阅读说明：本技术 根癌农杆菌介导的菊花‘神马’的转基因方法 (Agrobacterium tumefaciens-mediated chrysanthemum 'shenma' transgenic method ) 是由 亓帅 于晓艳 王爽 张小羽 徐宗大 王金娥 谌堪 于 2020-12-18 设计创作，主要内容包括：本发明公开了一种根癌农杆菌介导的菊花‘神马’的转基因方法,主要包括外植体预培养、菌液制备、共培养、延迟培养和筛选培养几个关键步骤。本发明对影响菊花‘神马’转化过程的多个因素进行了筛选和改良,通过优选外植体、提供足够时间的暗培养以及优化培养基的组成,从而提高了菊花转基因过程的再生转化率,提高了转化体系的稳定性,为后期在菊花中进行转基因功能验证和基因工程育种奠定了基础。(The invention discloses a transgenic method of agrobacterium tumefaciens-mediated chrysanthemum 'shenma', which mainly comprises the key steps of explant pre-culture, bacterial liquid preparation, co-culture, delayed culture and screening culture. The invention screens and improves a plurality of factors influencing the transformation process of the chrysanthemum, improves the regeneration transformation rate of the chrysanthemum in the transgenic process, improves the stability of a transformation system and lays a foundation for carrying out transgenic function verification and genetic engineering breeding in the chrysanthemum at the later stage by optimizing explants, providing dark culture for enough time and optimizing the composition of a culture medium.)

1. An agrobacterium tumefaciens-mediated chrysanthemum 'shenma' transgenic method is characterized by comprising the following steps:

(1) taking the young leaves of the aseptic chrysanthemum Mare seedlings as explants, inoculating the abaxial surfaces of the explants downwards to a pre-culture medium, and pre-culturing for 2d at 22-24 ℃ under the dark condition;

(2) activating and culturing the agrobacterium tumefaciens transformed with the recombinant expression vector, collecting thalli, and carrying out heavy suspension on the thalli by using 1/2MS liquid culture medium to prepare agrobacterium tumefaciens bacterial liquid;

(3) immersing the explant pre-cultured in the step (1) into agrobacterium liquid for infection for 8-12 min;

(4) sucking the redundant agrobacterium liquid on the infected explant, then inoculating the explant into a co-culture medium with the paraxial surface facing downwards, and co-culturing for 2d at 22-24 ℃ under the dark condition;

(5) washing the explant subjected to co-culture in the step (4) with a carbenicillin solution, washing with sterile water, inoculating the washed explant into a delay culture medium with the paraxial surface facing downwards, and performing delay culture for 4 days at 22-24 ℃ under a dark condition;

(6) transferring the explant after delayed culture with paraxial surface facing downwards to a screening differentiation culture medium, and screening and culturing at 22-24 deg.C under dark condition for 10 d; then transferring to an incubator with the illumination cycle of 16h/8h in darkness, and culturing at 22-24 ℃ until the seedlings are differentiated and emerge;

(7) when the height of the seedling is 1-2cm, cutting the seedling and inoculating the seedling into a rooting culture medium for culture to obtain a transgenic resistant plant.

2. The transgenic method of claim 1, wherein the pre-culture medium is an MS culture medium containing 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH is 5.8-6;

the co-culture medium is an MS culture medium containing 2mg/L of 6-BA, 0.5mg/L of NAA, 30g/L of sucrose and 6g/L of agar powder, and the pH value is 5.8-6;

the delay culture medium is an MS culture medium containing 400mg/L carbenicillin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6;

the screening differentiation culture medium is an MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6;

the rooting culture medium is an MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

3. The transgenic method according to claim 1, wherein in the step (1), young leaves with fully developed upper parts in 20-25 d-old chrysanthemum 'Neuma' aseptic seedlings are selected as explants.

4. The transgenic method of claim 1, wherein in step (1), young leaves of the chrysanthemum 'Neuma' aseptic seedlings are cut into 5mm x 5mm squares and petioles are cut into 5mm lengths on a clean bench.

5. The method for transferring gene of claim 1, wherein in step (2), the Agrobacterium tumefaciens is Agrobacterium tumefaciens GV 3101.

6. The method for transferring gene of claim 1, wherein in the step (2), the recombinant expression vector is pBI121 which is a vector plasmid into which a target gene is inserted.

7. The transgenic method according to claim 1, characterized in that in step (2), the activation and culture are specifically: putting Agrobacterium tumefaciens into a sterilized Erlenmeyer flask, adding 10ml YEB liquid culture medium containing 50mg/L kanamycin and 50mg/L rifampicin, and performing shake culture in a shake culture box at 28 ℃ and 200rpm overnight; 90ml of YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin was added to the flask, and cultured with shaking for 2 to 3 hours until OD600 reached 0.4 to 0.6.

8. The method of claim 1, wherein after the transgenic resistant plant is obtained in step (7), the method further comprises the step of performing genetic testing on the transgenic resistant plant.

9. Use of the transgenic method according to any one of claims 1 to 8 in chrysanthemum breeding.

Technical Field

The invention relates to the technical field of chrysanthemum transgenic, in particular to a transgenic method of agrobacterium tumefaciens-mediated chrysanthemum 'shenma'.

Background

Chrysanthemum morifolium (Chrysanthemum morifolium) is a perennial root herbaceous plant of Chrysanthemum of Compositae, is ten traditional famous flowers originally produced in China and is one of four cut flowers in the world, and the yield and value of the Chrysanthemum morifolium are always in the prostate in flowers in the world. The chrysanthemum is originated from China, is distributed all over the world, and has a long cultivation history of over 2500 years in China. The flower has various colors, beautiful patterns, rich varieties and higher ornamental characteristics, is widely applied to urban garden and park greening and the like, is one of important ornamental plants in gardens, and is deeply loved by people.

The chrysanthemum 'Shenma' is a white autumn chrysanthemum variety cultivated in Japan, has large flowers and pure white color, is an excellent cut chrysanthemum variety and is popular in the market. Meanwhile, the cut chrysanthemum 'shenma' is also a widely used material in the research of chrysanthemum molecular biology.

Due to the great limitation of species self genotype and seed source, the traditional chrysanthemum breeding method is difficult to succeed. The emergence of transgenic technology provides a new thought and approach for chrysanthemum breeding. The agrobacterium tumefaciens mediated transgenic system is one of the commonly used means for studying gene function of chrysanthemum and obtaining genetically modified organisms. The agrobacterium-mediated chrysanthemum transformation process is influenced by a plurality of factors such as a promoter, agrobacterium strain type, plant genotype, transformation receptor type, wound, chemical substances, pre-culture time, bacterium concentration and infection time, selection degree and time and the like.

At present, the chrysanthemum transgenic process still has great problems: (1) different documents describe conditions of regeneration and transformation systems of the cut chrysanthemum 'Neuma', which have great difference, such as the types of culture media, the types and concentrations of growth hormones and the like, so that the repeatability and the genetic transformation are poor; (2) after the explants are dedifferentiated into callus, the callus is easy to brown, die and the like, and the differentiation rate is low, so that the regeneration and transformation efficiency is low.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a transgenic method of agrobacterium tumefaciens mediated chrysanthemum. The invention screens and improves a plurality of factors influencing the transformation process of the chrysanthemum, thereby improving the regeneration transformation rate of the chrysanthemum in the transgenic process, improving the stability of a transformation system and laying a foundation for carrying out transgenic function verification and genetic engineering breeding in the chrysanthemum at the later stage.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, an agrobacterium tumefaciens-mediated chrysanthemum 'shenma' transgenic method is provided, which comprises the following steps:

(1) taking the young leaves of the aseptic chrysanthemum Mare seedlings as explants, inoculating the abaxial surfaces of the explants downwards to a pre-culture medium, and pre-culturing for 2d at 22-24 ℃ under the dark condition;

(2) activating and culturing the agrobacterium tumefaciens transformed with the recombinant expression vector, collecting thalli, and carrying out heavy suspension on the thalli by using 1/2MS liquid culture medium to prepare agrobacterium tumefaciens bacterial liquid;

(3) immersing the explant pre-cultured in the step (1) into agrobacterium liquid for infection for 8-12 min;

(4) sucking the redundant agrobacterium liquid on the infected explant, then inoculating the explant into a co-culture medium with the paraxial surface facing downwards, and co-culturing for 2d at 22-24 ℃ under the dark condition;

(5) washing the explant subjected to co-culture in the step (4) with a carbenicillin solution, washing with sterile water, inoculating the washed explant into a delay culture medium with the paraxial surface facing downwards, and performing delay culture for 4 days at 22-24 ℃ under a dark condition;

(6) transferring the explant after delayed culture with paraxial surface facing downwards to a screening differentiation culture medium, and screening and culturing at 22-24 deg.C under dark condition for 10 d; then transferring to an incubator with the illumination cycle of 16h/8h in darkness, and culturing at 22-24 ℃ until the seedlings are differentiated and emerge;

(7) when the height of the seedling is 1-2cm, cutting the seedling and inoculating the seedling into a rooting culture medium for culture to obtain a transgenic resistant plant.

Preferably, the pre-culture medium is an MS culture medium containing 2mg/L of 6-BA, 0.5mg/L of NAA, 30g/L of sucrose and 6g/L of agar powder, and the pH value is 5.8-6;

the co-culture medium is an MS culture medium containing 2mg/L of 6-BA, 0.5mg/L of NAA, 30g/L of sucrose and 6g/L of agar powder, and the pH value is 5.8-6;

the delay culture medium is an MS culture medium containing 400mg/L carbenicillin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6;

the screening differentiation culture medium is an MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6;

the rooting culture medium is an MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

Preferably, in the step (1), the young and tender leaves of 20-25 d-old chrysanthemum 'Mare' aseptic seedlings, the middle and upper parts of which are fully unfolded, are selected as explants.

Preferably, in the step (1), young leaves of the chrysanthemum 'shenma' aseptic seedlings are cut into squares of 5mm multiplied by 5mm on a clean bench, and petioles are cut into 5mm in length.

Preferably, in step (2), the agrobacterium tumefaciens is agrobacterium tumefaciens GV 3101.

Preferably, in the step (2), the recombinant expression vector is a vector plasmid into which a target gene is inserted and is pBI 121.

Preferably, in the step (2), the activating and culturing are specifically: putting Agrobacterium tumefaciens into a sterilized Erlenmeyer flask, adding 10ml YEB liquid culture medium containing 50mg/L kanamycin and 50mg/L rifampicin, and performing shake culture in a shake culture box at 28 ℃ and 200rpm overnight; 90ml of YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin was further added to the flask, and shake culture was performed for 2 to 3 hours until OD600 reached 0.4 to 0.6.

Preferably, after the transgenic resistant plant is obtained in the step (7), a step of performing genetic detection on the transgenic resistant plant is further included.

In a second aspect of the invention, the transgenic method is applied to breeding chrysanthemum.

The invention has the beneficial effects that:

(1) selection of explants: the seedling age of the chrysanthemum tissue culture seedling is the best of 20-25d, at the moment, the chrysanthemum grows most vigorously, and the meristematic capacity of leaves and petioles is strongest. Usually, the selected explant for chrysanthemum transformation is a leaf disc, but experiments show that the leaf disc and a petiole can be used as suitable infecting explants.

(2) Sufficient dark culture: the invention finds that dark culture is very important for callus induction and callus differentiation, and dark culture is needed from pre-culture to delayed culture in the early stage of transformation until ten days before screening culture.

(3) The explant placing mode is as follows: after the leaf disc and the petiole are cultured in the culture medium for a period of time, the phenomenon of backward curling often occurs, and in order to ensure that the leaf disc and the petiole contact the culture medium as much as possible, the leaf disc and the petiole are placed in a mode that the paraxial surface (namely the front surface) faces downwards in the whole culture process.

(4) The components of the culture medium: the differentiation of buds is a difficult step in the regeneration and transformation process of chrysanthemum, and the MS culture medium of 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder can induce the generation of callus and can induce the differentiation of buds through callus or without callus. The culture medium has high growth rate, and can raise the regeneration efficiency of explant and thus the conversion efficiency.

The method for transforming the chrysanthemum with the agrobacterium has the advantages of high regeneration and transformation rate, short time period and improvement of the efficiency and stability of chrysanthemum transgenosis.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background art, chrysanthemum is an ornamental plant which starts late in genetic engineering, the transformation process is influenced by various factors, and the low genetic transformation efficiency of chrysanthemum is one of the problems to be solved urgently in the genetic engineering research of chrysanthemum.

Based on the method, a plurality of factors influencing the chrysanthemum transformation process are screened and improved, so that the regeneration transformation rate of the chrysanthemum transgenic process is improved, and the stability of a transformation system is improved.

In one embodiment of the invention, the method for transforming chrysanthemum by using the agrobacterium tumefaciens-mediated exogenous gene comprises the following specific implementation steps:

(1) pre-culturing: selecting the young leaves fully developed at the middle upper part of the sterile chrysanthemum 'shenma' seedling with the age of 20-25d as explants, cutting the leaves into squares with the length of 5mm by using a scalpel in a clean bench, cutting the petioles into the length of 5mm, inoculating the cut leaves and the petioles into a pre-culture medium with the paraxial surfaces facing downwards, and placing the pre-culture medium in the dark at the temperature of 23 ℃ for pre-culture for 2 d. The culture medium is an MS culture medium of 2mg/L6-BA, 0.5mg/L LNAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

(2) Preparing bacterial liquid: the vector plasmid is pBI121, and the Agrobacterium tumefaciens strain is GV 3101. Taking out the prepared agrobacterium liquid from a refrigerator at the temperature of-80 ℃, placing the agrobacterium liquid in an ice box, and waiting for the agrobacterium liquid to melt. In a clean bench, 500. mu.l of the bacterial suspension was placed in a sterilized Erlenmeyer flask, 10ml of YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin was added thereto, and the mixture was cultured overnight at 28 ℃ with shaking in a shaking incubator at 200 rpm. The following morning, 90ml YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin was added to the flask, and shake culture was performed for 2-3 hours until OD₆₀₀Up to about 0.5. In the working table, the shaken 100ml of the bacterial solution is divided into two centrifuge tubes, centrifuged at 4 ℃ and 4000rpm for 8min, the thalli are collected, the supernatant is discarded, and 50ml of 1/2MS liquid culture medium is added for basic suspension for standby.

(3) Infection: taking out the leaf disc and petiole of flos Chrysanthemi which has been pre-cultured for 2 days in superclean bench, and placing in prepared OD₆₀₀And (3) infecting the agrobacterium liquid with the concentration of 0.5 for 10min, and continuously oscillating to ensure that the agrobacterium liquid is fully contacted with the explant.

(4) Co-culturing: after infection, taking out the leaf disc and the leafstalk, placing on sterile filter paper, sucking off redundant bacterial liquid, enabling the paraxial surface to face downwards, inoculating to a co-culture medium, and placing in the dark at 23 ℃ for culture for 2 d. The co-culture medium is an MS culture medium of 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

(5) And (3) delayed culture: in a clean bench, the leaf disc and the petiole which are co-cultured for 2 days are put into a carbenicillin solution of 400mg/L for 3 times, and then washed with sterile water for 2 times, and the bacteria solution and the explant are fully contacted by continuously shaking in the period. After cleaning, taking out the leaf disc and the leafstalk, placing on sterile filter paper to suck off redundant bacteria liquid and water, enabling the paraxial surface to face downwards, inoculating to a delayed culture medium, and placing in the dark at 23 ℃ for culture for 4 days. The delay culture medium is an MS culture medium containing 400mg/L carbenicillin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

(6) Screening and culturing: taking out the chrysanthemum leaf disc and the petiole which are cultured for 4 days in a delay way in a super-clean workbench, turning the chrysanthemum leaf disc and the petiole with the paraxial surface downward to a screening differentiation culture medium, and screening and culturing the chrysanthemum leaf disc and the petiole for 10 days at 23 ℃ in the dark. After 10 days, it was placed in an incubator with a light cycle of 16h/8h (light/dark), the temperature was still 23 ℃ during which the medium was changed every 2 weeks. The screening culture medium is MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6. At this stage, the explant can be further differentiated into buds and seedlings through the callus, or the explant is directly differentiated to emerge, and the emergence time is 20-50 days.

(7) Obtaining transgenic resistant plants: after screening culture, when the height of the resistant plant seedlings is 1-2cm, cutting the differentiated buds from the base part and inoculating the buds to a rooting culture medium for culture. The rooting culture medium is an MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6. The resistant bud starts to root after about 9 days, and grows into a complete plant after about 30 days. When the seedlings grow to a certain height, the seedlings can be moved into sandy soil for culture to obtain transgenic resistant plants, and then transgenic detection is carried out.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.

Example 1:

the method is carried out in Shandong agricultural university No. 7#518 laboratory of Taian city, Shandong province, provides a method for transforming chrysanthemum 'Nemao' by using agrobacterium tumefaciens-mediated exogenous genes, and mainly solves the problems of low explant regeneration and transformation efficiency and unstable transformation system in the chrysanthemum transgenic process. The exogenous gene transferred in the embodiment is a chrysanthemum SVP homologous gene, and correspondingly, the vector plasmid and the agrobacterium strain both contain the gene sequence.

The specific implementation steps are as follows:

(1) pre-culturing: selecting 25d seedling-old chrysanthemum 'shenma' aseptic seedling with fully-developed young leaf at the middle upper part as an explant, cutting the leaf into 5 mm-5 mm squares by using a scalpel in a clean bench, cutting a leaf stalk into 5mm in length, enabling the cut leaf and leaf stalk to face downwards proximally, inoculating the cut leaf and leaf stalk into a pre-culture medium, and placing the pre-culture medium in the dark at 23 ℃ for pre-culture for 2 d. The culture medium is an MS culture medium of 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

(2) Preparing bacterial liquid: the vector plasmid is pBI121, and the Agrobacterium tumefaciens strain is GV 3101. Taking out the prepared agrobacterium liquid from a refrigerator at the temperature of-80 ℃, placing the agrobacterium liquid in an ice box, and waiting for the agrobacterium liquid to melt. In a clean bench, 500. mu.l of the bacterial suspension was placed in a sterilized Erlenmeyer flask, 10ml of YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin was added thereto, and the mixture was cultured overnight at 28 ℃ with shaking in a shaking incubator at 200 rpm. The following morning, 90ml YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin was added to the flask, and shake culture was performed for 2-3 hours until OD₆₀₀Up to about 0.5. And subpackaging the shaken 100ml of bacterial liquid into two centrifuge tubes in a workbench, centrifuging at 4 ℃ and 4000rpm for 8min, collecting thalli, discarding supernatant, and adding 50ml of 1/2MS liquid culture medium for resuspension respectively to prepare the agrobacterium liquid for later use.

(3) Infection: taking out the leaf disc and petiole of flos Chrysanthemi which has been pre-cultured for 2 days in superclean bench, and placing in prepared OD₆₀₀And (3) infecting the agrobacterium liquid with the concentration of 0.5 for 10min, and continuously oscillating to ensure that the agrobacterium liquid is fully contacted with the explant.

(4) Co-culturing: after infection, taking out the leaf disc and the leafstalk, placing on sterile filter paper, sucking off redundant bacterial liquid, enabling the paraxial surface to face downwards, inoculating to a co-culture medium, and placing in the dark at 23 ℃ for culture for 2 d. The co-culture medium is an MS culture medium of 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

(5) And (3) delayed culture: in a clean bench, the leaf disc and the petiole which are co-cultured for 2 days are put into a carbenicillin solution of 400mg/L for 3 times, and then washed with sterile water for 2 times, and the bacteria solution and the explant are fully contacted by continuously shaking in the period. After cleaning, taking out the leaf disc and the leafstalk, placing on sterile filter paper to suck off redundant bacteria liquid and water, enabling the paraxial surface to face downwards, inoculating to a delayed culture medium, and placing in the dark at 23 ℃ for culture for 4 days. The delay culture medium is an MS culture medium containing 400mg/L carbenicillin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6.

(6) Screening and culturing: taking out the chrysanthemum leaf disc and the petiole which are cultured for 4 days in a delay way in a super-clean workbench, turning the chrysanthemum leaf disc and the petiole with the paraxial surface downward to a screening differentiation culture medium, and screening and culturing the chrysanthemum leaf disc and the petiole for 10 days at 23 ℃ in the dark. After 10 days, it was placed in an incubator with a light cycle of 16h/8h (light/dark), the temperature was still 23 ℃ during which the medium was changed every 2 weeks. The screening culture medium is MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 2mg/L6-BA, 0.5mg/L NAA, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6. At this stage, the explants are further differentiated to bud and form seedlings through the callus, and the seedling emergence time is 20-30 days.

(7) Obtaining transgenic resistant plants: after screening culture, when the height of the resistant plant seedlings is 1-2cm, cutting the differentiated buds from the base parts, and inoculating the buds to a rooting culture medium for culture. The rooting culture medium is an MS culture medium containing 400mg/L carbenicillin, 5mg/L kanamycin, 30g/L sucrose and 6g/L agar powder, and the pH value is 5.8-6. The resistant bud starts to root after about 9 days, and grows into a complete plant after about 30 days. And when the seedlings grow to a certain height, the seedlings can be moved into sandy soil for culture to obtain transgenic resistant plants, and PCR detection is carried out on the obtained transgenic resistant plants to identify whether the seedlings are transgenic positive seedlings.

According to statistics, the method of the embodiment has the callus induction rate of 98.9%, the germination rate of 30.3% and the seedling rate of 70.0%.

Comparative example 1:

the explant selection, infection mode and culture conditions were the same as in example 1 except that the types and concentrations of hormones added to the media of steps (1), (4), (5) and (6) were different. As shown in table 1 and table 2, the combination of 18 hormones including the present application was tested in total, and the callus induction rate, the germination rate and the seedling rate were counted:

table 1: influence of different hormone ratios of 6-BA and NAA on chrysanthemum callus induction and differentiation capacity

Table 2: influence of different hormone ratios of 6-BA and 2,4-D on chrysanthemum callus induction and differentiation capacity

In tables 1 and 2, the sprouting rate is the number of explants differentiated into adventitious buds/the number of explants induced to callus x 100%;

the seedling rate is the number of explants for seedling/the number of explants differentiated to adventitious buds multiplied by 100%.

As can be seen from Table 1, among 9 culture media with different concentration ratios using 6-BA and NAA as additives, when the concentration of 6-BA is 2mg/L, NAA and the concentration is 0.5mg/L, the culture media perform best overall performance, and the callus induction and differentiation of chrysanthemum explants are facilitated best.

As can be seen from Table 2, the adventitious buds differentiated from the transgenic chrysanthemum explants can not be successfully obtained by nine culture media with 6-BA and 2,4-D as additives and different hormone ratios, and the seedling rate is 0.

Therefore, the optimal callus induction and differentiation medium MS +2mg/L6-BA +0.5mg/LNAA of the chrysanthemum leaf can be determined.

Comparative example 2:

the infection mode, culture conditions and culture medium components are the same as those in example 1, and the difference is that in the step (1), explants are derived from sterile tissue culture seedlings with different seedling ages, and middle and upper leaves of sterile seedlings of 25d, 50d and 65d chrysanthemum 'Neuma' are respectively selected to perform agrobacterium transformation tests, so as to count the callus induction rate and the budding rate.

Table 3: influence of aseptic seedling age on the induction and differentiation of chrysanthemum 'shenma' callus

As shown in Table 3, the seedling age of the sterile seedlings increased, and the callus induction rate and the germination rate both showed a significant decline trend. Wherein, the sterile seedlings with the seedling age of 25 days have the highest healing rate and the highest germination rate which are respectively 98.0 percent and 24.5 percent and are far higher than the sterile seedlings with the seedling age of 50 days and 65 days. Therefore, the seedling age of aseptic seedlings has some influence on genetic transformation. The higher the seedling age of the leaf explant, the poorer the regeneration capability, and the negative correlation relationship between the leaf explant and the regeneration capability is shown. The chrysanthemum 20-25 days after inoculation is in the vigorous growth stage, the leaves are light green and are fully developed, and the optimal explant material taking time is the optimal time.

Comparative example 3:

the explant selection, infection mode, etc. were the same as in example 1, except that:

(1) pre-culturing: the illumination condition is 40-200 mu mol.m^-2s^-1，25℃；

(2) And (3) delayed culture: the illumination condition is 40-200 mu mol.m^-2s^-1，25℃；

(3) 10 days before screening culture: the illumination condition is 40-200 mu mol.m^-2s^-1，25℃。

According to statistics, the method of the comparative example 3 is adopted, the callus induction rate is 91.2%, the germination rate is 12.4%, and the seedling rate is 32.5%.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.