CN106811481B

CN106811481B - Genetic transformation method of sedum plants

Info

Publication number: CN106811481B
Application number: CN201710205829.9A
Authority: CN
Inventors: 徐文忠; 刘桓; 赵海霞
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2019-12-24
Anticipated expiration: 2037-03-31
Also published as: CN106811481A

Abstract

The invention discloses a genetic transformation method of sedum plants. The invention firstly protects a genetic transformation method of sedum plants, which sequentially comprises the following steps: (1) taking a stem section of the sedum plant, and inducing the formation of cluster buds to obtain a meristem block; (2) co-culturing the meristem block obtained in the step (1) with recombinant agrobacterium; the recombinant agrobacterium is obtained by introducing a plasmid with a target DNA molecule into starting agrobacterium; (3) taking the meristem block obtained in the step (2), and inducing germination; (4) taking the meristematic block of the shoot, inducing rooting. The method provided by the invention has the advantage of high genetic transformation rate, and has great application and popularization values for genetic transformation and genetic engineering research of sedum plants.

Description

Genetic transformation method of sedum plants

Technical Field

The invention relates to a genetic transformation method of sedum plants.

Background

Cadmium (Cd) has a severe toxic effect on all organisms. At present, the cadmium pollution form of soil in China is severe and tends to be aggravated year by year. Cadmium in soil has strong migration capacity, is easily absorbed by plants and enters human bodies through food chains, and seriously harms the health of people. Phytoremediation (phytoredation) is an ideal way for treating heavy metal pollution of cadmium and other soil. In recent years, more cadmium-enriched plants are researched such as arabidopsis thaliana, Thalassia serrulata, Sedum alfredii Hance and Sedum plumbizincicola. The cadmium hyperaccumulator is also a zinc hyperaccumulator. Compared with other cadmium super-enriched plants, the sedum plumbizincicola has the characteristics of strong cadmium enrichment capacity, large biomass and the like (Cao D, Zhang HZ, Wang YD, Zheng LN (2014) Accumulation and distribution characteristics of zinc and cadmium in the peraccumulator plant sedum plumbiguicola, B.environ.Contam. tox.93, 171-176.). In recent years, the research on the phytoremediation of the cadmium-contaminated soil by the rhodiola rosea is more, and the Cd content in the soil is reduced by 87.8% 7 years after the rhodiola rosea is planted (Deng L, Li Z, Wang J, Liu HY, Li N, Wu LH, Hu PJ, LuoYM, Christie P (2016) Long-term field phytoextraction of zinc/cadmium contained soil by seed plus binary anode under diffusion agricultural chemical strategies. int.J.Phytoendedia.18, 134-140.).

At present, the plant restoration of cadmium polluted soil by utilizing cadmium hyperaccumulator plants needs a large amount of investment for a long time, and large-scale commercial application is difficult to realize. The research on the Sedum plumbizincicola only stays in a small-scale test stage, and is difficult to be applied in practice. Therefore, the cultivation of the hyper-enriched engineering plant with economic benefits has important significance for the continuous phytoremediation of the cadmium-polluted soil. The molecular mechanism of cadmium enrichment and detoxification of super-enriched plants such as Sedum plumbizincicola and the like is deeply researched, and related genes of cadmium enrichment are excavated, so that theoretical basis and gene elements can be provided for obtaining engineering repair plants with cadmium super-enrichment. If the gene function of the Sedum plumbizincicola is to be researched, the premise is that the Sedum plumbizincicola can be subjected to gene manipulation. The establishment of a genetic transformation system of a hyper-enriched plant is a key means for revealing the mechanism of heavy metal absorption and transport molecules of the special habitat plants. Moreover, the Sedum plumbizincicola has high resistance and super-enrichment capacity only for cadmium and zinc, but does not have super-enrichment and high resistance for other heavy metals such as arsenic, copper, mercury and the like. If the genetic transformation of the Sedum plumbizincicola can be realized, the research of cadmium hyper-enrichment molecular mechanism and the excavation of related functional genes of plants can be greatly promoted, and Sedum plumbizincicola engineering plants with resistance and repair capability to multiple heavy metal complex pollution can be obtained.

Disclosure of Invention

The invention aims to provide a genetic transformation method of sedum plants.

The invention firstly protects a genetic transformation method of sedum plants, which sequentially comprises the following steps:

(1) taking a stem section of the sedum plant, and inducing the formation of cluster buds to obtain a meristem block;

(2) co-culturing the meristem block obtained in the step (1) with recombinant agrobacterium; the recombinant agrobacterium is obtained by introducing a plasmid with a target DNA molecule into starting agrobacterium;

(3) taking the meristem block obtained in the step (2), and inducing germination;

(4) taking the meristematic block of the budding, and inducing the rooting.

In the step (1), the induction of cluster bud formation is carried out on a bud induction medium; the bud induction medium is (a1), (a2) or (a3) as follows:

(a1) a medium containing 6-BA and NAA;

(a2) a medium containing 0.1 mg/L6-BA and 1.0mg/L NAA;

(a3) MS solid medium containing 0.1 mg/L6-BA and 1.0mg/L NAA.

In the step (1), the culture time for inducing the formation of the cluster buds may be 3 to 6 weeks.

In the step (1), in the process of inducing the formation of the cluster buds, subculture is carried out once per week, and a bud induction culture medium is adopted.

In step (1), the culture conditions for inducing the formation of the cluster buds may specifically be: at 25 ℃ 14 hours light/10 hours dark.

In the step (1), the preparation method of the stem section comprises the following specific steps: taking the stem of the sedum plant, sterilizing and longitudinally cutting into stem sections. Each stem segment has 1 stalk node. The length of each stem segment is about 1 cm. The sterilization method specifically comprises the following steps: washing with tap water → disinfecting with 75% (volume ratio) alcohol water solution for 30s → washing with sterile water for 3 times, then blotting with sterile filter paper → washing with 0.1g/100mLHgCl₂The aqueous solution was soaked for 6 minutes (with gentle shaking) → rinsed with sterile water at least 4 times and then blotted dry with sterile filter paper.

In the step (1), after the formation of the cluster buds is induced, stem tissue blocks with good growth conditions and dense cluster buds (40 per stem node) are selected, the leaves of the cluster buds are cut off, and the cluster buds are further cut into blocks (small blocks about 1 cm), namely the meristem blocks.

The plasmid has a hygromycin resistance gene thereon. The hygromycin resistance gene can be specifically a hygromycin phosphotransferase gene (HPT gene). The plasmid may specifically be the pSN1301 plasmid. The plasmid can be specifically a recombinant plasmid obtained by inserting a target DNA molecule into the multiple cloning site of the pSN1301 plasmid.

The starting agrobacterium is agrobacterium tumefaciens, and specifically can be agrobacterium tumefaciens C58.

In the step (2), the meristem blocks are infected with the infection solution and then co-cultured. The infection liquid is a liquid phase containing the recombinant agrobacterium. OD of the staining solution_600nmSpecifically, it may be 2.0. The infection liquid can be specifically a liquid phase obtained by resuspending the recombinant agrobacterium thallus by using an MS liquid culture medium containing 100 mu M AS. The infection can be specifically soaking at room temperature for 30min (shaking once every 2-3 min). In the step (2), the co-culture is carried out on a co-culture medium.

The co-cultivation medium may be specifically (d1), (d2) or (d3) as follows:

(d1) a medium comprising 6-BA, 2,4-D and AS;

(d2) a medium containing 0.5 mg/L6-BA, 1.5 mg/L2, 4-D and 100mg/L AS;

(d3) MS solid medium containing 0.5 mg/L6-BA, 1.5 mg/L2, 4-D and 100mg/L AS.

In the step (2), the co-cultivation conditions may specifically be: dark culture was carried out at 25 ℃ for 3 days.

In the step (3), inducing germination is carried out on a screening culture medium; the screening medium is any one of the following (b1) to (b 9):

(b1) a medium containing 6-BA and NAA;

(b2) a medium containing 0.1 mg/L6-BA and 1.0mg/L NAA;

(b3) MS solid culture medium containing 0.1 mg/L6-BA and 1.0mg/L NAA;

(b4) a medium comprising hygromycin, 6-BA and NAA;

(b5) a culture medium containing 20mg/L hygromycin, 0.1 mg/L6-BA and 1.0mg/L NAA;

(b6) MS solid culture medium containing 20mg/L hygromycin, 0.1 mg/L6-BA and 1.0mg/L NAA;

(b7) a culture medium containing hygromycin, cefamycin, 6-BA and NAA;

(b8) a culture medium containing 20mg/L hygromycin, 600mg/L cefamycin, 0.1 mg/L6-BA and 1.0mg/L NAA;

(b9) MS solid culture medium containing 20mg/L hygromycin, 600mg/L cephamycin, 0.1 mg/L6-BA and 1.0mg/L NAA.

In the step (3), the culture time for inducing the sprouting may be: culturing until the stem height of the seedling reaches 1 cm.

In the step (3), the culture conditions for inducing germination may specifically be: at 25 ℃ 14 hours light/10 hours dark.

In the step (3), in the process of inducing the buds, subculturing once a week, and adopting a screening culture medium.

In the step (4), the induced rooting is carried out on a root induction culture medium; the root induction medium is (c1), (c2) or (c3) as follows:

(c1) a medium comprising hygromycin and cefamycin;

(c2) a culture medium containing 30mg/L hygromycin and 600mg/L cefamycin;

(c3) MS solid medium containing 30mg/L hygromycin and 600mg/L cephamycin.

In the step (3), the culture conditions for inducing rooting may specifically be: at 25 ℃ 14 hours light/10 hours dark.

In the step (3), in the process of inducing rooting, subculturing once a week adopts a root induction culture medium.

The invention also provides a method for preparing a meristem mass of a sedum plant, comprising the following steps: taking a stem section of the sedum plant, and inducing the formation of cluster buds to obtain a meristem block; said inducing the formation of multiple shoots is carried out on any of said shoot-inducing media above. Induction ofThe culture period for cluster bud formation may be 3-6 weeks. In the process of inducing the formation of cluster buds, subculture is carried out once a week by using a bud induction culture medium. The culture conditions for inducing the formation of the cluster buds may specifically be: at 25 ℃ 14 hours light/10 hours dark. The preparation method of the stem section comprises the following steps: taking the stem of the sedum plant, sterilizing and longitudinally cutting into stem sections. Each stem segment has 1 stalk node. The length of each stem segment is about 1 cm. The sterilization method specifically comprises the following steps: washing with tap water → disinfecting with 75% (volume ratio) alcohol water solution for 30s → washing with sterile water for 3 times, then blotting with sterile filter paper → washing with 0.1g/100mLHgCl₂The aqueous solution was soaked for 6 minutes (with gentle shaking) → rinsed with sterile water at least 4 times and then blotted dry with sterile filter paper. After inducing the formation of cluster buds, selecting cluster buds with good growth condition and dense cluster buds (1)>40/stem node), cutting off the leaves of the cluster buds, and further cutting the cluster buds into blocks (small blocks about 1 cm), namely the meristem blocks.

The pH of any of the above media may specifically be 5.8.

The invention also provides a kit for genetic transformation of sedum plants, which comprises any one of the bud induction culture medium and any one of the screening culture medium. The kit also comprises the root induction culture medium. The kit further comprises the co-culture medium described in any one of the above. The kit may further comprise agrobacterium, specifically agrobacterium tumefaciens, more specifically agrobacterium tumefaciens C58. The kit can also comprise a starting plasmid, specifically an expression vector with a hygromycin resistance gene, more specifically an expression vector with a hygromycin phosphotransferase gene (HPT gene), and more specifically a pSN1301 plasmid.

The invention also provides a kit for preparing a meristem mass of a sedum plant, which comprises any one of the above bud induction culture media.

Any one of the above Crassulaceae plants is Sedum alfredii Hance or Sedum plumbizincicola. The sedum alfredii hance can be specifically a non-hyper-enriched ecological sedum alfredii hance.

The invention firstly induces the stem nodes of the Sedum plumbizincicola and the non-super-enriched Sedum alfredii Hance to generate a large number of cluster buds, so that a plurality of apical meristems are formed on the explant material, and the dense bud primordium is used as an agrobacterium infection object, thereby successfully establishing a genetic transformation system of the Sedum plant.

The method or the kit provided by the invention has the advantage of high genetic transformation rate, and has great application and popularization values for genetic transformation and genetic engineering research of sedum plants.

Drawings

FIG. 1 is a photograph showing the results of example 1.

FIG. 2 is a photograph showing the results of example 2.

FIG. 3 shows the survival results of the explants of example 2.

FIG. 4 is a schematic diagram of the elements of the pSN1301 plasmid.

FIG. 5 is a photograph of the stem tissue piece of Sedum kamtschaticum, a photograph during the culture on a screening medium, a photograph of a single meristem piece cultured on a root-inducing medium, and a photograph of GUS staining thereof in example 3.

FIG. 6 is a photograph of a plant of Sedum alfredii Hance cultured on a root-inducing medium for 3-5 weeks and a photograph of a plant cultured in a culture medium for 2 weeks.

FIG. 7 is a photograph showing the results of individual identifications of 5 lines of Sedum alfredii Hance.

FIG. 8 is a photograph of the stem segments of Sedum plumbizincicola in example 3 and the stem segments after culturing for various periods of time.

FIG. 9 is a photograph of rhodiola crenulata cultured on the screening medium for 2 weeks.

FIG. 10 is a photograph of rhodiola crenulata cultured on a root-inducing medium for 2 to 3 weeks.

FIG. 11 is a photograph of a single meristem mass of Sedum plumbizincicola cultured on root inducing medium for 3-5 weeks and a photograph of GUS staining thereof.

FIG. 12 is a photograph of a single bud of Sedum plumbizincicola and a photograph of GUS staining thereof.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The function of the cefuroxime is to prevent the massive growth of agrobacterium. The culture medium consists of 2 parts by volume of flower fertilizer, 1 part by volume of perlite and 1 part by volume of vermiculite. 6-BA is called 6-benzylamino adenine. NAA is known as 1-naphthylacetic acid. 2,4-D is called 2, 4-dichlorophenoxyacetic acid. AS is collectively known AS acetosyringone.

Sedum plumbizicola (Sedum plumbizicola): the Sedum plumbizincicola is a cadmium hyper-enrichment plant. Reference documents: wu LH, Liu YJ, Zhou SB, Guo FG, Bi D, Guo XH, Baker AJM, Smith JAC, Luo YM.2013.Sedum flumubicicola X.H. Guo et S.B. Zhou ex L.H.Wu (Crassulaceae) a new species from Zhoujiang Provice, China.plant systems and Evolution 299: 487-.

The Sedum alfredii used in the examples is non-hyper-enriching ecotype Sedum alfredii (non-hyperaccumulating ecotype), which is "Sedum alfredii (non-hyperaccumulating ecotype, NHE) in the reference: reference documents: peng, J.S., Ding, G.G., Meng, S.Y., Yi, H.Y., and Gong, J.M (2017). Enhanced metal tolerance coatings with a heterologous variation in SpMTL, a metal-like protein from the peraccumulator reduced cellulose. plant Cell Environment. doi:10.1111/pce.12929.

Example 1 optimization of Cluster bud Induction parameters

The method comprises the following steps of taking a stem section of sedum alfredii hance as an explant, and inducing cluster buds:

1. collecting fresh stems of sedum alfredii hance, and sequentially carrying out the following steps:

washing with tap water → disinfecting with 75% (volume ratio) alcohol water solution for 30s → washing with sterile water for 3 times, then blotting with sterile filter paper → washing with 0.1g/100mLHgCl₂The aqueous solution was soaked for 6 minutes (with gentle shaking) → rinsed with sterile water at least 4 times and then blotted dry with sterile filter paper.

2. After completion of step 1, the stem was cut longitudinally into stem segments of about 1cm in length (each stem segment having 1 stalk node).

3. And (3) placing the stem segments obtained in the step (2) on a bud induction culture medium for culturing for 3 weeks (subculture is carried out once a week, the same bud induction culture medium is adopted, and the culture conditions are that the temperature is 25 ℃, the light is 14 hours and the darkness is 10 hours), and then counting the number of buds generated by induction of each stem segment.

The bud induction medium is MS solid medium (pH5.8) containing 6-BA and NAA. The 6-BA concentration, NAA concentration in the different shoot induction media and the number of shoots induced per stem segment on average after 3 weeks of culture in step 3 are shown in Table 1and FIG. 1. Three replicates were set up, with at least 9 explants cultured per shoot induction medium in each replicate.

The results showed that the combination of 0.1 mg/L6-BA and 1.0mg/L NAA induced the formation of clumpy shoots most efficiently, with an average number of shoots induced per stem segment of 4.40. Thus, the combination of 0.1 mg/L6-BA and 1.0mg/L NAA was identified as the optimal hormone combination for inducing cluster shoot production.

TABLE 1 Effect of phytohormones on the Induction of clustered shoots of Sedum alfredii Hance

6-BA(mg/L)	NAA(mg/L)	Average number of shoots induced per stem segment
			0.1	0.1	1.30±0.70f
0.1	0.5	2.11±0.17cde
			0.1	1.0	4.40±0.74a
0.1	1.5	1.74±0.33de
			0.5	0.1	1.36±0.31e
0.5	0.5	2.50±0.29bcd
			0.5	1.0	2.83±0.07bc
0.5	1.5	2.37±0.27bcde
			1.0	0.1	3.37±0.70ab
1.0	0.5	3.06±1.12bc
			1.0	1.0	2.43±1.08bcd
1.0	1.5	3.41±0.57ab

The result of the above steps is the same as the result of the sedum alfredii hance.

Example 2 optimization of screening parameters

1. Same as in step 1 of example 1.

2. Same as in step 2 of example 1.

3. And (3) placing the stem segments obtained in the step (2) on a bud induction culture medium for culturing for 3 weeks (subculture is carried out once a week, the bud induction culture medium is adopted, and the culture conditions are 25 ℃, 14-hour light/10-hour dark).

The shoot induction medium was MS solid medium (pH5.8) containing 0.1mg/L of 6-BA and 1.0mg/L of NAA.

4. After step 3, the stem segments with the cluster buds are taken, the leaves are cut off, and the stem segments are placed on a screening medium for 3 weeks (the same screening medium is adopted once per week; the culture conditions are 25 ℃, 14 hours of light/10 hours of dark).

The screening medium was MS solid medium (pH5.8) containing antibiotics, 0.1 mg/L6-BA and 1.0mg/L NAA. When the antibiotic is hygromycin (Hyg), the concentration in the selection medium is 5mg/L, 10mg/L, 20mg/L, 30mg/L or 40 mg/L. When the antibiotic is kanamycin (Kan), the concentration in the screening medium is 50mg/L, 100mg/L, 150mg/L, 200mg/L or 250 mg/L.

In step 4, the photograph after 3 weeks of culture is shown in FIG. 2. The clustered shoots of the sedum alfredii hance are sensitive to hygromycin, and the clustered shoots die completely when the concentration of the hygromycin is more than 20 mg/L. The clustered shoots of sedum alfredii hance have strong resistance to kanamycin, and the growth state of the clustered shoots is still good when the concentration of kanamycin is 250 mg/L.

The survival of explants after 1, 2 and 3 weeks of culture in step 4 when the antibiotic was hygromycin is shown in FIG. 3. With increasing hygromycin concentration and longer treatment time, the survival rate of explants decreases dramatically. When the concentration of hygromycin reached 20mg/L, the survival rate after 1 week of culture was 29.63%, and the survival rates after 2 and 3 weeks of culture were 7.41% and 0, respectively.

Example 3 genetic transformation

The plant expression vector used was the pSN1301 plasmid. The pSN1301 plasmid is obtained by modifying on the basis of pCAMBIA1301 plasmid, and has GUS reporter gene driven by CaMV 35S promoter. The literature describing the pSN1301 plasmid is: guo, j.; dai, x.; xu, w.; ma, M.Overexpression GSH1and AsPCS1 minor ingredients the tolerance and accumulation of calcium and aromatic in Arabidopsis thaliana. Chemosphere 2008,72, 1020. 1026.). A schematic diagram of the elements of the pSN1301 plasmid is shown in FIG. 4. In fig. 4: LB stands for T-DNA insertion left border; RB represents the right border of the T-DNA insertion; HPT represents hygromycin phosphotransferase gene; 35S P represents the CaMV35 promoter; MCS represents the multiple cloning site; GUS represents a beta-glucuronidase gene.

Preparation of invasion dye solution

1. The pSN1301 plasmid was introduced into Agrobacterium tumefaciens C58 to obtain recombinant Agrobacterium.

2. The recombinant Agrobacterium was inoculated into 5ml of LB liquid medium containing 50mg/L rifampicin and 100mg/L kanamycin and cultured overnight at 28 ℃ at 200 rpm.

3. Taking the bacterial liquid obtained in the step 2, transferring to 40ml LB liquid culture medium containing 50mg/L rifampicin and 100mg/L kanamycin, culturing at 28 ℃ and 200rpm to OD_600nmThe value reaches 0.5-1.0.

4. After completion of step 3, the precipitate was collected by centrifugation at 4000g for 10 min.

5. Taking the precipitate obtained in the step 4, and re-suspending the precipitate with 5ml of MS liquid culture medium containing 100 mu MAS to obtain the infection solution (OD of the infection solution)_600nmIs 2.0).

Second, genetic transformation of Sedum alfredii Hance

1. Preparation of meristem Block

(1) Same as in step 1 of example 1.

(2) Same as in step 2 of example 1.

(3) And (3) placing the stem segments obtained in the step (2) on a bud induction culture medium for culturing for 3 weeks (subculture is carried out once a week and the bud induction culture medium is adopted, and the culture conditions are 25 ℃, 14-hour light/10-hour dark).

(4) And (3) after the step (3) is finished, selecting stem tissue blocks which have good growth conditions and are formed by densely clustering the cluster buds (>40 per stem node), cutting off leaves of the cluster buds, and further cutting the cluster buds into small blocks of about 1cm to obtain the meristem blocks.

A photograph of a tissue mass of a stem segment (before cutting off the leaves of the cluster buds) is shown in the left panel of FIG. 5A. A photograph of a tissue mass of a stem segment (after cutting off the leaves of the cluster buds) is shown in the right panel of FIG. 5A.

2. Genetic transformation

(1) Taking the meristem blocks prepared in the step 1 (three times of repeated tests are carried out, about 50 meristem blocks are taken each time), placing the meristem blocks in the infection solution prepared in the step one, soaking for 30min at room temperature (shaking once every 2-3 min), and then taking out and sucking the meristem blocks by using sterile filter paper.

(2) After the completion of step (1), the meristem blocks were taken and placed on a co-culture medium and cultured in the dark at 25 ℃ for 3 days.

Co-culture medium: MS solid medium (pH5.8) containing 0.5 mg/L6-BA, 1.5 mg/L2, 4-D and 100mg/L AS.

(3) And (3) after the step (2) is finished, taking the meristem block, soaking the meristem block in sterile water containing 300mg/L of cefuroxime for 30min under the aseptic condition, then washing the meristem block with the sterile water containing 300mg/L of cefuroxime for 3 to 4 times, and then putting the meristem block on sterile filter paper for blotting.

(4) After the step (3) is completed, the meristem blocks are taken and transferred to a screening medium for culture (once a week, the screening medium is adopted, and the culture conditions are 25 ℃, 14-hour light/10-hour dark).

Screening a culture medium: MS solid medium (pH5.8) containing 20mg/L hygromycin, 600mg/L cephamycin, 0.1 mg/L6-BA and 1.0mg/L NAA.

During the above culture, the meristematic block which has not completed genetic transformation becomes white or brown, and the meristematic block which has successfully completed genetic transformation maintains green buds.

The photograph after 2 weeks of culture is shown in the left of FIG. 5B, and the photograph after 6 weeks of culture is shown in the right of FIG. 5B.

The photograph of a single meristem block after 6 weeks of culture (left panel) and the photograph of its GUS staining (right panel) are shown in FIG. 5C.

After 6 weeks of culture, statistics were carried out on the number of meristematic blocks used for genetic transformation as denominator, and on average 57.67% of the meristematic blocks had hygromycin resistance, 82.35% of the meristematic blocks having hygromycin resistance were GUS staining positive (having GUS gene expression), and the genetic transformation efficiency was 47.49%.

(5) In the step (4), when the height of the seedling stem reaches 1cm, the meristem blocks are taken and transferred to a root induction culture medium for culture (the root induction culture medium is adopted once per week, and the culture conditions are 25 ℃, 14 hours of light/10 hours of darkness). Root induction medium: MS solid medium (pH5.8) containing 30mg/L hygromycin and 600mg/L cephamycin.

The photograph of a single meristem block cultured for 2-3 weeks (left panel) and the photograph of its GUS staining (right panel) are shown in FIG. 5D.

The photographs of the plants cultured for 3-5 weeks are shown in FIG. 6A.

And transferring the plant with well developed root into a culture medium for cultivation. The photograph after 2 weeks of cultivation is shown in FIG. 6B.

(6) From FIG. 5D, it was found that not all shoots on the same meristem mass could be stained blue, probably due to false positive shoots induced by part of the mosaic tissue. The clustered shoots aggregated in a single meristem mass were separated and each shoot was placed on a root-inducing medium for further culture (root-inducing medium was used for each subculture once a week; culture conditions: 25 ℃ C., 14 hours light/10 hours dark). False positive buds die after whitening, and the survival buds are all GUS positive staining (with GUS gene expression). Root induction medium: MS solid medium (pH5.8) containing 30mg/L hygromycin and 600mg/L cephamycin.

(7) A plurality of strains are obtained in the steps, and 5 strains are randomly selected for identification (rhodiola southeast without genetic transformation at the same period is used as a control, and a wild type is expressed by WT). The 5 lines are represented by L1, L2, L3, L4 and L5, respectively.

GUS staining was performed on roots, stems and leaves of the plants. The photograph is shown in FIG. 7A. The roots, stems and liquid of 5 strains are all positive in GUS staining, and the roots, stems and leaves of WT are all negative in GUS staining. The results showed that the GUS gene had integrated into the plant genome and was stably expressed in each line.

Genomic DNA of the plants was extracted and the GUS gene was identified by PCR (forward primer 5'-GGTGGGAAAGCGCGTTACAAG-3'; reverse primer 5'-CGGTGATACATATCCAGCCAT-3'). The results are shown in FIG. 7B. About 1.5kb of GUS gene fragment was detected in all 5 transgenic lines, and no corresponding GUS gene fragment was amplified in WT.

And (3) identifying the copy number of the GUS gene in the plant by using real-time PCR. The experiment was performed by using a standard curve method of absolute quantification using a single copy of GUS gene on the pSN1301 plasmid as an internal reference gene and genomic DNA (50. mu.g) of the plant as a template. The results are shown in FIG. 7C. The copy number of the GUS gene varied between strains, with the highest copy number in L5 (126 copies/. mu.g DNA) and the lowest copy number in L1 (46 copies/. mu.g DNA). The results showed that the GUS gene had integrated into the plant genome and was stably expressed in each line.

Third, genetic transformation of Sedum plumbizincicola

Replacing the sedum alfredii with the sedum plumbizinci, culturing for 6 weeks in the step (3) in the step 1, and the other steps are the same as the step two.

FIG. 8 shows four photographs from left to right of the stem segments obtained in step 1 (2), the stem segments obtained in step 1 (3) after 1 week of culture, the stem segments obtained in step 1 (3) after 2 weeks of culture, and the stem segments obtained in step 1 (3) after 3 weeks of culture.

FIG. 9 is a photograph of the left image of step 2 (4) after 2 weeks of culture, and a partially enlarged view of a meristem mass in the left image.

FIG. 10, the left is a photograph of the root induction medium cultured for 2 to 3 weeks in (5) of step 2, and the right is a partial enlarged view of a meristem mass in the left.

FIG. 11, photograph of a single meristem mass cultured on root inducing medium for 3-5 weeks in (5) of step 2 (left panel) and photograph of GUS staining thereof (right panel).

FIG. 12 is a photograph of a single shoot in step (6) (left panel) and GUS-stained photograph thereof (right panel).

In the step (4) of the step 2, statistics is carried out after 6 weeks of culture, and the genetic transformation efficiency of the Sedum plumbizincicola is about 10%.

SEQUENCE LISTING

<110> institute of plant of Chinese academy of sciences

<120> a genetic transformation method of crassulaceae plants

<130> GNCYX170702

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<400> 1

ggtgggaaag cgcgttacaa g 21

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<400> 2

cggtgataca tatccagcca t 21

Claims

1. A genetic transformation method of sedum plants sequentially comprises the following steps:

(1) taking a stem section of the sedum plant, inducing the formation of cluster buds, selecting a stem section tissue block which has good growth condition and is formed by densely clustering the cluster buds, cutting off the leaves of the cluster buds, and further cutting the cluster buds into blocks to obtain a meristem block;

(4) taking a meristematic block of the gemmation, and inducing the rooting;

in the step (1), the induction of cluster bud formation is carried out on a bud induction medium; the bud induction medium is (a1) or (a2) as follows: (a1) a medium containing 0.1 mg/L6-BA and 1.0mg/L NAA; (a2) MS solid culture medium containing 0.1 mg/L6-BA and 1.0mg/L NAA;

in the step (2), the plasmid has a hygromycin resistance gene;

in the step (3), inducing germination is carried out on a screening culture medium; the screening medium is any one of the following (b1) to (b 4): (b1) a culture medium containing 20mg/L hygromycin, 0.1 mg/L6-BA and 1.0mg/L NAA; (b2) MS solid culture medium containing 20mg/L hygromycin, 0.1 mg/L6-BA and 1.0mg/L NAA; (b3) a culture medium containing 20mg/L hygromycin, 600mg/L cefamycin, 0.1 mg/L6-BA and 1.0mg/L NAA; (b4) MS solid culture medium containing 20mg/L hygromycin, 600mg/L cephamycin, 0.1 mg/L6-BA and 1.0mg/L NAA.

2. The method of claim 1, wherein: the induced rooting is carried out on a root induction culture medium; the root induction medium is (c1), (c2) or (c3) as follows:

(c1) a medium comprising hygromycin and cefamycin;

(c2) a culture medium containing 30mg/L hygromycin and 600mg/L cefamycin;

(c3) MS solid medium containing 30mg/L hygromycin and 600mg/L cephamycin.

3. The method of claim 1 or 2, wherein: the Crassulaceae plant is Sedum alfredii or Sedum plumbizincicola.

4. A kit for genetic transformation of crassula plants comprising the shoot induction medium and the selection medium of claim 1.

5. The kit of claim 4, wherein: the kit further comprises a root induction medium as described in claim 2.

6. The kit of claim 4 or 5, wherein: the Crassulaceae plant is Sedum alfredii or Sedum plumbizincicola.