orchid categories, Cymbidium kanran is mainly distributed in Yunnan, Fujian, Guangdong and Guangxi of China. As a potted ornamental plant, Cymbidium kanran is known as one of the "four traditional famous orchids in Yunnan" together with Cymbidium goeringii, Cymbidium kanran var. parviflorum and Cymbidium kanran var. tonkinense, and is one of the excellent traditional cultivation varieties and representative varieties of Cymbidium kanran in Yunnan. With its high ornamental value and rich cultural connotation, the economic value of Cymbidium kanran has been rising year by year. However, many traits of Cymbidium kanran, such as leaf color and stress resistance, are difficult to break through by traditional breeding methods, so it is crucial to establish an efficient genetic transformation and regeneration system. In the present technology, the effects of pre-culture time, bacterial liquid concentration, infection method, infection time and co-culture time on the transformation rate and regeneration rate of Cymbidium kanran rhizomes are tested. On this basis, a construction method of a Cymbidium kanran genetic transformation system is developed. SUMMARY

[0003] In order to overcome the problems in the prior art, the present application provides a construction method of a Cymbidium kanran genetic transformation system.

[0004] To achieve the above-mentioned purpose, the present application is realized by the following technical scheme: The construction method of the Cymbidium kanran genetic transformation system comprises the following steps: S1, taking mature Cymbidium kanran seeds, disinfecting and then sowing in a germination culture medium to obtain rhizomes; S2, taking the rhizomes, selecting healthy rhizomes with a length of 1-1.2 cm, and inoculating in a proliferation culture medium to obtain sufficient rhizome transformation receptors and control materials; S3, preparing an infection liquid: adding Agrobacterium EHA105 containing a GFP marker gene into a YEB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and oscillating in a 28℃, 200 rpm shaker overnight to make the cell suspension have an optical density OD of 0.4-1.0 at 600 nm, and adding 100 umol / L acetyl-syringone for standby; S4, genetic transformation: after the transformation receptor is cut with a sterile scalpel, it is placed in the infection liquid and transferred to a shaking incubator for co-culture; after the co-culture is completed, the long-bacterium transformation is washed with sterile water containing 30 mg / L meropenem trihydrate, and the washed rhizome is placed in a regeneration medium added with 15 mg / L meropenem trihydrate, wherein the differentiation regeneration medium comprises 1 / 2MS+3.0 mg / L BA, 2 mg / L NAA, 10 ml coconut milk; S5, regeneration culture: the transformed rhizome is selected, washed twice with sterile water, treated once with sterile water containing 30 mg / L meropenem, then dried on sterile filter paper, then all the treated rhizomes are placed on a regeneration medium containing 15 mg / L meropenem, and finally the rhizomes are placed in a culture room for light culture for 30 days; wherein the regeneration medium comprises 1 / 2MS+4.0 mg / L BA, 0.2 mg / L NAA, 15 mg / L Mer.

[0005] Further, in the S1 step, the germination medium comprises 1 / 2MS+2.0 mg / L 6-BA, 1.5 mg / L NAA, 45 g / L trehalose, 7 g / L agar, and pH=5.8.

[0006] Further, in the S1 step, the germination culture condition is 25±1℃, dark culture for 10 days.

[0007] Further, in the S2 step, the proliferation medium comprises 1 / 2MS+4.0 mg / L 6-BA, 0.2 mg / L NAA.

[0008] Further, in the S2 step, the proliferation culture condition is 25±1℃, dark culture for 10-15 days.

[0009] Through the above technical solution, the present application can at least achieve the following beneficial effects: The present application establishes a high-efficiency genetic transformation and regeneration system of Cymbidium 'Zhu San Lan', which lays a foundation for the further development of the verification of related functional genes of the high-efficiency genetic transformation and regeneration system of Cymbidium 'Zhu San Lan' and the transgenic breeding of Cymbidium. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is the plasmid map in the present application; Figure 2 is a graph showing the influence of the concentration of hygromycin on Cymbidium 'Zhu San Lan' in the present application; Figure 3is the rhizome fluorescence reaction graph of the invention under different dark culture time, in which a is the rhizome fluorescence reaction graph after 5 days of dark culture, b is the rhizome fluorescence reaction graph after 10 days of dark culture, and c is the rhizome fluorescence reaction graph after 15 days of dark culture; Figure 4 is the influence graph of the concentration of Agrobacterium liquid on the fluorescence effect and fluorescence rate of Cymbidium 'Zhu San Lan' in the invention, in which a is the rhizome fluorescence reaction graph of OD 600 =0.4, b is the rhizome fluorescence reaction graph of OD 600 =0.6, c is the rhizome fluorescence reaction graph of OD 600 =0.8, and d is the rhizome fluorescence reaction graph of OD 600 =1.0; Figure 5 is the rhizome fluorescence graph under different infection time and methods in the invention, in which a is the fluorescence reaction rate under different infection time and methods, b is the rhizome fluorescence reaction graph under 20 min of infection, c is the rhizome fluorescence reaction graph under 40 min of infection, and d is the rhizome fluorescence reaction graph under 60 min of infection; Figure 6 is the influence graph of co-culture time on genetic transformation efficiency in the invention, according to single factor variance analysis, different letters represent significant differences between treatments (P<0.05); Figure 7 is the growth difference graph of rhizome of Cymbidium 'Zhu San Lan' under different pre-culture time in the invention, in which a is 5 days of dark culture, b is 10 days of dark culture, and c is 15 days of dark culture; Figure 8 is the growth difference graph of Cymbidium 'Zhu San Lan' rhizome before and after vacuum treatment in the invention, in which a is 40 min of oscillation, and b is 40 min of oscillation and 30 min of vacuum treatment of rhizome; Figure 9 is the growth difference graph of rhizome of Cymbidium 'Zhu San Lan' under different co-culture time in the invention, in which a is 1 day of co-culture, b is 3 days of co-culture, and c is 5 days of co-culture; Figure 10 is the fluorescence detection graph of rhizome of Cymbidium 'Zhu San Lan' in the invention; Figure 11 is the resistant plant of Cymbidium 'Zhu San Lan' in the invention; Figure 12 is the fluorescence detection graph of the resistant plant of Cymbidium 'Zhu San Lan' in the invention; Figure 13 is the PCR detection graph of the resistant plant of Cymbidium 'Zhu San Lan' in the invention; Figure 14 is the relative expression amount graph of GFP of Cymbidium 'Zhu San Lan' in the invention, in which WT represents untransformed plants, GFP represents transgenic plants, and * represents significant difference. DETAILED DESCRIPTION

[0011] Unless otherwise indicated, the materials, reagents used in the present application (except for plasmid vectors) are commercially available. EXAMPLE

[0012] 1. Materials and Methods 1.1 Plant material The test material was rhizome of Cymbidium kanran 'Zhu San Lan'. The rhizome induction method was as follows: Cymbidium kanran 'Zhu San Lan' self-crossed seeds were sowed aseptically on germination medium. The medium was 1 / 2MS medium with 3% sucrose and 0.7% agar (pH 5.8). The germination medium formula was 1 / 2MS + 2.0 mg / L 6-benzylaminopurine (6-BA) + 1.5 mg / L naphthalene acetic acid (NAA). The seeds of Cymbidium kanran 'Zhu San Lan' used in the present study were collected from the Orchid Base of the Flower Research Institute of the Yunnan Academy of Agricultural Sciences (Yuxi, China), and the induced rhizomes were preserved in the laboratory of the Flower Research Institute of the Yunnan Academy of Agricultural Sciences. In the present study, a 1-1.2 cm healthy rhizome was selected for stable propagation, and the obtained rhizome was used as the test material.

[0013] 1.2 Related medium In the present application, two types of medium, i.e., propagation medium and regeneration medium, were mainly used, and both of them were composed of basic medium and plant growth regulators. The types and components of the medium are shown in the following table (Table 1). In the present application, the pH of all the media was 5.8, and the medium was sterilized at 121°C for 30 min.

[0014] Table 1. Medium type and component table 1.3 Plasmid vector and strain The plasmid vector used in the present study was preserved by the Flower Research Institute of the Yunnan Academy of Agricultural Sciences. The vector was overexpression vector pCAMBIA1300-GFP containing green fluorescent reporter gene (GFP) and plant selection marker gene HPT II (Hygromycin Gesistant Gene, hygromycin resistance gene) map Figure 1 The strain used in the test was E. coli competent DH5a and Agrobacterium competent EHA105.

[0015] The plasmid used in the present application was preserved by the Flower Research Institute of the Yunnan Academy of Agricultural Sciences. The plasmid contained green fluorescent marker gene GFP and selection marker gene HPT II (Hygromycin Phosphotransferase) encoding hygromycin Figure 1 In the present study, Agrobacterium tumefaciens EHA105 strain was used for transfection.

[0016] 1.4 Hygromycin selection test The 1-2 cm long and well-grown rhizomes of 'Zhu San Lan' were selected as experimental materials and inoculated on the proliferation medium containing different concentrations of hygromycin (20, 30, 40, 50, 60 mg / L). Each concentration was inoculated with 30 rhizomes, and the experiment was repeated 3 times. The mortality rate was determined after 14 days of culture at 25 °C under a light cycle of 14 h light / 10 h dark.

[0017] 1.5 Screening of the optimal concentration of the bacteriostatic agent To obtain the effects of meropenem on Agrobacterium and rhizome growth, respectively, the concentration of meropenem was set to 0, 10, 20, 30, and 40 mg / L. First, different concentrations of meropenem were added to the liquid YEB medium containing Agrobacterium, which was then cultured at 28 °C and 200 rpm for 48 h. The concentration of the Agrobacterium liquid at OD 600 was then determined to obtain the inhibitory effect of different concentrations of meropenem on Agrobacterium growth. The materials were inoculated on the medium containing different concentrations of meropenem, with 30 rhizomes inoculated in each concentration and the experiment repeated 3 times. The survival rate and regeneration rate ((proliferation number / number of live explants) x 100%) were calculated based on the survival number and proliferation number after 30 days of culture at 25 °C under a light cycle of 14 h light / 10 h dark, thereby obtaining the effects of meropenem on rhizome growth.

[0018] 1.6 Infection of Agrobacterium The 1-2 cm long and well-grown rhizomes were inoculated on the proliferation medium and pre-cultured (dark culture) for a certain period of time (5, 10, and 15 days) at room temperature (25 °C) as the receptor for genetic transformation. Before Agrobacterium infection, 3-5 deep (1 mm) wounds were made on the surface of the rhizomes after dark culture for a certain period of time using a sterile scalpel. The rhizomes with wounds were placed in the infection liquid (OD 600 = 0.4, 0.6, 0.8, and 1.0) and shaken at 200 rpm for 20, 40, and 60 min and vacuumed for 10, 20, and 30 min. Then, the bacterial liquid on the surface of the rhizomes was absorbed with filter paper, and the rhizomes were inoculated on the co-culture medium for co-culture (1, 3, and 5 d) at 25 °C in the dark. Each treatment group was inoculated with 60 explants, and the experiment was repeated 3 times. After sterilization, the GFP fluorescence was observed, and the number of rhizome fluorescence responses was counted to obtain the transient expression rate (transient expression rate = number of rhizome fluorescence responses / 60 x 100%).

[0019] 1.7 Design of the regeneration experiment The rhizomes showing green fluorescence were treated with sterile water containing 30 mg / L meropenem once, washed with sterile water twice, and dried with sterile filter paper. Then the treated rhizomes were placed on regeneration medium containing 15 mg / L meropenem and 50 mg / L hygromycin (Hyg) for alternating culture, and the medium was replaced every 7 days to ensure the screening effect of hygromycin and the inhibition effect of meropenem on Agrobacterium. After 30 days of light culture, the number of surviving rhizomes, the number of proliferations, the survival rate, and the regeneration rate ((the number of proliferations / the number of living explants) x 100%) were counted. When the rhizomes grew to about 4-5 cm long, they were inoculated into differentiation medium for differentiation.

[0020] 2.8 Green fluorescent protein (GFP) fluorescence detection The degermed rhizomes were observed for GFP fluorescence, the green fluorescent protein signal was detected with a Leica fluorescence microscope MZ16 (excitation wavelength 440-490 nm), and pictures were taken.

[0021] 2.9 Molecular biology detection of resistant plants DNA of rhizomes of resistant plants was extracted using the Plant Genome Extraction Kit (Magen), and the GFP gene was amplified using 2x EasyTaq® PCR SuperMix (TransGen, Beijing, China) with 1300-F: 5'-GCCATTTCGCCTTTTC-3' and GFP-R: 5'-GTAGCGCGTGAGACTG-3' as primers (primers synthesized by Kunming Qikexiao Biotechnology Co., Ltd.). The PCR product was subjected to 1% agarose gel electrophoresis to observe whether the target fragment was 411 bp. For qRT-PCR analysis, total RNA was extracted from the leaves of resistant plants using the High-purity RNA Extraction Kit TransZol Up Plus RNA Kit (TransGen, Beijing, China), and the RNA was reverse transcribed using the One-step cDNA Synthesis Kit TransScriptOne-Step gDNA Removal and Cdna Synthesis SuperMix (TransGen, Beijing, China) with incubation at 42°C for 15 min and inactivation at 85°C for 5 s. The reverse-transcribed cDNA was used as a template, and primers ACTIN-F: 5'-ATGGCCGACGGTGAAGAAAT-3' and ACTIN-R: 5'-CAGCAAAACCAGCCTTGACC-3' were used as internal reference genes. qRT-PCR was performed using the TransStart Top Green qPCR SuperMix (TransGen), and the reaction system was as follows. The experimental protocol was as follows: preheating at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, 55°C for 30 s, and 72°C for 10 s.

[0022] 2.10 Statistical analysis SPSS 24.0 software and Excel 2019 were used for single-factor analysis of variance, and sample comparisons were made using LSD and Duncan's range test for multiple comparisons. The mean was represented as "mean ± standard deviation".

[0023] 2. Results and analysis 2.1 Effect of hygromycin on rhizomes of Cymbidium kanran 'Zhusanlan' The mortality rate of rhizomes of Cymbidium kanran 'Zhusanlan' on different concentrations of hygromycin medium was analyzed Figure 2), it can be seen that with the increase of the concentration of hygromycin, the mortality rate of C. japonicum ‘Zhu San Lan’ is also increasing, and when the concentration of hygromycin reaches 60 mg / L, the mortality rate of C. japonicum ‘Zhu San Lan’ reaches 100% (Table 1). Plants are extremely sensitive to hygromycin. In order to ensure the screening effect of hygromycin and at the same time to reduce the toxic effect on plants, the selection of the screening concentration of hygromycin in the experiment should be slightly lower than the critical lethal concentration. And when the concentration of hygromycin is slightly lower than the critical lethal concentration of 50 mg / L, the mortality rate of C. japonicum ‘Zhu San Lan’ is close to 100%, which is 95.56%±5.09. Therefore, in genetic transformation, the best screening concentration of hygromycin for C. japonicum ‘Zhu San Lan’ is 50 mg / L.

[0024] Table 2 Effect of hygromycin on rhizomes of C. japonicum ‘Zhu San Lan’ Note: The data is expressed as mean ± standard deviation. According to one-way ANOVA, different letters indicate significant differences between treatments (P<0.05).

[0025] 2.2 Selection of the best concentration of C. japonicum ‘Zhu San Lan’ bacteriostatic agent From the results of Table 3, it can be seen that the higher the concentration of meropenem, the lower the concentration of Agrobacterium, and the survival rate and regeneration rate of C. japonicum ‘Zhu San Lan’ rhizomes are inversely proportional to the concentration of meropenem. When the concentration of meropenem is 20 mg / L, it can successfully inhibit the growth of Agrobacterium. When the concentration of meropenem is 20 mg / L, the survival rate of rhizomes is 75.56%±3.85, and the regeneration rate is 116.16%±2.31. When the concentration of meropenem reaches 30 mg / L, the survival rate and regeneration rate of C. japonicum ‘Zhu San Lan’ rhizomes have dropped sharply, which are 6.67%±3.33 and 0.00%±00.00, respectively. Therefore, the best concentration of meropenem as a bacteriostatic agent for C. japonicum ‘Zhu San Lan’ is also 20 mg / L.

[0026] Table 3 Inhibition effect of different concentrations of Mer on the growth of C. japonicum ‘Zhu San Lan’ rhizomes and Agrobacterium Note: The data is expressed as mean ± standard deviation. According to one-way ANOVA, different letters indicate significant differences between treatments (P<0.05).

[0027] 2.3 Study on the influencing factors of transformation efficiency of C. japonicum ‘Zhu San Lan’ 2.3.1 Effect of dark incubation time on transformation efficiency of C. japonicum ‘Zhu San Lan’ After the rhizomes of the three different dark incubation times were infected and degermed, GFP fluorescence observation was carried out Figure 3During 5 days of dark culture, the fluorescence effect of the rhizomes was not obvious, with only a weak fluorescence reaction observed at the tips of some rhizomes. The fluorescence reaction of the rhizomes after 10 and 15 days of dark culture was much stronger. Therefore, the number of rhizomes exhibiting fluorescence at different dark culture times was further counted. The results showed significant differences in the fluorescence rate of the rhizomes under different dark culture times. The fluorescence rate of the rhizomes after 5 days of pre-culture was less than 50%, while the fluorescence rate of the rhizomes after 10 or 15 days of pre-culture reached 90% (Table 4). Therefore, based on the fluorescence rate, the optimal pre-culture time for the genetic transformation of *Cymbidium goeringii* 'Zhu Sha Lan' is 10 or 15 days.

[0028] Table 4. Effect of pre-cultivation time on the conversion efficiency of Cymbidium goeringii 'Zhushalan' Note: Data are expressed as mean ± standard deviation. One-way ANOVA showed significant differences between the different letter representations (P < 0.05).

[0029] 2.3.2 Effect of Agrobacterium concentration on the transformation efficiency of Cymbidium goeringii 'Zhushalan' Using different concentrations of Agrobacterium (OD) 600 Infection experiments were conducted on rhizomes pre-cultured for 10 days using infection solutions prepared with concentrations of 0.4, 0.6, 0.8, and 1.0 (μF = 0.4, 0.6, 0.8, and 1.0). The infection time was consistently 200 rpm for 40 min with shaking. The results showed that the fluorescence produced by different concentrations of Agrobacterium was relatively strong in rhizomes, with no significant difference, and there was no significant difference in fluorescence rate between different bacterial concentrations. Figure 4 Rhizomes exhibiting green fluorescence accounted for over 90% (Table 5). Therefore, in the genetic transformation of 'Zhu Sha Lan', the concentration of Agrobacterium tumefaciens in the culture was within the OD range. 600 Within the range of 0.4-1.0, there is no significant effect on fluorescence rate.

[0030] Table 5. Effect of Agrobacterium tumefaciens concentration on the genetic transformation efficiency of Cymbidium goeringii 'Zhushalan'.

[0031] Note: Data are expressed as mean ± standard deviation. One-way ANOVA showed significant differences between treatments represented by different letters (P < 0.05).

[0032] 2.3.3 Effects of Agrobacterium infection time and method on the transformation efficiency of Cymbidium goeringii 'Zhushalan' Rhizomes that had been cultured in the dark for 10 days were placed in an Agrobacterium solution with an OD concentration of [missing information]. 600=0.6, and shaken for 20 min, 40 min, and 60 min, respectively, at 28 °C and 200 rpm. The results showed that there was no significant difference in the fluorescence rate of the rhizomes of C. ‘Zhusanlan’ under different infection times, and the number of rhizomes producing green fluorescence was as high as more than 90%. However, the rhizomes infected for 40 min had the best fluorescence effect Figure 5 To further explore whether vacuuming can help the genetic transformation of C. ‘Zhusanlan’, vacuuming was performed for 10 min, 20 min, and 30 min after 40 min of shaking. The results showed that the fluorescence rate (99.44% ± 0.96) was significantly higher than that of other treatments when shaking for 40 min and vacuuming for 30 min (Table 6). Therefore, different infection times have no significant effect on the transformation rate during the genetic transformation of C. ‘Zhusanlan’, but the combination of multiple infection methods can improve the transformation efficiency of C. ‘Zhusanlan’ to some extent.

[0033] Table 6 Effect of Agrobacterium infection time and method on the genetic transformation efficiency of C. ‘Zhusanlan’ Note: S20 represents shaking for 20 min; S40 represents shaking for 40 min; S60 represents shaking for 60 min; S40, V10 represents shaking for 40 min followed by vacuuming for 10 min; S40, V20 represents shaking for 40 min followed by vacuuming for 20 min; S40, V30 represents shaking for 40 min followed by vacuuming for 30 min. The data are presented as mean ± standard deviation. According to one-way ANOVA, different letters indicate significant differences between treatments (P < 0.05).

[0034] 2.3.4 Effect of co-cultivation time on the transformation efficiency of C. ‘Zhusanlan’ The pre-cultured rhizomes for 10 days were infected with Agrobacterium solution with a concentration of OD 600 =0.6 for 40 min, followed by different co-cultivation times. The results showed that the fluorescence rate was the highest (91.11 ± 1.92%) when co-cultivated for 3 days, followed by co-cultivation for 5 days (82.22 ± 1.92%), and the fluorescence rate was the lowest (77.78 ± 0.96%) when co-cultivated for 1 day (Table 7). The fluorescence rate of the rhizomes after 3 days of co-cultivation was significantly higher than that after 1 day and 5 days of co-cultivation Figure 6 Therefore, 3 days is the best co-cultivation time for C. ‘Zhusanlan’.

[0035] Table 7 Effect of co-cultivation time on the genetic transformation efficiency of C. ‘Zhusanlan’

[0036] Note: Data are expressed as mean ± standard deviation. According to one-way ANOVA, different letters indicate significant differences between treatments (P < 0.05).

[0037] 2.4 Establishment of the regeneration system of Cymbidium ‘Zhu Shalan’ after transformation 2.4.1 Selection of the optimal dark incubation time for rhizome regeneration of Cymbidium ‘Zhu Shalan’ To determine the effect of pre-culture (dark incubation) time on rhizome regeneration of Cymbidium ‘Zhu Shalan’ during the genetic transformation process, we measured the survival number and regeneration number of rhizomes under different dark incubation times after 30 days of transformation. The survival number and survival rate reached the highest at 10 days of dark incubation, which were 59.67% ± 0.58 and 99.44% ± 0.96, respectively, and were significantly higher than those at 5 days (46.67) and 15 days (42.67) of dark incubation (Table 8). This indicated that too short or too long dark incubation time was not conducive to the survival of rhizomes, and 10 days of dark incubation could maximize the survival of rhizomes. In terms of regeneration frequency, the rhizomes pre-cultured for 5 days and 15 days were significantly lower than those pre-cultured for 10 days, and the rhizomes pre-cultured for 10 days had the highest regeneration frequency (155.34%) (Table 8). The above results showed that the rhizomes pre-cultured for 10 days had the highest survival rate, and the survival number and regeneration frequency were much higher than those of the other two groups. Finally, we compared the growth status of rhizomes under different dark incubation times (Fig. 4). There were no obvious proliferative bud points when the rhizomes were pre-cultured for 5 days, and the rhizomes showed extensive browning when they were pre-cultured for 15 days. In general, the rhizomes of Cymbidium ‘Zhu Shalan’ grew best when they were pre-cultured for 10 days. Therefore, 10 days of pre-culture was the optimal pre-culture time for the regeneration of Cymbidium ‘Zhu Shalan’ after transformation, under which the survival number, survival rate, proliferation number, regeneration rate, and growth status of rhizomes were all optimal, which could effectively promote the regeneration and growth of rhizomes. Figure 7

[0038] Table 8 Effect of dark incubation time on the regeneration of Cymbidium ‘Zhu Shalan’ after transformation Note: Data are expressed as mean ± standard deviation. According to one-way ANOVA, different letters indicate significant differences between treatments (P < 0.05).

[0039] 2.4.2 Selection of the optimal bacterial solution concentration for rhizome regeneration of Cymbidium ‘Zhu Shalan’ In the process of plant genetic transformation, the concentration of Agrobacterium solution has a very important influence on the regeneration of transformed plants. In this study, too high or too low concentration of Agrobacterium would affect the survival and regeneration of rhizomes of Cymbidium ‘Zhu Shalan’, and the survival number and survival rate, as well as the proliferation number and regeneration rate of rhizomes were all the highest when the bacterial solution concentration was OD 600 ​=0.6 (Table 9). When the concentration of bacterial solution was higher than 0.6, both the survival rate and the regeneration rate of rhizomes decreased significantly, indicating that too high concentration of bacterial solution might inhibit the growth of rhizomes. This is enough to show that the regeneration ability of rhizomes is the best when the concentration of Agrobacterium is OD 600 =0.6. Therefore, in order to maximize the survival rate and regeneration frequency of rhizomes of Cymbidium ‘Zhu San Lan’ after transformation, the concentration of Agrobacterium should be controlled at about OD 600 =0.6.

[0040] Table 9 Effect of bacterial solution concentration on the regeneration of Cymbidium ‘Zhu San Lan’ after transformation Note: Data are expressed as mean ± standard deviation. According to one-way ANOVA, different letters indicate significant differences between treatments (P < 0.05).

[0041] 2.4.3 Selection of the best infection time and method for the regeneration of rhizomes of Cymbidium ‘Zhu San Lan’ In order to explore the regeneration status of rhizomes of Cymbidium ‘Zhu San Lan’ under different treatment times and methods, we counted and compared the survival number and proliferation number of rhizomes under different treatment times and methods. The results, as shown in Table 10, are that both the survival number and survival rate and the proliferation number and regeneration rate are the highest at 40 min of shaking (S40), which are 59.67 ± 0.58 and 99.44 ± 0.96% and 92.67 ± 2.08 and 155.34 ± 4.99%, respectively. The survival status and regeneration ability of 20 min of shaking (S20) are slightly lower than those of S40 but the difference is not significant. The survival rate and regeneration rate of 60 min of shaking (S60) are significantly lower than those of S40, indicating that too long shaking is not conducive to the survival and value-added regeneration of rhizomes. In addition, when the vacuum time is increased (e.g., S40, V10; S40, V20; S40, V30), the survival number, proliferation number, survival rate and regeneration rate all decrease significantly, indicating that vacuum treatment has a great negative impact on the growth and regeneration of rhizomes. Moreover, after the vacuum treatment, the growth of Agrobacterium is difficult to be inhibited in the recovery process of rhizomes compared with that of rhizomes without vacuum treatment ( Figure 8 ). In the process of genetic transformation of Cymbidium ‘Zhu San Lan’, although the vacuum method greatly affects the survival and proliferation regeneration of rhizomes. Therefore, 20 - 40 min of shaking will be more conducive to the recovery and regeneration of rhizomes of Cymbidium ‘Zhu San Lan’ after genetic transformation.

[0042] Table 10 Effect of infection time and method on the regeneration of Cymbidium ‘Zhu San Lan’ after transformation Note: S20 means shaking for 20 min; S40 means shaking for 40 min; S60 means shaking for 60 min; S40, V10 means shaking for 40 min and vacuuming for 10 min; S40, V20 means shaking for 40 min and vacuuming for 20 min; S40, V30 means shaking for 40 min and vacuuming for 30 min.

[0043] Data were expressed as mean ± standard deviation. Different letters indicated significant difference between treatments (P < 0.05) according to one-way ANOVA.

[0044] 2.4.4 Selection of optimal co-cultivation time for Cymbidium ‘Zhu Shalan’ rhizome regeneration During genetic transformation, too short co-cultivation time of Cymbidium ‘Zhu Shalan’ rhizome with Agrobacterium would lead to unsuccessful transformation or low transformation rate, while too long co-cultivation time would affect rhizome growth and even cause death. We recovered the rhizomes co-cultivated for different days for 30 d, and counted and compared their survival number and proliferation number. The specific data are shown in Table 11. In terms of survival number and survival rate, both were higher when co-cultivated for 1 d and 3 d, being 98.89% ± 0.96 and 99.44% ± 0.96, respectively, indicating that shorter co-cultivation time (1 d or 3 d) could maximize the survival of rhizomes. When co-cultivated for 5 d, the survival number and survival rate decreased significantly, indicating that too long co-cultivation time might inhibit rhizome growth. In terms of proliferation number and regeneration rate, both were best when co-cultivated for 1 d, being 94.33% ± 3.05 and 159.00% ± 5.12, respectively. When co-cultivated for 3 d, the proliferation number and regeneration rate were slightly lower than those of 1 d, but the difference was not significant. When co-cultivated for 5 d, the proliferation number and regeneration rate decreased significantly, indicating that too long co-cultivation time was not conducive to rhizome proliferation and regeneration. In terms of rhizome growth state (Fig. 11), the rhizomes co-cultivated for 1 d and 3 d grew well and showed no obvious difference. When co-cultivation was prolonged to 5 d, the rhizomes began to brown and die in large areas. In summary, too long co-cultivation time might increase the metabolic burden of cells, inhibit rhizome growth and regeneration, and even cause browning and death. Therefore, the optimal co-cultivation time for Cymbidium ‘Zhu Shalan’ rhizome regeneration after genetic transformation was 1 d or 3 d. Figure 9

[0045] Table 11 Effect of co-cultivation time on regeneration of Cymbidium ‘Zhu Shalan’ after transformation Note: Data were expressed as mean ± standard deviation. Different letters indicated significant difference between treatments (P < 0.05) according to one-way ANOVA.

[0046] ​2.5 Identification of Cymbidium 'Zhu Shalan' Transgenic Plants First, the possible positive transformation plants were preliminarily screened and subcultured by fluorescence GFP at the initial stage of transformation Figure 10 ). Through further screening by antibiotics, resistant plants of Cymbidium 'Zhu Shalan' were obtained Figure 11 ). The resistant plants were identified again by fluorescence GFP Figure 12 ), and the result showed that no fluorescence reaction was detected in the resistant plants. Further, PCR Figure 13 ) and qRT-PCR detection Figure 14 ) were carried out using the resistant plants as experimental materials, and the target band and the difference in the relative expression amount of the GFP gene were detected in the 21 resistant plants. Finally, 21 transgenic positive plants of Cymbidium 'Zhu Shalan' were obtained from 180 rhizomes of Cymbidium 'Zhu Shalan', and the transformation rate was 11.67%.

[0047] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A method for constructing a genetic transformation system for *Cymbidium goeringii*, characterized in that: It comprises the following steps: S1, taking the Cymbidium seed, disinfecting and then sowing in the germination culture medium to obtain the rhizome; S2, taking the rhizome, cutting into 1-1.2cm small segments, inoculating in the proliferation culture medium to obtain the transformation receptor and the control material; S3, preparing the infection liquid: adding the Agrobacterium EHA105 containing the GFP marker gene into the YEB liquid medium containing 50mg / L kanamycin and 25mg / L rifampicin, oscillating in the 28℃, 200rpm shaker overnight, making the cell suspension liquid density OD at 600nm between 0.4-1.0, and adding 100umol / L acetyl-syringone for standby; S4, gene transformation: using the sterile scalpel to cut the transformation receptor, placing it in the infection liquid, transferring to the shaker for co-culture; after the co-culture, washing the long bacteria transformation body with the sterile water containing 30mg / L meropenem trihydrate, and placing the washed rhizome in the regeneration culture medium containing 15mg / L meropenem trihydrate, wherein the differentiation regeneration culture medium comprises 1 / 2MS+3.0mg / L BA, 2mg / L NAA, 10ml coconut milk; S5, regeneration culture: selecting the transformed rhizome, washing twice with sterile water, treating once with the sterile water containing 30mg / L meropenem, then drying on the sterile filter paper, then placing all the treated rhizomes on the regeneration culture medium containing 15mg / L meropenem, and finally placing the rhizomes in the culture room for illumination culture for 30 days; wherein the regeneration culture medium comprises 1 / 2MS+4.0mg / L BA, 0.2mg / L NAA, 15mg / L Mer.

2. The method according to claim 1, wherein: In the S1 step, the germination culture medium comprises 1 / 2MS+2.0mg / L 6-BA, 1.5mg / L NAA, 45g / L trehalose, 7g / L agar, pH=5.

8.

3. The method according to claim 1, wherein the method is characterized by: In the S1 step, the germination culture condition is 25±1℃, dark culture for 10 days.

4. The method according to claim 1, wherein the method is characterized by: In the S2 step, the proliferation culture medium comprises 1 / 2MS+4.0mg / L 6-BA, 0.2mg / L NAA.

5. The method according to claim 1, wherein the method is characterized by: In the S2 step, the proliferation culture condition is 25±1℃, dark culture for 10-15 days.

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