LU600835B1 - Method for efficient agrobacterium-mediated genetic transformation of euphorbia hirta - Google Patents

Abstract

The present invention discloses a method for efficient Agrobacterium-mediated genetic transformation of Euphorbia hirta, belonging to the field of plant genetic engineering. The method primarily includes the following steps: 1) activation and cultivation of Agrobacterium; 2) seed sterilization and material selection; 3) callus induction; 4) preparation of infection solution: cultivate Agrobacterium in YEB liquid medium, add it into MS liquid medium, adjust the bacterial liquid to OD600 = 0.3-0.9, then add 100 μmol/L acetosyringone and 100 μ mol/L MgCl2, and shake well; 5) infection; 6) co-cultivation; 7) washing and recovery cultivation; 8) screening, differentiation, and root induction; 9) plant regeneration and identification. Through the above steps, a successful genetic transformation system for Euphorbia hirta has been established, achieving a positive transformation rate of up to 76.19%. This invention holds significant importance and value for the rapid creation of transgenic Euphorbia hirta germplasm. (Fig.1)

Description

DESCRIPTION LU600835

METHOD FOR EFFICIENT AGROBACTERIUM-MEDIATED GENETIC

TRANSFORMATION OF EUPHORBIA HIRTA

TECHNICAL FIELD

The invention relates to the technical field of plant genetic engineering, in particular to a method for efficient Agrobacterium-mediated genetic transformation of Euphorbia hirta.

BACKGROUND

Euphorbia hirta is widely distributed in tropical and subtropical regions and contains a rich array of chemical components such as terpenoids, flavonoids, and alkaloids. It exhibits various pharmacological effects including anti-allergic, anti-anxiety, anti- inflammatory, sedative, analgesic, anti-malarial, antioxidant, and anti-cancer properties.

Recognized as an important medicinal plant, it is included in the Pharmacopoeia of the

People's Republic of China in 2010. Current research on Euphorbia hirta primarily focuses on the identification of basic biological characteristics, extraction of chemical components, and analysis of pharmacological activities. Cultivating high-yield, high- quality, and disease-resistant varieties of Euphorbia hirta is a pressing issue in current research. However, the scarcity of germplasm resources and the slow progress in breeding pose significant challenges. At present, genetic engineering technology is one of the most efficient methods for developing new plant varieties. Yet, there have been no reports on transgenic technology for Euphorbia hirta, nor any published articles on transgenic research related to this plant.

Plant genetic transformation methods can be categorized into three types based on their implementation. The first category involves vector-mediated transformation systems, where the target gene is linked to a vector plasmid, which then integrates the gene into the plant genome. Agrobacterium is commonly used as a medium in this method.

Agrobacterium is a type of Gram-negative bacterium prevalent in soil, primarily adhering to plant root surfaces and surviving on nutrients from the roots. It contains a plasmid known as the Ti plasmid, whose T-DNA can integrate into the plant genome and express, making it widely used as a genetic transformation medium.

The second category does not use any vector but directly introduces foreign genes/600835 into recipient cells through physical or chemical methods, including the gene gun method and electroporation. The gene gun method uses compressed air to propel gold particles coated with foreign genes into the cell nucleus, facilitating gene transfer. This method is advantageous due to its wide range of recipients, including callus, suspension cells, and immature embryos, but it is costly and has low transformation efficiency. The third category uses the plant's own reproductive cells as a medium for genetic transformation, including methods like the pollen tube pathway, ovule injection, and germ cell immersion.

The most common among these is the pollen tube pathway method, which utilizes the pollen tube formed during plant pollination to inject foreign DNA into recipient cells, achieving gene transformation through the development of the fertilized egg. This method is simple and does not rely on tissue culture technology, but it is labor-intensive and has low transformation efficiency. Each genetic transformation method has its own advantages and disadvantages, and achieving ideal transformation results requires selecting the appropriate method based on the experimental objectives and material differences. Currently, Agrobacterium-mediated genetic transformation is favored for its stable expression of foreign genes regardless of integration site, minimal disruption to the host genome stability, stable transformation efficiency, low cost, and simplicity of operation. With a deeper understanding of the mechanisms behind Agrobacterium- mediated transformation, the method continues to be refined. However, a systematic and efficient Agrobacterium-mediated transgenic method for Euphorbia hirta, an important

Chinese medicinal herb, has yet to be established.

SUMMARY

The objective of the present invention is to provide a method for efficient

Agrobacterium-mediated genetic transformation of Euphorbia hirta, addressing the issues present in the existing technology. By optimizing key factors such as the concentration of the infection solution, infection time, co-culture duration, and post-transformation callus resistance screening, a successful genetic transformation system for Euphorbia hirta has been established. This effectively resolves the inadequacies of the current

Agrobacterium-mediated genetic transformation system for Euphorbia hirta.

To achieve the above objective, the invention provides the following technick/600835 scheme: the invention provides a method for efficient Agrobacterium-mediated genetic transformation of Euphorbia hirta, including the following steps: using sterile Euphorbia hirta seedling leaves as explants, inoculating them onto a callus induction medium for callus induction; the callus induction medium includes the following components: MS liquid medium + Naphthaleneacetic Acid (NAA) + 6-

Benzylaminopurine (6-BA) + sucrose + agar; immersing the induced callus in an Agrobacterium infection solution, after infection, discarding the infection solution and transferring the callus to a co-culture medium for cultivation; the co-culture medium includes the following components: MS liquid medium + sucrose + agar + NAA + 6-BA + Acetosyringone (AS) + Timentin; after co-culturing, transferring the callus to a recovery medium for recovery cultivation, followed by screening and differentiation on a differentiation medium, and root induction on a rooting medium, after rooting, transplanting to obtain mature Euphorbia hirta plants; the recovery medium includes the following components: MS liquid medium + sucrose + agar + NAA + 6-BA + Timentin; the differentiation medium includes the following components: MS liquid medium + sucrose + agar powder + NAA + 6-BA + AS + Timentin + Kanamycin; the rooting medium includes the following components: MS liquid medium + Indole-3-butyric acid (IBA) + sucrose + agar + potato.

Preferably, the callus induction medium includes the following component concentrations: MS liquid medium + 0.5 mg/L NAA + 1.5 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar; the callus induction conditions are: 28°C in dark culture.

Preferably, the Agrobacterium infection solution is prepared by centrifuging the

Agrobacterium recombinant bacterial fermentation broth to obtain the bacterial pellet, resuspending it in MS liquid medium to an ODs00 of 0.3-0.9, and adding 100 pmol/L AS and 100 pmol/L magnesium chloride.

Preferably, the Agrobacterium recombinant strain is constructed by transferring the rubber tree HbCBF 1 gene into an expression vector to create a recombinant vector, which is then transformed into Agrobacterium. The nucleotide sequence of the HbCBF1 gene is as shown in SEQ ID NO: 5.

Preferably, the infection time is 20 min.

Preferably, the co-culture medium includes the following component concentrationsY800835

MS liquid medium + 30 g/L sucrose + 8 g/L agar + 0.5 mg/L NAA + 2 mg/L 6-BA + 100

MM/L AS + 300 mg/L Timentin; the co-culture conditions are: 22°C in dark culture for 3 days.

Preferably, the recovery medium includes the following component concentrations:

MS liquid medium + 30 g/L sucrose + 8 g/L agar + 0.5 mg/L NAA + 2 mg/L 6-BA + 300 mg/L Timentin; the differentiation medium includes the following component concentrations: MS liquid medium + 30 g/L sucrose + 0.8 g/L agar powder + 0.5 mg/L NAA + 2 mg/L 6-BA + 100 uM/L AS + 300 mg/L Timentin + 150 mg/L Kanamycin; the rooting medium includes the following component concentrations: MS liquid medium + 1.5 mg/L IBA + 30 g/L sucrose + 3.5 g/L agar + 20 g/L potato.

Preferably, after culturing on the recovery medium under 5000 lux weak light for 7 days, the callus is transferred to the differentiation medium for 14 days, followed by transfer to the rooting medium for root induction.

Preferably, when root induction begins and the seedlings reach 5-10 cm in height, they are transplanted into vermiculite and cultured indoors for 7 days, then moved outdoors to grow into mature Euphorbia hirta plants. The vermiculite is soaked with 1/2

MS solution before use, and during cultivation, the vermiculite is watered with tap water.

The 1/2 MS solution includes the following component concentrations: 1/2 MS liquid medium + 30 g/L sucrose.

Preferably, the sterile Euphorbia hirta seedlings are obtained by aseptically inoculating Euphorbia hirta seeds onto MS solid medium for cultivation.

The invention discloses the following technical effects: by using Euphorbia hirta callus as the transformation receptor and optimizing key factors such as bacterial infection concentration, infection time, and co-culture duration, as well as conducting resistance concentration screening on post-transformation callus, the invention successfully establishes an efficient Agrobacterium-mediated genetic transformation method for Euphorbia hirta. This method successfully obtains transformed

Euphorbia hirta seedlings, overcoming the current lack of genetic engineering techniques for Euphorbia hirta and achieving a breakthrough from zero to one.

The invention provides substantial experimental evidence for establishing a geneti&/600835 transformation system for Euphorbia hirta or other herbaceous plants in the

Euphorbiaceae family, offering significant value for rapidly creating transgenic Euphorbia hirta germplasm.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain the embodiments of the present invention or the technical scheme in the prior art more clearly, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without creative work for ordinary people in the field.

Fig. 1 is a PCR amplification electrophoregram of HbCBF1 gene; M is a standard

DNA molecule, and 1-4 are samples;

Fig. 2 is a map of HbCBF1 recombinant plasmid vector;

Fig. 3 shows the effect of different concentrations of antibiotics (kanamycin) on the browning rate of callus;

Fig. 4 shows the effect of different concentrations of Agrobacterium GV3101 on callus transformation efficiency;

Fig. 5 shows the effect of different infection time of Agrobacterium GV3101 on callus transformation efficiency;

Fig. 6 shows the influence of different co-culture time on callus transformation efficiency;

Fig. 7 is the GUS staining identification diagram of some transgenic TO plants of

HbCBF1; the first row is negative for GUS staining, and the second row is positive for

GUS staining;

Fig. 8 is an electrophoregram of PCR identification of partial transgenic TO plants of

HbCBF1; Lane 1-3 are 2k Marker, wild-type Euphorbia hirta samples and negative control (ddH20) respectively, and lanes 4-18 are 1-15 TO generation single plant samples of

Euphorbia hirta.

DESCRIPTION OF THE INVENTION LUB00835

A number of exemplary embodiments of the present invention will now be described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

It should be understood that the terminology described in the present invention is only for describing specific embodiments and is not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

Unless otherwise specified, 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 invention relates. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the contents of this specification shall prevail.

It is obvious to those skilled in the art that many improvements and changes can be made to the specific embodiments of the present invention without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the invention. The description and embodiment of that present invention are exemplary only.

The terms "including", "comprising", "having" and "containing" used in this article are all open terms, which means including but not limited to.

Embodiment 1

A method for efficient Agrobacterium-mediated genetic transformation of Euphorbia hirta includes the following steps: 1) Activation and Cultivation of Agrobacterium

Extract RNA from rubber trees and reverse transcribe it into cDNA. Use this cDNA/600835 as a template to amplify the HbCBF1 gene from rubber trees using specific primers (SEQ

ID NO.1 and SEQ ID NO.2). The result is shown in Fig. 1: the amplified HbCBF1 fragment size is 696 bp (SEQ ID NO.5). Construct the pCAMBIA2301-HbCBF1 vector using homologous recombination, as shown in Fig. 2, the recombinant plasmid vector size is approximately 13,664 bp; transform the correctly sequenced recombinant plasmid into

Agrobacterium GV3101 (pSoup-p19) and inoculate it onto YEB solid medium, and culture at 28°C for three days. Pick single colonies from the transformation plate for PCR verification and inoculate them into 2 mL of YEB liquid medium. Activate at 28°C and 200 rpm until ODeoo = 0.7, transfer the culture to fresh YEB liquid medium at a 1:20 dilution, placed in a 200 mL triangular flask, and expanded under the above conditions. When

ODeoo=0 .8, it is introduced into a 50 mL centrifuge tube, and centrifuged at 5000 g/min to collect bacteria; YEB liquid = 1: 20 was diluted, the YEB liquid medium contains: 0.5% beef extract, 0.1% yeast extract, 0.5% peptone, 0.5% sucrose, 0.4% magnesium sulfate, mg/L rifampicin, and kanamycin at concentrations of 10 mg/L, 30 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, or 300 mg/L.

PCR verification system: Primer-F 0.5 pL, Primer-R 0.5 pL, cDNA 1 pL (200 ng/uL), 2x Rapid Taq Master Mix 25 pL, ddH20 23 pL. Program: 98°C for 5 min, 98°C for 30 s, 55°C for 10 s, 72°C for 1 min (To 2 Steps, 35 cycles), 72°C for 1 min, and hold at 4°C. 2) Seed Sterilization and Material Selection

Take 30-50 dried wild-type Euphorbia hirta seeds, remove surface impurities, and place them in a 1.5 mL centrifuge tube. Add 50% chlorine-containing disinfectant and sterilize for 5 min. Then in a sterile environment, discard the disinfectant and rinse the seeds with sterile water 5-7 times until the water is clear. Last, use a micropipette to transfer the seeds onto MS medium and culture in a constant-temperature light incubator at 28°C and 10,000 lux (16 h light/8 h dark). Healthy seeds will germinate within 3-7 days.

After the sterile seedlings grow for a period, cut their leaves into approximately 0.5 cm x 0.5 cm pieces using a sterilized scalpel for subsequent infection steps. 3) Callus Induction

Inoculate the cut leaf pieces from step 2) onto callus induction medium (MS liquid medium + 0.5 mg/L NAA + 1.5 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar). Inoculate 10 explants per dish and culture in a constant temperature illumination incubator at 28°C in the dark.

Observe callus induction every 7 days and subculture every 14 days; set kanamycly/600835 concentrations at 10, 30, 50, 100, 150, and 300 mg/L, transfer vigorously growing callus cultured on induction medium for about 14 days to MS medium containing kanamycin, observe callus growth every 7 days and subculture every 14 days, after 30 days, record callus growth and browning mortality.

Results are shown in Fig. 3: at 10 mg/L kanamycin, the browning rate is only 7%, as kanamycin concentration increases, the browning rate rises, reaching 100% at 150 mg/L.

To achieve optimal screening and cost efficiency, 150 mg/L is selected as the critical concentration for transgenic callus screening. 4) Preparation of Infection Solution

Centrifuge the bacterial pellet from step 1) at 5000 rpm and 4°C for 10 min. Discard the supernatant and resuspend the pellet in MS liquid medium to adjust OD600 to 0.3, 0.6, and 0.9. Add 100 umol/L acetosyringone and 100 pmol/L magnesium chloride, mix well, and let stand in the dark for 1 h to prepare the infection solution. 5) Infection

Immerse the 0.5 x 0.5 cm leaf pieces from step 2) in the Agrobacterium infection solution from step 4. Incubate at 30°C and 100 rpm for 20 min to ensure uniform infection.

In order to explore the influence of the concentration of bacterial liquid on the transformation effect of the calli of Euphorbia hirta, the invention sets three agrobacteria with different concentrations as in step 4) to carry out treatment experiments. Results in

Fig. 4 show that the ODeoo = 0.6 group had the highest transformation efficiency and GUS expression, with most positive callus stained dark blue. However, the transformation efficiency is low in the treatment groups of ODs00=0.3 and ODs00=0.9, the number of GUS- positive calli in the treatment group of ODeoo=0.3 is less, and the culture medium is polluted by excessive propagation of Agrobacterium in the treatment group of ODs00=0.9, and the number of surviving calli is less. Thus, ODeoo = 0.6 is the optimal concentration; at the same time, the infection time of Agrobacterium is also one of the key factors affecting the transformation effect. The infection time should be long enough to make the callus fully contact with the bacterial liquid. The infection time is another critical factor.

Insufficient time leads to low transformation efficiency, while excessive time causes callus death. Three treatments (10 min, 20 min and 30min) are set up in the experiment.

Results in Fig. 5 show that when Agrobacterium with ODeoo=0.6 is selected 16600835 different time, the genetic transformation efficiency of the three treatments was similar, all between 30%-40%, after one-way analysis of variance, the transformation efficiency is significantly highest when the infection time is 20 min, so it is confirmed that 20 min is the best treatment time of Agrobacterium. 6) Co-Cultivation

After infection, discard the infection solution and blot excess liquid from the material with sterile filter paper. Transfer the material to co-culture medium and incubate in the dark at 22°C. The co-culture medium contains: MS liquid medium + 30 g/L sucrose + 8 g/L agar + 0.5 mg/L NAA + 2 mg/L 6-BA + 100 uM/L acetosyringone + 300 mg/L Timentin, pH = 5.8.

Agrobacterium needs to contact with plant tissues for a period of time to realize the introduction and integration of foreign genes, so it is necessary to co-culture the infected callus with Agrobacterium in the dark, which is generally not less than 16 h, but it can't last too long, too long co-culture time will lead to the excessive reproduction of

Agrobacterium and the death of plant tissues. Therefore, choosing the appropriate co- culture time is one of the important factors affecting the transformation efficiency. Three co-culture times (2 d, 3 d and 4 d) are set for the experiment. Results as shown in Fig. 6, the transformation efficiency of the treatment group co-cultured for 2 d and 3 d is similar, the transformation efficiency of the treatment group co-cultured for 3 d is slightly higher than that of the treatment group co-cultured for 2 d, and their GUS staining area is similar, while the transformation efficiency of the treatment group co-cultured for 4 d is low. The observation of experimental materials showed that the callus in the co-culture group for 4 d overflowed in a large area, which led to the death of most of the callus, indicating that the risk of Agrobacterium contamination increased with the extension of co-culture time.

Therefore, co-culture for 3 d is the best time. 7) Washing and Recovery Cultivation

After co-cultivation, wash the materials with sterile ddH2O, and then soak them in distilled water containing Timentin for 8 min at 30°C and 100 rpm to inhibit excessive

Agrobacterium proliferation. Blot the surface liquid with sterile filter paper and transfer the materials to recovery medium for weak light (5000 lux) recovery cultivation for 7 days; the recovery medium contains: MS liquid medium + 30 g/L sucrose + 8 g/L agar + 0.5 mg/L

NAA + 2 mg/L 6-BA + 300 mg/L Timentin, pH = 5.8. 8) Screening, Differentiation, and Root Induction

After the co-culture of the transformed callus in step 7) is completed, it is resumed/600835 on the culture medium containing 300 mg/L of temetin for 7 d, and the callus after the resumption of culture is completely transferred to the differentiation medium containing kanamycin in clean bench, and screened under the conditions of light intensity of 5000 lux and temperature of 26°C, and then transferred to the rooting medium for induction and rooting after 14 d. During screening, decide whether to further screen or subculture based on bud formation, if many buds form, continue screening; if few buds form, proceed directly to subculture. Adjust the kanamycin concentration in the bud medium to a final concentration of 50 mg/L; if buds are few, remove GA and Timentin; if buds are abundant, maintain the medium and only adjust kanamycin concentration.

Where, differentiation medium: MS liquid medium + 30 g/L sucrose + 0.8 g/L agar + 0.5 mg/L NAA + 2 mg/L 6-BA + 100 uM/L acetosyringone + 300 mg/L Timentin + 150 mg/L kanamycin; rooting medium: MS liquid medium + 1.5 mg/L IBA + 30 g/L sucrose + 3.5 g/L agar + 20 g/L potato powder. 9) Seedling Formation and Identification

When differentiated buds grow to 2-3 cm, cut them and transfer to rooting medium for root induction. Once seedlings reach about 8 cm, wash off the medium from the roots and transplant into vermiculite soaked with 1/2 MS solution. Water with tap water and cultivate indoors for 7 days before moving outdoors, perform PCR identification of transgenic plants; where, the 1/2 MS solution contains: 1/2 MS liquid medium + 30 g/L sucrose, adjusted to pH = 5.8 with sterile ddH2O; for GUS staining, place transgenic plant leaves or callus in 1.5 mL centrifuge tubes. Use wild-type as a negative control and previously obtained positive transgenic tobacco leaves as a positive control. Add prepared GUS staining solution to submerge the plant tissue, incubate in the dark at 37°C for 12 h, then discard the staining solution and add 75% ethanol for decolorization.

Observe and record staining results after complete decolorization. Due to growth rate variations among TO-generation Euphorbia hirta individuals, mature transformed plants are obtained in two batches, totaling 42 plants. Randomly selected 20 plants are stained, with results shown in Fig. 7: 12 plants are GUS-positive, yielding a 60% positive rate.

At the same time, the invention using wild-type and water as negative and blank controls, respectively, perform PCR amplification of the transformed plant genomic DNA with specific primers for the target gene (SEQ ID NO.3 and SEQ ID NO.4). The target fragment size is 539 bp.

As shown in Fig. 8, no amplification bands are observed in wild-type and negative600835 controls (ddH20), while clear target bands of the correct size are amplified in samples 1- 9 and 11-15. This confirms the integration of the HbCBF1 gene into the Euphorbia hirta genome. Statistical analysis shows that 32 plants produced the target band, with a positive rate of approximately 76.19%.

The sequence of SEQ ID NO.1 is: 5'-taagctagaagegtgtctagaATGGATGTTTTCCCTCAATATTCTG-3; the sequence of SEQ ID NO.2 is: 5'-gtacttatattttttactagt TATAGAAAAACTCCATAGTGACACATCAG-3; the sequence of SEQ ID NO.3 is:

HbCBF1-F: 5-CGCAAGACCCTTCCTCTA-3’;

The sequence of SEQ ID NO .4 is:

HbCBF1-R: 5-GCTGCTGCCTTCCTAATG-3’;

The sequence of SEQ ID NO.5 is:

ATGGATGTTTTCCCTCAATATTCTGACTCACTTCCTTTTGCCACTCATTCCTGC

TCACTTCACTACCCAGAATCTTCTACATTGTCTGATACCTGCAGTGCTCTTCGTGCG

AACCTTTCCGATGAGGAAGTCTTGTTAGCTTCCAGCTATCCCAAGAAGCGCGCAG

GCAGAAAGAAGTTCCGGGAGACACGCCATCCTATTTATCGTGGGGTCCGTCGGAG

GAACTCCGGGAAGTGGGTTTGTGAAATTAGAGAACCCAACAAGAAATCAAGAATAT

GGTTGGGGACTTTCCCCACGGAGGAAATGGCTGCTAGGGCCCACGACGTGGCGG

CGCTGGCTCTCAGAGGACGGTCTGCCTGCTTGAATTTTGCGGACTCGTCATGGCG

ACTTCCTGTACCTGCGTCCAGAGAAGCAAAGGACATTAGGAAGGCAGCAGCAGAG

GCGGCGATGGCGTTCCAGCCGGAGGGGACAGAAGGGTTTTCTGGAGAATTAAAA

CAGGAAAATAAGTGGACGACAGAGTCAGCGCCGGAGGATGTGTTTTATATGGACG

AAGAGGCGGTTTTTGCAATGCCGGGACTACTGGCATCTATGGCAGAAGGGATGCT

ACTTCCTCCACCTCAGTGCGTTGCAGGGAGCGGAGGAGAAGACGGAGAAATGGAT

GCAGCTGATGTGTCAC TATGGAGTTTTTCTATAA.

The above-mentioned embodiments only describe the preferred mode of the invention, and do not limit the scope of the invention. Under the premise of not departing from the design spirit of the invention, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the protection scope determined by the claims of the invention.

SEQUENCE LISTING 0600835

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<INSDSeq_division>PAT</INSDSeq_division> LU600835 <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..694</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q10"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>atggatgtittccctcaatattetgactcacttccettttgccactcattectgctcacttcactacccagaatettctacattg tctgatacctgcagtgctcticgtgecgaacctitccgatgaggaagtctigttagceticcagctatcccaagaagcgcegcaggcagaaagaagttceg ggagacacgccatcctatttatcgtggggtcegtcggaggaactccgggaagtgggtitgtgaaattagagaacccaacaagaaatcaagaatat ggttggggactttccccacggaggaaatggctgctagggceccacgacgtggeggcegctggctctcagaggacggtcetgcectgcttgaatittgecgga ctcgtcatggcegacttcctgtacctgegtccagagaagcaaaggacattaggaaggceagcagceagaggceggcgatggcegticcagecggaggg gacagaagggttitctggagaattaaaacaggaaaataagtggacgacagagtcagcgccggaggatgtgttttatatggacgaagaggceggtttt tgcaatgccgggactactggcatctatggcagaagggatgctacttectccacctcagtgcgttgcagggagcggaggagaagacggagaaatg gatgcagctgatgtgtcactatggagttttictataa</INSDSeq_sequence> </INSDSeq> </SequenceData> </ST26SequenceListing

Claims (9)

CLAIMS LU600835

1. A method for efficient Agrobacterium-mediated genetic transformation of Euphorbia hirta, cmprising the following steps: using sterile Euphorbia hirta seedling leaves as explants, inoculate the explants onto a callus induction medium for callus induction; the callus induction medium comprises the following components: MS liquid medium + Naphthaleneacetic Acid (NAA) + 6- Benzylaminopurine (6-BA) + sucrose + agar; immersing the induced callus in an Agrobacterium infection solution, after infection, discarding the infection solution and transferring the callus to a co-culture medium for cultivation; the co-culture medium comprises the following components: MS liquid medium + sucrose + agar + NAA + 6-BA + Acetosyringone (AS) + Timentin; after co-cultivation, transferring the callus to a recovery medium for recovery cultivation, followed by screening and differentiation on a differentiation medium, and root induction on a rooting medium, after rooting, transplanting to obtain mature Euphorbia hirta plants; the recovery medium comprises the following components: MS liquid medium + sucrose + agar + NAA + 6-BA + Timentin; the differentiation medium comprises the following components: MS liquid medium + sucrose + agar powder + NAA + 6-BA + AS + Timentin + Kanamycin; the rooting medium comprises the following components: MS liquid medium + Indole-3-butyric acid (IBA) + sucrose + agar + potato; the Agrobacterium infection solution is prepared by centrifuging the Agrobacterium recombinant bacterial fermentation broth to obtain the bacterial pellet, resuspending the bacterial pellet in MS liquid medium to an ODeoo of 0.3-0.9, and adding 100 pmol/L AS and 100 pmol/L magnesium chloride.

2. The method according to claim 1, characterized in that the callus induction medium comprises the following component concentrations: MS liquid medium + 0.5 mg/L NAA +

1.5 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar; the callus induction conditions are: 28°C in dark culture.

3. The method according to claim 1, characterized in that the Agrobacteriutt600885 recombinant strain is constructed by transferring the rubber tree HbCBF1 gene into an expression vector to create a recombinant vector, the recombinant vector is then transformed into Agrobacterium; the nucleotide sequence of the HbCBF1 gene is as shown in SEQ ID NO: 5.

4. The method according to claim 1, characterized in that the infection time is 20 min.

5. The method according to claim 1, characterized in that the co-culture medium comprises the following component concentrations: MS liquid medium + 30 g/L sucrose + 8 g/L agar + 0.5 mg/L NAA + 2 mg/L 6-BA + 100 uM/L AS + 300 mg/L Timentin; the co-culture conditions are: 22°C in dark culture for 3 days.

6. The method according to claim 1, characterized in that the recovery medium comprises the following component concentrations: MS liquid medium + 30 g/L sucrose + 8 g/L agar + 0.5 mg/L NAA + 2 mg/L 6-BA + 300 mg/L Timentin; the differentiation medium comprises the following component concentrations: MS liquid medium + 30 g/L sucrose + 0.8 g/L agar powder + 0.5 mg/L NAA + 2 mg/L 6-BA + 100 uM/L AS + 300 mg/L Timentin + 150 mg/L Kanamycin; the rooting medium comprises the following component concentrations: MS liquid medium + 1.5 mg/L IBA + 30 g/L sucrose + 3.5 g/L agar + 20 g/L potato.

7. The method according to claim 1, characterized in that after 7 days of weak light (5000 lux) cultivation on the recovery medium, the callus is transferred to the differentiation medium for 14 days, followed by transfer to the rooting medium for root induction.

8. The method according to claim 1, characterized in that when root induction begins and the seedlings reach 5-10 cm in height, they are transplanted into vermiculite and cultured indoors for 7 days, then moved outdoors to grow into mature Euphorbia hirta plants; the vermiculite is soaked with 1/2 MS solution before use, and during cultivation, the vermiculite is watered with tap water, the 1/2 MS solution comprises the following component concentrations: 1/2 MS liquid medium + 30 g/L sucrose.

9. The method according to claim 1, characterized in that the sterile Euphorbia hirt&1600835 seedlings are obtained by aseptically inoculating Euphorbia hirta seeds onto MS solid medium for cultivation.