NL2033006B1 - High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation - Google Patents

High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation Download PDF

Info

Publication number
NL2033006B1
NL2033006B1 NL2033006A NL2033006A NL2033006B1 NL 2033006 B1 NL2033006 B1 NL 2033006B1 NL 2033006 A NL2033006 A NL 2033006A NL 2033006 A NL2033006 A NL 2033006A NL 2033006 B1 NL2033006 B1 NL 2033006B1
Authority
NL
Netherlands
Prior art keywords
medium
culture
leaves
leaf
vitro
Prior art date
Application number
NL2033006A
Other languages
Dutch (nl)
Inventor
Tang Li
Li Xiaolong
Liu Yongqiang
Cheng Yuxing
Luo Yinghan
Xu Shiqiong
Li Jiale
Wang Yiping
Ye Yuntian
Tang Haoru
Original Assignee
Univ Sichuan Agricultural
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Univ Sichuan Agricultural filed Critical Univ Sichuan Agricultural
Application granted granted Critical
Publication of NL2033006B1 publication Critical patent/NL2033006B1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
    • A01H6/7409—Fragaria, i.e. strawberries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Abstract

A high-frequency regeneration method of Fragarl'a ananassa in vitro leaves and an application method thereof in genetic transformation are provided. The specific steps 5 are as follows: step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragarl'a ananassa grown on a l/2MS medium well as materials and transferring the aseptic seedlings to a preculture medium for preculture, step 2: material cutting and sampling, in sterile environment, taking out the strawberry materials grown on the preculture medium in the step 1, cutting off the leaves thereof 10 and placing into a wet culture dish, cutting and separating stem base tissues of the leaves into about 0.5 cm >< 0.5 cm leaf discs, step 3: differentiation culture, paving the leaf discs obtained in the step 2 on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium.

Description

HIGH-FREQUENCY REGENERATION METHOD OF FRAGARIA ANANASSA
IN VITRO LEAVES AND APPLICATION METHOD THEREOF IN GENETIC
TRANSFORMATION
TECHNICAL FIELD
[01] The present invention relates to the field of plant genetic engineering, and in particular to a high-frequency regeneration method of Fragaria ananassa in vitro leaves and an application method thereof in genetic transformation.
BACKGROUND ART
[02] Strawberry is a kind of plant belonging to Fragaria I. genus, Rosaceae. Due to delicious fruit and abundant vitamins and other nutrients, strawberry is popular among consumers. Therefore, strawberry has also been widely cultivated as an excellent horticultural plant. With the continuous promotion of strawberry cultivation techniques and the continuous expansion of strawberry cultivation area, the demand for excellent varieties of strawberry is also ever-increasing. Therefore, the breeding of preferable strawberry varieties is the inner demand of strawberry industry development, and is one of the main tasks in the strawberry scientific research. Wild strawberry (Fragaria vesca) is used as the major material for strawberry breeding research.
Fragaria ananassa 1s the most widely cultivated strawberry species in agricultural production. However, Fragaria vesca is a diploid strawberry species, while Fragaria ananassa 1s an octoploid strawberry species. The difference in genetic background between the two species results in that the research results of gene function of
Fragaria vesca cannot be directly applied in the variety breeding of Fragaria ananassa. On the other hand, with the development of molecular biology and bioinformatics, important breakthroughs have been also achieved in the genomic research of Fragaria ananassa. The publication of genomic information of Fragaria ananassa has pushed the research of strawberry to the post-genome era, making it possible to directly study the gene function of Fragaria ananassa. The verification on biological functions of one or more strawberry genes by studies can further provide theoretical basis for strawberry variety breeding. Genetic transformation of strawberry is the basis for the studies on its gene function. Genetic transformation of strawberry, as an important biotechnology, is combined with molecular biological technology (e.g., gene editing technology) to specifically change the genome of strawberry, so as to systematically study the biological functions of specific genes and their effects on agronomic traits of strawberry. Therefore, the development and establishment of a genetic transformation system with Fragaria ananassa as a Receptor system are important for the studies on the gene function of Fragaria ananassa and direct application thereof in the new varieties breeding of Fragaria ananassa.
[03] Agrobacterium-mediated genetic transformation is the main method to obtain transgenic strawberry plants. Genetic transformation system of Fragaria ananassa has been gradually improved in recent years. In this system, Fragaria ananassa in vitro leaves are mainly used as explants as a transformation receptor. The nucleic acid fragments to be verified are transferred into plant cells by means of the transformation of explant callus via Agrobacterium infection, and the genetically transformed plants are formed relying on totipotent differentiation of plant cells. However, there are still many problems in such a system, such as diversity and complexity of culture media, difficulty in regeneration of the transformed callus, and shortage of the obtained genetically transformed plants. The regeneration efficiency of the Fragaria ananassa leaves directly affects the transgenic efficiency, and is the key factor to determine the success of strawberry genetic transformation or not. Therefore, there is an urgent need to establish a high-frequency regeneration system of Fragaria ananassa in vitro leaves capable of being applied in genetic transformation of Fragaria ananassa.
SUMMARY
[04] The objective of the present invention is to overcome the shortcomings existing in the prior art and to propose a high-frequency regeneration method of
Fragaria ananassa in vitro leaves and an application method thereof in genetic transformation.
[05] To achieve the above objective, the present invention adopts the following technical solutions:
[06] a method for high-frequency regeneration of Fragaria ananassa in vitro leaves includes the following specific steps:
[07] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 3-5 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 18-24 d for further use;
[08] step 2: material cutting and sampling, in sterile environment, taking out the strawberry aseptic seedlings grown on the strawberry material preculture medium in the step 1, cutting off the leaves thereof and placing into a wet culture dish, cutting leaflet base of the strawberry leaves into 0.5 cm x 0.5 cm leaf discs with a sharp scalpel or special puncher;
[09] step 3: differentiation culture, paving the leaf discs obtained in the step 2 on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs 1s in sufficient contact with the regeneration medium; and culturing the paved culture medium for 21 d in a culture room, then observing adventitious buds obtained by differentiation, and continuously culturing for 21 d to obtain a large number of adventitious buds.
[10] The present invention further provides an application method of the high-frequency regeneration method of Fragaria ananassa in vitro leaves in genetic transformation, and specific steps are as follows:
[11] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 3-5 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 18-24 d; then taking out the strawberry materials grown on the strawberry preculture medium, cutting oft the leaves and placing into a wet culture dish, and cutting leaflet of the strawberry leaves into 0.5 cm x 0.5 cm leaf discs with a sharp scalpel or special puncher; paving the leaf discs on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and preculturing the paved culture medium for 3 d in a culture room;
[12] step 2: strain preparation: transforming a plasmid vector into a GV310I
Agrobacterium engineering strain by a heat shock method, and obtaining a GV3101 monoclonal Agrobacterium containing the correct plasmid vector through solid medium screening and PCR means; inoculating the monoclonal Agrobacterium onto a 1 ml YEP medium containing antibiotics, and performing shaking culture for 2 d at 27-30°C and 200 rpm until the medium is seen turbid; pipetting the turbid bacterial solution into a 25 ml YEP medium in sterile environment for shaking culture, wherein the culture environment is 27°C and the shaking speed is 200 rpm; adding 20ul acetosyringone solution (100 mM) when the bacterial solution is cultured to OD600 = 0.1-0.15, and continuously culturing the bacterial solution for 3 h and then taking out, and performing rotary centrifugation for 5 min at an accelerated speed of 2000 g; discarding supernatant and adding to a 25 ml MS liquid medium containing 100 uM acetosyringone, and gently shaking to obtain suspended bacterial cells for further use;
[13] step 3: material infection: taking out the leaf discs in the step 1 and placing into a wet culture dish, pouring the bacterial solution prepared in the step 2 into the culture dish containing the leaf discs and shaking gently such that the bacterial solution and the leaf discs are fully contacted and mixed for infection, where the infection time is 30-40 min; shaking the culture dish once every other 5 min during the infection process until the end of infection, and transferring the leaves to the in vitro leaf regeneration medium containing acetosyringone for dark culture for 3 d as a co-culture;
[14] step 4: material differentiation, regeneration and resistance screening:
transferring the leaf discs in the co-culture medium in the step 3 to an in vitro leaf regeneration medium containing antibacterial antibiotics for 7 d as a delayed screening culture, and finally transferring the leaf discs in the delayed screening culture medium to an in vitro leaf regeneration medium containing antibacterial antibiotics and 5 screening antibiotics for screening culture for 56 d to obtain robust transgenic resistant buds.
[15] Preferably, a Cambia1301 vector is selected as the plasmid vector in the step 2; the vector has a protein capable of being stained by GUS and a resistance protein against Hygromycin screening, which is capable of being used in the tissue staining identification and resistance selection at the later period of genetic transformation.
[16] Preferably, the antibacterial antibiotics in the step 4 have the following types and concentrations: Timentin: 250 mg/L and Carbenicillin: 250mg/L.
[17] Preferably, if the Cambial301 vector is used in the step 2, and type and concentration of the screening antibiotics in the step 4 are as follows: Hygromycin B
Smg/L, and in the step 4, the explants need to be transferred into a new screening medium every other 14 d of the screening culture, and meanwhile, the concentration of
Hygromycin B is gradually increased to 7 mg/L.
[18] Compared with the prior art, the present invention has the beneficial effects:
[19] 1, in the present invention, pre-treated aseptic strawberry leaves are used as explants, and the number of the pre-treated aseptic strawberry leaves is increased by 3 folds, thereby providing a large number of leaf explants as raw materials for the late differentiation;
[20] 2, compared with the single use of a differentiation medium, the material is subjected pretreatment culture in the present invention, thus increasing the regeneration efficiency of leaf explants; moreover, the regeneration efficiency of adventitious buds can be up to more than 90%, which is much higher than that in other existing technical solutions;
[21] 3, since the differentiation medium proposed in the present invention can achieve the high-frequency differentiation of the Fragaria ananassa in vitro leaves,
the present invention can be applied to genetic transformation of Fragaria ananassa, thus achieving a good regeneration differentiation effect and obtaining transgenic strawberry strains.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[22] To make the objectives, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to detailed examples. It should be understood that the detailed examples described herein are illustrative only, but are not construed as limiting the present invention.
[23] Example 1
[24] A method for high-frequency regeneration of Iragaria ananassa in vitro leaves is provided, and the specific steps are as follows:
[25] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 3 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 18 d for further use;
[26] step 2: material cutting and sampling, in sterile environment, taking out the strawberry materials grown on the strawberry material preculture medium in the step 1, cutting off the leaves thereof and placing into a wet culture dish, cutting leaflet base of the strawberry leaves into 0.5 cm x 0.5 cm leaf discs with a sharp scalpel or special puncher;
[27] step 3: differentiation culture, paving the leaf discs obtained in the step 2 on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and culturing the paved culture medium for 21 d in a culture room, then observing adventitious buds obtained by differentiation, and continuously culturing for 21 d to obtain a large number of adventitious buds.
[28] Two culture media involved in the high-frequency regeneration process of the
Fragaria ananassa in vitro leaves are characterized in that
[29] a medium for preculture: MS medium (pH = 5.6-6.0)+6-BA(0.5mg/L)y+IBA(0. Img/L)+ sucrose (30 g/L) + agar (7 g/L);
[30] differentiation medium for leaf discs differentiation: MS medium (pH= 5.6-6.0)+TDZ(4 mg/L)+IBA(0.5 mg/L }+sucrose (30 g/L)+agar (7g/L),
[31] abbreviations and CAS number:
[32] 6-BA: 6-BENZYLAMINOPURINE (CAS: 1214-39-7)
[33] IBA: INDOLE-3-BUTYRIC ACID (CAS: 133-32-4)
[34] TDZ: THIDIAZURON (CAS: 51707-55-2).
[35] The present invention further provides an application method of the high-frequency regeneration method of Fragaria ananassa in vitro leaves in genetic transformation, and specific steps are as follows:
[36] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 3 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 18 d; then taking out the strawberry materials grown on the strawberry preculture medium, cutting off the leaves and placing into a wet culture dish, and cutting leaflet of the strawberry leaves into 0.5 cm * 0.5 cm leaf discs with a sharp scalpel or special puncher; paving the leaf discs on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and preculturing the paved culture medium for 3 d in a culture room;
[37] step 2: strain preparation: transforming a plasmid vector into a GV3101
Agrobacterium engineering strain by a heat shock method, and obtaining a GV3101 monoclonal Agrobacterium containing the correct plasmid vector through solid medium screening and PCR means; inoculating the monoclonal Agrobacterium onto a
1 ml YEP medium containing antibiotics, and performing shaking culture for 2 d at 27°C and 200 rpm until the medium is seen turbid; pipetting the turbid bacterial solution into a 25 ml YEP medium in sterile environment for shaking culture, wherein the culture environment is 27°C and the shaking speed is 200 rpm; adding 20ul acetosyringone solution (100 mM) when the bacterial solution is cultured to 0D600= 0.1-0.15, and continuously culturing the bacterial solution for 3 h and then taking out, and performing rotary centrifugation for 5 min at an accelerated speed of 2000 g; discarding supernatant and adding to a 25 ml MS liquid medium containing 100 uM acetosyringone, and gently shaking to obtain suspended bacterial cells for further use;
[38] step 3: material infection: taking out the leaf discs in the step 1 and placing into a wet culture dish, pouring the bacterial solution prepared in the step 2 into the culture dish containing the leaf discs and shaking gently such that the bacterial solution and the leaf discs are fully contacted and mixed for infection, where the infection time is 30 min; shaking the culture dish once every other 5 min during the infection process until the end of infection, and transferring the leaves to the in vitro leaf regeneration medium containing acetosyringone for dark culture for 3 d as a co-culture;
[39] step 4: material differentiation, regeneration and resistance screening: transferring the leaf discs in the co-culture medium in the step 3 to an in vitro leaf regeneration medium containing antibacterial antibiotics for 7 d as a delayed screening culture, and finally transferring the leaf discs in the delayed screening culture medium to an in vitro leaf regeneration medium containing antibacterial antibiotics and screening antibiotics for screening culture for 56 d to obtain robust transgenic resistant buds.
[40] A Cambial301 vector was selected as the plasmid vector in the step 2; the vector had a protein capable of being stained by GUS and a resistance protein against
Hygromycin screening, which was capable of being used in the tissue staining identification and resistance selection at the later period of genetic transformation. The types and concentrations of antibacterial antibiotics in the step 4 were as follows:
Timentin: 250 mg/L and Carbenicillin: 250 mg/L. If the Cambial301 vector was used in the step 2, and type and concentration of the screening antibiotics in the step 4 were as follows: Hygromycin B 5 mg/L, and in the step 4, the explants needed to be transferred into a new screening medium every other 14 d of the screening culture, and meanwhile, the concentration of Hygromycin B was gradually increased to 7 mg/L.
[41] Example 2
[42] A method for high-frequency regeneration of Fragaria ananassa in vitro leaves is provided, and the specific steps are as follows:
[43] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 4 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 21 d for further use;
[44] step 2: material cutting and sampling, in sterile environment, taking out the strawberry materials grown on the strawberry material preculture medium in the step 1, cutting off the leaves thereof and placing into a wet culture dish, cutting leaflet base of the strawberry leaves into 0.5 cm x 0.5 cm leaf discs with a sharp scalpel or special puncher;
[45] step 3: differentiation culture, paving the leat discs obtained in the step 2 on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and culturing the paved culture medium for 21 d in a culture room, then observing adventitious buds obtained by differentiation, and continuously culturing for 21 d to obtain a large number of adventitious buds.
[46] The present invention further provides an application method of the high-frequency regeneration method of Fragaria ananassa in vitro leaves in genetic transformation, and specific steps are as follows:
[47] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 4 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 21 d; then taking out the strawberry materials grown on the strawberry preculture medium, cutting off the leaves and placing into a wet culture dish, and cutting leaflet of the strawberry leaves into 0.5 cm * 0.5 cm leaf discs with a sharp scalpel or special puncher; paving the leaf discs on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and preculturing the paved culture medium for 3 d in a culture room;
[48] step 2: strain preparation: transforming a plasmid vector into a GV3101
Agrobacterium engineering strain by a heat shock method, and obtaining a GV3101 monoclonal Agrobacterium containing the correct plasmid vector through solid medium screening and PCR means; inoculating the monoclonal Agrobacterium onto a 1 ml YEP medium containing antibiotics, and performing shaking culture for 2 d at 29°C and 200 rpm until the medium is seen turbid; pipetting the turbid bacterial solution into a 25 ml YEP medium in sterile environment for shaking culture, wherein the culture environment is 27°C and the shaking speed is 200 rpm; adding 20ul acetosyringone solution (100 mM) when the bacterial solution is cultured to 0D600= 0.1-0.15, and continuously culturing the bacterial solution for 3 h and then taking out, and performing rotary centrifugation for 5 min at an accelerated speed of 2000 g; discarding supernatant and adding to a 25 ml MS liquid medium containing 100 uM acetosyringone, and gently shaking to obtain suspended bacterial cells for further use;
[49] step 3: material infection: taking out the leaf discs in the step 1 and placing into a wet culture dish, pouring the bacterial solution prepared in the step 2 into the culture dish containing the leaf discs and shaking gently such that the bacterial solution and the leaf discs are fully contacted and mixed for infection, where the infection time is 35 min; shaking the culture dish once every other 5 min during the infection process until the end of infection, and transferring the leaves to the in vitro leaf regeneration medium containing acetosyringone for dark culture for 3 d as a co-culture;
[50] step 4: material differentiation, regeneration and resistance screening: transferring the leaf discs in the co-culture medium in the step 3 to an in vitro leaf regeneration medium containing antibacterial antibiotics for 7 d as a delayed screening culture, and finally transferring the leaf discs in the delayed screening culture medium to an in vitro leaf regeneration medium containing antibacterial antibiotics and screening antibiotics for screening culture for 56 d to obtain robust transgenic resistant buds. [S1] A Cambial301 vector was selected as the plasmid vector in the step 2; the vector had a protein capable of being stained by GUS and a resistance protein against
Hygromycin screening, which was capable of being used in the tissue staining identification and resistance selection at the later period of genetic transformation. The types and concentrations of antibacterial antibiotics in the step 4 were as follows:
Timentin: 250 mg/L and Carbenicillin: 250 mg/L. If the Cambial301 vector was used in the step 2, and type and concentration of the screening antibiotics in the step 4 were as follows: Hygromycin B 5mg/L, and in the step 4, the explants needed to be transferred into a new screening medium every other 14 d of the screening culture, and meanwhile, the concentration of Hygromycin B was gradually increased to 7 mg/L.
[52] Example 3
[53] A method for high-frequency regeneration of Fragaria ananassa in vitro leaves is provided, and the specific steps are as follows:
[54] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 5 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 24 d for further use;
[55] step 2: material cutting and sampling, in sterile environment, taking out the strawberry materials grown on the strawberry material preculture medium in the step 1, cutting off the leaves thereof and placing into a wet culture dish, cutting leaflet base of the strawberry leaves into 0.5 cm x 0.5 cm leaf discs with a sharp scalpel or special puncher; [S6] step 3: differentiation culture, paving the leat discs obtained in the step 2 on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and culturing the paved culture medium for 21 d in a culture room, then observing adventitious buds obtained by differentiation, and continuously culturing for 21 d to obtain a large number of adventitious buds. [S7] The present invention further provides an application method of the high-frequency regeneration method of Fragaria ananassa in vitro leaves in genetic transformation, and specific steps are as follows:
[58] step 1: material pretreatment, in sterile environment, serving aseptic seedlings of Fragaria ananassa grown on a 1/2MS medium well as materials, cutting and separating cluster buds of the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud points and 5 surrounding leaves thereof and transferring onto a strawberry material preculture medium for culturing in a culture room for 24 d; then taking out the strawberry materials grown on the strawberry preculture medium, cutting off the leaves and placing into a wet culture dish, and cutting leaflet of the strawberry leaves into 0.5 cm x 0.5 cm leaf discs with a sharp scalpel or special puncher; paving the leaf discs on the surface of an in vitro leaf regeneration medium such that the periphery of the leaf discs is in sufficient contact with the regeneration medium; and preculturing the paved culture medium for 3 d in a culture room;
[59] step 2: strain preparation: transforming a plasmid vector into a GV3101
Agrobacterium engineering strain by a heat shock method, and obtaining a GV3101 monoclonal Agrobacterium containing the correct plasmid vector through solid medium screening and PCR means; inoculating the monoclonal Agrobacterium onto a 1 ml YEP medium containing antibiotics, and performing shaking culture for 2 d at 30°C and 200 rpm until the medium is seen turbid; pipetting the turbid bacterial solution into a 25 ml YEP medium in sterile environment for shaking culture, wherein the culture environment is 27°C and the shaking speed is 200 rpm; adding 20ul acetosyringone solution (100 mM) when the bacterial solution is cultured to 0D600= 0.1-0.15, and continuously culturing the bacterial solution for 3 h and then taking out, and performing rotary centrifugation for 5S min at an accelerated speed of 2000 g; discarding supernatant and adding to a 25 ml MS liquid medium containing 100 uM acetosyringone, and gently shaking to obtain suspended bacterial cells for further use;
[60] step 3: material infection: taking out the leaf discs in the step 1 and placing into a wet culture dish, pouring the bacterial solution prepared in the step 2 into the culture dish containing the leaf discs and shaking gently such that the bacterial solution and the leaf discs are fully contacted and mixed for infection, where the infection time is 40 min; shaking the culture dish once every other 5 min during the infection process until the end of infection, and transferring the leaves to the in vitro leaf regeneration medium containing acetosyringone for dark culture for 3 d as a co-culture;
[61] step 4: material differentiation, regeneration and resistance screening: transferring the leaf discs in the co-culture medium in the step 3 to an in vitro leaf regeneration medium containing antibacterial antibiotics for 7 d as a delayed screening culture, and finally transferring the leaf discs in the delayed screening culture medium to an in vitro leaf regeneration medium containing antibacterial antibiotics and screening antibiotics for screening culture for 56 d to obtain robust transgenic resistant buds.
[62] A Cambia1301 vector was selected as the plasmid vector in the step 2; the vector had a protein capable of being stained by GUS and a resistance protein against
Hygromycin screening, which was capable of being used in the tissue staining identification and resistance selection at the later period of genetic transformation. The types and concentrations of antibacterial antibiotics in the step 4 were as follows:
Timentin: 250 mg/L and Carbenicillin: 250 mg/L. If the Cambial301 vector was used in the step 2, and type and concentration of the screening antibiotics in the step 4 were as follows: Hygromycin B 5 mg/L, and in the step 4, the explants needed to be transferred into a new screening medium every other 14 d of the screening culture, and meanwhile, the concentration of Hygromycin B was gradually increased to 7 mg/L.
[63] In the present invention, the pre-treated aseptic strawberry leaves are used as explants, and the number of the pre-treated aseptic strawberry leaves is increased by 3 folds, thereby providing a large number of leaf explants as raw materials for the late differentiation. Compared with the single use of a differentiation medium, the material is subjected pretreatment culture in the present invention, thus increasing the regeneration efficiency of leaf explants; Moreover, the regeneration efficiency of adventitious buds can be up to more than 90%, which is much higher than that in other existing technical solutions; Since the differentiation medium proposed in the present vention can achieve the high-frequency differentiation of the Fragaria ananassa in vitro leaves, the present invention can be applied to genetic transformation of Fragaria ananassa, thus achieving a good regeneration differentiation effect and obtaining transgenic strawberry strains.
[64] What are described above are detailed preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any equivalent replacement or change made by a person skilled in the art based on the technical solution and improvement concept of the present invention within the technical scope disclosed herein shall be covered within the protection scope of the present invention.

Claims (5)

Conclusies L Hogefrequentieregenereerwerkwijze voor in vitro-bladeren van Fragaria ananassa, waarbij specifieke stappen als volgt zijn: stap 1: het voorbehandelen van materiaal, in steriele omgeving, het verschaffen van aseptische zaailingen van Fragaria ananassa die gegroeid zijn op een 1/2MS-medium evenals materialen, het snijden en scheiden van clusterknoppen van de aseptische zaailingen met een aseptische scalpel, en uiteindelijk het behouden van de clusterknoppunten en 3-5 omringende bladeren daarvan en het overbrengen op een aardbeimateriaalvoorkweekmedium voor het kweken in een kweekkamer gedurende 18- 24 d voor verder gebruik; stap 2: het snijden en bemonsteren van materiaal, in steriele omgeving, het uitnemen van de aseptische aardbeizaailingen die gegroeid zijn op het aardbeimateriaalvoorkweekmedium in de stap 1, het afsnijden van de bladeren daarvan en het plaatsen in een natte kweekschaal, het in 0,5x0,5 cm bladschijven snijden van bladbasis van de aardbeibladeren met een scherpe scalpel of speciale perforator, stap 3: het differentiëren van kweek, het plaveien van de bladschijven die in de stap 2 verkregen zijn op het oppervlak van een in vitro-bladregeneratiemedium zodanig dat de rand van de bladschijven in voldoende contact is met het regeneratiemedium; en het kweken van het geplaveide kweekmedium gedurende 21 d in een kweekkamer, vervolgens het observeren van onvoorziene knoppen die middels differentiatie verkregen zijn, en het ononderbroken kweken gedurende 21 d om een groot aantal onvoorziene knoppen te verkrijgen.Conclusions L High-frequency regeneration method for in vitro leaves of Fragaria ananassa, where specific steps are as follows: Step 1: Pre-treatment of material, in sterile environment, to provide aseptic seedlings of Fragaria ananassa grown on 1/2MS medium as well as materials, cutting and separating cluster buds from the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster buds and 3-5 surrounding leaves thereof and transferring them to a strawberry material pre-culture medium for growing in a growth chamber for 18-24 d for further usage; step 2: cutting and sampling material, in sterile environment, taking out the aseptic strawberry seedlings grown on the strawberry material pre-cultivation medium in step 1, cutting the leaves thereof and placing it in a wet culture dish, placing it in 0.5x0 cutting .5 cm leaf discs from leaf base of the strawberry leaves with a sharp scalpel or special perforator, step 3: differentiating culture, paving the leaf discs obtained in step 2 on the surface of an in vitro leaf regeneration medium such that the edge of the leaf blades is in sufficient contact with the regeneration medium; and culturing the cobbled culture medium for 21 d in a growth chamber, then observing adventitious buds obtained by differentiation, and culturing continuously for 21 d to obtain a large number of adventitious buds. 2. Toepassingswerkwijze van de hogefrequentieregenereerwerkwijze van in vitro- bladeren van Fragaria ananassa in genetische transformatie volgens conclusie 1, waarbij specifieke stappen als volgt zijn: stap 1: het voorbehandelen van materiaal, in steriele omgeving, het verschaffen van aseptische zaailingen van Fragaria ananassa die gegroeid zijn op een 4MS-medium evenals materialen, het snijden en scheiden van clusterknoppen van de aseptische zaailingen met een aseptische scalpel, en uiteindelijk het behouden van de clusterknoppunten en 3-5 omringende bladeren daarvan en het overbrengen op een aardbeimateriaalvoorkweekmedium voor het kweken in een kweekkamer gedurende 18-The application method of the high-frequency regeneration method of in vitro leaves of Fragaria ananassa in genetic transformation according to claim 1, wherein specific steps are as follows: Step 1: Pre-treating material, in sterile environment, to provide aseptic seedlings of Fragaria ananassa grown are on a 4MS medium as well as materials, cutting and separating cluster buds from the aseptic seedlings with an aseptic scalpel, and finally retaining the cluster bud tips and 3-5 surrounding leaves thereof and transferring them to a strawberry material preculture medium for growing in a growth chamber during 18- 24 d; vervolgens het uitnemen van de aardbeimaterialen die gegroeid zijn op het PM, het snijden van de bladeren daarvan en het plaatsen in een natte kweekschaal, het in 0,5x0,5 cm bladschijven snijden van een blaadje van de aardbeibladeren met een scherpe scalpel of speciale perforator; het plaveien van de bladschijven op het oppervlak van een in vitro-bladregeneratiemedium zodanig dat de rand van de bladschijven in voldoende contact is met het regeneratiemedium; en het voorkweken van het geplaveide kweekmedium gedurende 3 d in een kweekkamer; stap 2: het bereiden van een stam: het transformeren van een plasmidevector in een GV3101-modificatiestam van Agrobacterium middels een warmteschokwerkwijze, en het verkrijgen van een monoklonale GV3101-Agrobacterium die de correcte plasmidevector bevat door vastmediumscreening en PCR-middelen; het inoculeren van de monoklonale Agrobacterium op een 1 mL YEP-medium dat antibiotica bevat; en het uitvoeren van schudkweek gedurende 2 d bij 27-30 °C en 200 rpm tot het medium troebel wordt gezien; het pipetteren van de troebele bacterieoplossing in een 25 mL YEP-medium in steriele omgeving voor schudkweek, waarbij de kweekomgeving 27 °C en de schudsnelheid 200 rpm is; het toevoegen van 20 ul acetosyingonoplossing (100 mM) wanneer de bacterieoplossing gekweekt wordt tot OD600 = 0,1-0,15, en het ononderbroken kweken van de bacterieoplossing gedurende 3 h en vervolgens het uitnemen, en het uitvoeren van rotatiecentrifugatie gedurende 5 min bij een versnelde snelheid van 2000 g; het weggooien van supernatant en het toevoegen aan een 25 ml vloeibaar MS-medium dat 100 uM acetosyringon bevat, en het voorzichtig schudden om gesuspendeerde bacteriecellen voor verder gebruik te verkrijgen; stap 3: het infecteren van materiaal: het uitnemen van de bladschijven in de stap | en het plaatsen in een natte kweekschaal, het gieten van de bacterieoplossing die bereid is in de stap 2 in de kweekschaal die de bladschijven bevat en het voorzichtig schudden zodanig dat de bacterieoplossing en de bladschijven volledig in contact zijn en gemengd worden voor infectie, waarbij de infectietijd 30-40 min is; het schudden van de kweekschaal eenmaal per 5 min tijdens het infectieproces tot het einde van infectie, en het overbrengen van de bladeren naar het in vitro-regeneratiemedium dat acetosyringon bevat voor donkere kweek gedurende 3 d als een co-kweek; stap 4: het differentiëren van materiaal, regeneratie en resistentiescreening: het overbrengen van de bladschijven in het co-kweekmedium in de stap 3 naar een in vitro- regeneratiemedium dat antibacteriële antibiotica bevat gedurende 7 d als een uitgesteldescreeningkweek, en uiteindelijk het overbrengen van de bladschijven in het uitgesteldescreeningkweekmedium naar een in vitro-bladregeneratiemedium dat antibacteriële antibiotica en screeningantibiotica bevat voor het screenen van kweek gedurende 56 d om robuuste transgene resistente knoppen te verkrijgen.24 d; then taking out the strawberry materials grown on the PM, cutting the leaves from them and placing them in a wet culture dish, cutting a leaf from the strawberry leaves into 0.5x0.5 cm leaf slices with a sharp scalpel or special perforator ; paving the leaf disks on the surface of an in vitro leaf regeneration medium such that the edge of the leaf disks is in sufficient contact with the regeneration medium; and pre-cultivating the cobblestone culture medium for 3 d in a growth chamber; step 2: preparing a strain: transforming a plasmid vector into a GV3101 modification strain of Agrobacterium by a heat shock method, and obtaining a monoclonal GV3101 Agrobacterium containing the correct plasmid vector by solid medium screening and PCR means; inoculating the monoclonal Agrobacterium on a 1 mL YEP medium containing antibiotics; and performing shake culture for 2 d at 27-30°C and 200 rpm until the medium is seen to be cloudy; pipetting the turbid bacterial solution into a 25 mL YEP medium in sterile shaking culture environment, wherein the culture environment is 27°C and the shaking speed is 200 rpm; adding 20 µl of acetosyingone solution (100 mM) when the bacterial solution is grown to OD600 = 0.1-0.15, and continuously culturing the bacterial solution for 3 h and then taking it out, and performing rotational centrifugation for 5 min at an accelerated speed of 2000 g; discarding supernatant and adding to a 25 ml liquid MS medium containing 100 µM acetosyringone and shaking gently to obtain suspended bacterial cells for further use; step 3: infecting material: extracting the leaf discs in step | and placing in a wet culture dish, pouring the bacteria solution prepared in the step 2 into the culture dish containing the leaf disks, and shaking gently so that the bacteria solution and the leaf disks are in full contact and mixed for infection, whereby the infection time is 30-40 min; shaking the culture dish once every 5 min during the infection process until the end of infection, and transferring the leaves to the in vitro regeneration medium containing acetosyringone for dark culture for 3 d as a co-culture; step 4: differentiating material, regeneration and resistance screening: transferring the leaf disks in the co-culture medium in the step 3 to an in vitro regeneration medium containing antibacterial antibiotics for 7 d as a delayed screening culture, and finally transferring the leaf disks in the delayed screening culture medium to an in vitro leaf regeneration medium containing antibacterial antibiotics and screening antibiotics for screening culture for 56 d to obtain robust transgenic resistant buds. 3. Toepassingswerkwijze van de hogefrequentieregeneratiewerkwijze van in vitro- bladeren van Fragaria ananassa in genetische transformatie volgens conclusie 2, waarbij een Cambia1301-vector geselecteerd wordt als de plasmidevector in de stap 2; waarbij de vector een eiwit heeft dat in staat is gekleurd te worden middels GUS en een resistentie-eiwit tegen hygromycinescreening, dat in staat is gebruikt te worden in de weefselkleuringsidentificatie en resistentiescreening in de latere periode van genetische transformatie.The method of using the high-frequency regeneration method of in vitro leaves of Fragaria ananassa in genetic transformation according to claim 2, wherein a Cambia1301 vector is selected as the plasmid vector in the step 2; wherein the vector has a protein capable of being stained by GUS and a resistance protein to hygromycin screening capable of being used in the tissue staining identification and resistance screening in the later period of genetic transformation. 4. Toepassingswerkwijze van de hogefrequentieregeneratiewerkwijze van in vitro- bladeren van Fragaria ananassa in genetische transformatie volgens conclusie 2, waarbij de antibacteriële antibiotica in de stap 4 de volgende types en concentraties hebben: timentine: 250 mg/L en carbenicilline: 250 mg/L.The method of using the high-frequency regeneration method of in vitro leaves of Fragaria ananassa in genetic transformation according to claim 2, wherein the antibacterial antibiotics in the step 4 have the following types and concentrations: timentin: 250 mg/L and carbenicillin: 250 mg/L. 5. Toepassingswerkwijze van de hogefrequentieregeneratiewerkwijze van in vitro- bladeren van Fragaria ananassa in genetische transformatie volgens conclusie 2, waarbij als de Cambia1301-vector gebruikt wordt in de stap 2, en de screeningantibiotica in de stap 4 het volgende type en de volgende concentratie hebben: hygromycine B 5 mg/L, en in de stap 4 de explanten elke 14 d van de screeningkweek overgebracht dienen te worden in een nieuw screeningmedium, en ondertussen de concentratie van hygromycine B geleidelijk verhoogd wordt tot 7 mg/L.The application method of the high-frequency regeneration method of in vitro leaves of Fragaria ananassa in genetic transformation according to claim 2, wherein as the Cambia1301 vector is used in the step 2, and the screening antibiotics in the step 4 have the following type and concentration: hygromycin B 5 mg/L, and in the step 4 the explants should be transferred every 14 d of the screening culture into a new screening medium, meanwhile the concentration of hygromycin B is gradually increased to 7 mg/L.
NL2033006A 2021-11-15 2022-09-12 High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation NL2033006B1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111347332.3A CN114097614A (en) 2021-11-15 2021-11-15 High-frequency regeneration method for in vitro leaves of cultivated strawberries and application method of high-frequency regeneration method in genetic transformation

Publications (1)

Publication Number Publication Date
NL2033006B1 true NL2033006B1 (en) 2023-06-09

Family

ID=80395287

Family Applications (1)

Application Number Title Priority Date Filing Date
NL2033006A NL2033006B1 (en) 2021-11-15 2022-09-12 High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation

Country Status (2)

Country Link
CN (1) CN114097614A (en)
NL (1) NL2033006B1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755086A (en) * 2017-01-16 2017-05-31 四川农业大学 A kind of genetic transforming method of Agrobacterium tumefaciens mediated strawberry efficient stable
CN108094198A (en) * 2017-12-07 2018-06-01 陕西师范大学 A kind of Strawberry Leaves in-vitro regeneration method
CN113584072A (en) * 2021-07-29 2021-11-02 上海市农业科学院 Construction method of genetic transformation system of strawberries

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104195171B (en) * 2014-09-16 2016-11-09 江苏农林职业技术学院 Fructus Fragariae Ananssae efficient fast and stable gene transformation method
CN110387384B (en) * 2019-09-03 2022-09-13 上海市农业科学院 Agrobacterium tumefaciens-mediated genetic transformation method for peach smoked strawberries

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755086A (en) * 2017-01-16 2017-05-31 四川农业大学 A kind of genetic transforming method of Agrobacterium tumefaciens mediated strawberry efficient stable
CN108094198A (en) * 2017-12-07 2018-06-01 陕西师范大学 A kind of Strawberry Leaves in-vitro regeneration method
CN113584072A (en) * 2021-07-29 2021-11-02 上海市农业科学院 Construction method of genetic transformation system of strawberries

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AMJAD MASOOD HUSAINI: "Pre and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants", PLANT CELL REPORTS, SPRINGER, BERLIN, DE, vol. 29, no. 1, 3 December 2009 (2009-12-03), pages 97 - 110, XP019779597, ISSN: 1432-203X *

Also Published As

Publication number Publication date
CN114097614A (en) 2022-03-01

Similar Documents

Publication Publication Date Title
CN113621631A (en) Mevalonate kinase gene RKMK and application thereof
CN109293757B (en) Phyllostachys pubescens PeTCP10 protein with function of controlling blade curling and application thereof
CN104762314A (en) Screening marker gene-deletable plant expression vector and use thereof
CN107267528A (en) A kind of tobacco axillary bud adjusting and controlling growth gene NtMOC1 and its cloning process and application
NL2033006B1 (en) High-frequency regeneration method of fragaria ananassa in vitro leaves and application method thereof in genetic transformation
CN104531656B (en) A kind of Phophomannose isomerase gene and its application from chlorella
CN116926092B (en) Pantothenate kinase gene RkPank and application thereof
CN109136259A (en) A kind of watermelon High-efficient Genetic Transformation and transgenic plant identification method
CN110106171A (en) Long-chain non-coding RNA and its application in regulation plant frigostabile
CN111961677A (en) Method for regulating and controlling chrysanthemum flower type and/or leaf shape and/or plant type and application
Wan et al. Wheat (Triticum aestivum L.)
CN116732053B (en) Heat-resistant regulating gene of flowering cabbage and application thereof
Arencibia et al. Sugarcane (Saccharum spp.)
KR100523758B1 (en) Efficient method of Agrobacterium-mediated transformation in Chinese cabbage
CN113755519B (en) Multi-antibody screening poplar polygene genetic transformation method
CN108070026B (en) Chrysanthemum CmTFL1a gene and application thereof
Reski Physcomitrella patens as a novel tool for plant functional genomics
CN110106172A (en) A kind of long-chain non-coding RNA and its application in regulation plant frigostabile
CN114149993B (en) lncRNA for regulating and controlling content of soluble sugar in plants and application thereof
CN110423753B (en) Root knot specific promoter T106-P induced by root knot nematode and application
KR100894323B1 (en) Method for Producing Available Recombinant Protein by Culturing Adventitious Root of Transformed Plants
JP4472344B2 (en) Plastid transformation method using microbial recombination enzyme
KR100869565B1 (en) Rapid Production of transgenic hairy roots using the activation tagging system
CN114891802A (en) Application of OsDUF6 gene and encoding protein thereof in salt tolerance breeding of rice
KR100529005B1 (en) Transgenic lily expressing chalcone synthase antisense gene and preparative method thereof