NL2035132B1 - GENETIC TRANSFORMATION METHOD OF E. UROPHYLLA x E. GRANDIS DH3213 - Google Patents
GENETIC TRANSFORMATION METHOD OF E. UROPHYLLA x E. GRANDIS DH3213 Download PDFInfo
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- 230000002068 genetic effect Effects 0.000 title claims abstract description 41
- 241000404037 Eucalyptus urophylla Species 0.000 title claims abstract description 37
- 238000011426 transformation method Methods 0.000 title claims abstract description 11
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- 238000000034 method Methods 0.000 claims abstract description 25
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 18
- 235000021240 caseins Nutrition 0.000 claims description 18
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- 238000007400 DNA extraction Methods 0.000 claims description 3
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- AZZMGZXNTDTSME-JUZDKLSSSA-M cefotaxime sodium Chemical compound [Na+].N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 AZZMGZXNTDTSME-JUZDKLSSSA-M 0.000 claims description 3
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- 241000589155 Agrobacterium tumefaciens Species 0.000 abstract description 10
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000006100 Corymbia <angiosperm> Species 0.000 description 1
- 244000165963 Eucalyptus camaldulensis Species 0.000 description 1
- 240000006361 Eucalyptus saligna Species 0.000 description 1
- 240000007002 Eucalyptus tereticornis Species 0.000 description 1
- 235000019134 Eucalyptus tereticornis Nutrition 0.000 description 1
- 241000299576 Eucalyptus urophylla x Eucalyptus grandis Species 0.000 description 1
- 241000219926 Myrtaceae Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
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Classifications
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- 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
- A01H5/04—Stems
-
- 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
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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- 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
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
- C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
Disclosed is a genetic transformation method of E. urophyl/a x E. grandis DH3213. The high- efficiency and rapid adventitious bud regeneration and transformation method for a fine cultivated variety E. urophylla x E. grandis DH3213 mediated by Agrobacterium tumefaciens solves problems that a genetic transformation system is not established in the existing fine cultivated variety for Eucalyptus including E. urophyl/a x E. grandis DH3213 and a genetic transformation period of Eucalyptus is long. The present disclosure breaks through the establishment of a genetic transformation system of the fine cultivated variety of Eucalyptus, and may be directly used for the creation of a new cultivated variety DH3213 of Eucalyptus by modification and gene editing. Compared with other systems, the time for the established genetic transformation system is shortened by 1/3 - 1/2, and the system of the present disclosure may be used for gene function analysis of important characters of Eucalyptus. The method has a short period, high efficiency, simple procedure, convenient operation, and reliable result.
Description
GENETIC TRANSFORMATION METHOD OF E. UROPHYLLA x E. GRANDIS DH3213
FIELD OF TECHNOLOGY
The present disclosure belongs to the technical field of plant transgenesis, and in particular to a genetic transformation method of Eucalyptus urophylla x Eucalyptus grandis (E. urophylla x
E. grandis) DH3213.
Eucalyptus is a generic term for plants of the genera Eucalyptus, Angophora, and
Corymbia of the family Myrtaceae, native to Australia and islands nearby. Due to rapid growth, good adaptability, and fine wood quality, Eucalyptus can be used for the production of high- quality paper pulp, wood, firewood, essential oil, etc. Eucalyptus has been widely planted in tropical and subtropical regions of China at present with a cultivation area reaching 5,467,400 hectares.
The application of a plant genetic engineering technology in the forestry makes up the difficulties and defects, namely, the conventional breeding period is long, directional improvement is difficult to be controlled, and distant hybridization or interspecies hybridization is difficult to be realized, which thus provides a very good way for genetic improvement of trees and cultivation of new varieties of forest trees. At present, most plants are subjected to genetic transformation mediated by Agrobacterium tumefaciens to obtain a transgenic plant. The establishment of a stable and efficient genetic transformation system becomes a prerequisite for developing a forest transgenic breeding and gene function verification system. Due to the importance of Eucalyptus, a plurality of Eucalyptus genetic transformation systems such as
Eucalyptus tereticornis, E. urophylla x E. grandis, Eucalyptus saligna, and Eucalyptus camaldulensis have been successfully established. Moreover, some of the genetic transformation systems have been cultivated to obtain transgenic plants with the characters of reduced lignin, resistance to insect, herbicide, cold, salt, and bacterial wilt, and early flowering.
But these studies are mainly based on sexual materials or non-cultivated species. However, it is still difficult to break through a bottleneck of difficult regeneration for fine cultivars mainly cultivated in southern China, difficult to establish an effective genetic transformation system, and unable to conduct cultivation research on a new germplasm of a fine cultivated variety of
Eucalyptus by transgenesis and gene editing. Meanwhile, the established genetic transformation system of Eucalyptus has a long transformation period, and the whole process lasts 4-6 months, which affects the identification of gene functions and cultivation of new germplasms.
The fine cultivated variety E. urophylla x E. grandis DH3213 planted in southern China is a hybrid of Eucalyptus urophylla and Eucalyptus grandis bred in the Dongmen Forest Farm of
Guangxi; the fine cultivated variety shows a high growth speed and high outturn percentage in multi-point tests of Guangdong and Guangxi, has an annual average growth of diameter at breast height of 3.1 cm, an annual average growth of tree height of 4.4 m, and an annual average accumulated growth of 32.8 m3/hm2, has a straight and full trunk and a vigorous growth vigor, and belongs to a fast-growing clone with a good paper making performance. Further transgenic and gene editing breeding of the fine cultivated variety may maintain a good character and introduce new characters such as resistance to insect and herbicide, so as to obtain a new germplasm and meet needs of Eucalyptus production.
The present disclosure aims to solve the problems that genetic engineering improvement and gene editing technology may not be conducted as there exists no genetic transformation system to the major fine cultivated varieties of Eucalyptus in southern China at present, and
Eucalyptus lacks a stable and efficient genetic transformation system at present. Therefore, the present disclosure provides an Agrobacterium tumefaciens-mediated genetic transformation method for a superior clone E. urophylla x E. grandis DH3213 and provides a basis for further improvement. The method is easy to implement, rapid, and efficient.
The present disclosure takes the superior clone E. urophylla x E. grandis DH3213 cultivated in southern China as a material. Starting with the development on the formula of the most crucial adventitious bud induction medium for inducing regeneration and the optimization of an Agrobacterium transformation program, the efficiency of adventitious bud induction is improved and time for adventitious bud induction is simultaneously shortened via explant material selection, development of an adventitious bud induction culture medium, and optimization of an Agrobacterium transformation and transgenic plant screening program, etc, and by exogenously adding silver nitrate and hydrolysed casein. Meanwhile, a high-efficiency, rapid, and stable genetic transformation system of the superior clone E. urophylla x E. grandis
DH3213 is established by optimizing the genetic transformation process, which lays a foundation for further developing transgenesis and gene editing breeding.
The present disclosure utilizes a stem internode obtained after dark culture of propagation buds of the fine cultivated variety E. urophylla x E. grandis DH3213 as explants. An induction period of an adventitious bud is shortened by exogenously adding hydrolysed casein and silver nitrate. Mediated by Agrobacterium tumefaciens, an exogenous gene is transferred into the superior clone E. urophylia x E. grandis DH3213, which provides a basis for the further improvement of growth and other characters of E. urophylla x E. grandis DH3213, and a technology for the commercial use of transgenesis or gene editing of a new germplasm of E. urophylla x E. grandis DH3213.
A genetic transformation method of E. urophylla x E. grandis DH3213, comprising the following steps:
S 1: taking sterile plants of E. urophylla x E. grandis DH3213 as a propagation material, culturing the buds on a propagation medium for 20-25 d, and then culturing in the dark for 10 d to obtain an albino shoot;
S 2:taking a stem of the albino shoot, removing a terminal bud and a resting bud, and cutting a stem segment with a length of 0.5 - 1.0 cm with a removed lateral bud primordium as an explant; and inoculating on an adventitious bud induction culture medium for pre-culture for 3d;
S 3:taking out a GV3101 Agrobacterium strain carrying a pBI121 plasmid containing a GUS gene and a target gene, culturing and activating the strain on a resistance screening plate, picking a single colony, transferring the single colony into an LB liquid culture medium, and culturing until an ODsos value of a bacterial solution is 0.4 - 0.7; and centrifuging the bacterial solution, removing a supernatant, and adding a resuspension solution to resuspend bacteria precipitated until an ODsoe value of a bacterial solution is 0.4 - 0.7;
S 4: taking out the stem segment explant precultured for 3 d in step S2, placing and soaking the stem segment explant into the bacterial solution resuspended in step S3 for 10 min, and during the soaking period, slightly shaking the solution to fully contact Agrobacterium cells with the explant; then transferring the explant to a sterile dry filter paper, and absorbing a surface bacterial solution; and then transferring the explant to a co-culture medium for dark culture for 3 d;
S 5:washing the co-cultured stem segment explant once by using a 200 mg.I"! cefotaxime sodium solution, then washing the explant by using sterile water, transferring the explant to a screening culture medium for screening culture, replacing the fresh culture medium every 2 weeks, and removing an browning calli, where an obvious green tiny bud may be observed after replacing the culture medium for 2 - 3 times;
S 6:transferring the explant with the green tiny bud to a propagation medium containing 100 mg.I"" of Cef + 50 mg.l of Tmt + 15 mg.I"! of Kan for adventitious bud induction and elongation culture, culturing the explant for 2 weeks, and marking and cutting a part of leaves for DNA extraction when a part of resistant buds are elongated to 2 - 3 cm and have 4 - 6 leaves;
S7:taking a leaf of the resistant bud, extracting DNA, and performing a PCR amplification by using gene specific primers NPTI/ and GUS; where a line amplifying each target band at the same time is proved to be a transgenic line stably integrated into a nuclear genome of
E. urophylla x E. grandis DH3213; further determining the detected line by using GUS staining to ensure an expression of a GUS gene, and further verifying the line as a transgenic line; and
S 8: performing propagation culture on the identified transgenic plant on the propagation medium containing 100 mg.I"* of Cef + 50 mg.I"* of Tmt + 15 mg." of Kan, cutting a robust bud seedling with a length of 2 - 3 cm after propagation to a certain number, transferring the bud seedling to a rooting culture medium for culture, inducing the bud seedling to a rooting seedling, and exercising and transplanting the rooting seedling in time.
Preferably, the propagation medium includes a modified MS culture medium + 0.3 mg.I"* of
BAP + 0.05 mg.I"" of NAA + 30 g.I'"" of sucrose + 6.0 gl’ of agar, where the modified MS culture medium is obtained by reducing the content of ammonium nitrate in an MS culture medium by half.
Preferably, the adventitious bud induction culture medium includes: WPM + 0.25 mg.I"" of
TDZ + 0.1 mg.l of NAA + 0.1 g.I'* of hydrolysed casein + 0.5 mg." of AgNO; + 30 g.I"" of sucrose + 6 gl’ of agar, with a pH of 5.8.
Preferably, the resuspension solution includes: 1/2MS + 200 uM of AS + 30 g.I"" of sucrose, with a pH of 5.4.
Preferably, the co-culture medium includes: WPM + 0.25 mg.r' of TDZ + 0.1 mg.I"" of NAA + 0.1 g.I'" of hydrolysed casein + 200 uM of AS + 30 g.I"* of sucrose + 6 g.I"! of agar, with a pH of 5.8.
Preferably, the screening culture medium is the adventitious bud induction culture medium containing 100 mg.lt of Cef + 50 mg.I"! of Tmt + 15 mg.I"* of Kan, and the adventitious bud induction culture medium includes: WPM + 0.25 mg. of TDZ + 0.1 mg." of NAA + 0.1 g.l of hydrolysed casein + 0.5 mg.I"* of AgNO: + 30 gl of sucrose + 6 g.I"" of agar, with a pH of 5.8.
Preferably, the rooting culture medium includes: 1/2MS culture medium + 0.3 mg. of IBA +20.0g.l of sucrose + 6.0 g.I'* of agar + 0.05 g.I'' of activated carbon.
Preferably, in the genetic transformation method, the plant material is cultured at a temperature of 23+2°C, and the culture is performed at an illumination intensity of 100-300 umol.m2.s" for 16 h/d except the dark culture.
Preferably, the resistance screening plate in step S3 is an LB plate containing 20 mg.! of rifampicin and 50 mg.I"! of kanamycin.
The present disclosure has the following advantages:
The present disclosure provides a method for constructing a genetic transformation system of a fine cultivated variety E. urophylla x E. grandis DH3213. A target exogenous gene is introduced into a stem segment of fine cultivated variety E. urophylla x E. grandis DH3213 explant by means of genetic engineering, and subjected to callus induction, adventitious bud differentiation culture and propagation, and rooting culture to obtain a new E. urophylla x E. grandis strain with a target exogenous gene for controlling biological characters. The construction method provided by the present disclosure has characteristics of good repeatability and high success rate, and a transformation rate of 2.67%. The establishment of a genetic transformation system of the fine cultivated variety E. urophyila x E. grandis DH3213 opens up a new way for further improving the variety.
The construction method provided by the present disclosure constructs a genetic transformation system with a stem segment as a starting material, improves a transformation success rate, and shortens transformation time, may analyse a gene function at a specific stage of tree growth, explores basic problems of woody plant biology, and thus provides a stable and standard operational process for quick and efficient identification of Eucalyptus functional genes. 5 The construction method provided by the disclosure further improves an induction efficiency of Eucalyptus adventitious buds. Since in the prior art, the induction efficiency of
Eucalyptus adventitious buds is low and the period is long. Clumpy adventitious buds may be successfully induced in 30-40 days by adding 0.1 9.1 of hydrolysed casein and 0.5 mg.I"" of
AgNO: into an adventitious bud induction culture medium, an adventitious bud induction rate exceeds 90%, a large amount of clumpy buds may be formed, achieving rapid propagation. The method herein overcomes technical problems of low efficiency and low speed of propagation in the prior art, and provides a technical basis for other improved varieties of E. urophylla x E. grandis.
The construction method provided by the present disclosure may simultaneously improve disease resistance and wind resistance of a fine cultivated variety E. urophylia x E. grandis
DH3213 by transferring a gene for improving a specific target character directed to the existing slightly weak disease resistance and wind resistance. The construction method herein further increases a cultivation area and a yield, at the same time may also bring new characters such as insect resistance, herbicide resistance, etc., obtains a new germplasm, cultivates a transgenic fine cultivated variety with a higher cultivation yield, better quality, and stronger capability of coping with biotic and abiotic stresses, and realizes a great breakthrough of a forest biotechnology in Eucalyptus.
The high-efficiency and rapid adventitious bud regeneration and transformation method of the fine cultivated variety E. urophylla x E. grandis DH3213 mediated by Agrobacterium tumefaciens herein solves problems that a genetic transformation system is not established in the existing cultivated variety of Eucalyptus including E. urophylla x E. grandis DH3213 and a genetic transformation period of Eucalyptus is long. The present disclosure breaks through the establishment of a genetic transformation system of the fine cultivated variety of Eucalyptus, and may be directly used for the creation of a new cultivated variety DH3213 of Eucalyptus by transgenesis and gene editing. Compared with other systems, the time of the established genetic transformation system is shortened by 1/3-1/2, and the system may be used for gene function analysis of important characters of Eucalyptus. The method has a short period, high efficiency, simple procedure, convenient operation, and reliable result.
FIG. 1 shows an effect of different types of basic culture mediums on adventitious bud regeneration; note: lower case letters represent an analysis result of a significant difference by an ANOVA analysis, the same letters indicate no significant difference, but the different letters represent a significant difference (p < 0.05), the same below;
FIG. 2 shows an effect of different concentrations of hydrolysed casein on adventitious bud regeneration;
FIG. 3 shows an effect of different concentrations of NAA on adventitious bud regeneration;
FIG. 4 shows an effect of different concentrations of silver nitrate on adventitious bud regeneration;
FIG. 5 shows a process of an adventitious bud induction regeneration of E. urophylla x E. grandis DH3213, where, a: a freshly-cut stem segment is placed on an adventitious bud induction culture medium; b: culturing on the adventitious bud induction culture medium for 10 days; c: culturing on the adventitious bud induction culture medium for 30 days; d: transferring to a propagation medium for culturing for 20 days; e: transferring a single callus to the propagation medium; and f: transferring to a rooting culture medium for 20 days;
FIG. 8 shows an effect of different concentrations of kanamycin on adventitious bud regeneration;
FIG. 7 shows an effect of different bacterial strain types on transformation efficiency;
FIG. 8 shows an effect of different concentrations of a bacterial strain on transformation efficiency;
FIG. 9 shows an effect of different infection time on transformation efficiency;
FIG. 10 shows an effect of different co-culture time on transformation efficiency;
FIG. 11 shows an effect of different pre-culture time on transformation efficiency;
FIG. 12 shows an effect of added different concentrations of acetosyringone on transformation efficiency; and
FIG. 13 shows a PCR test and GUS staining of a transgenic line of E. urophylla x E. grandis
DH3213; note: different numbers represent different lines, W is a control plant, and + is a pBI121 plasmid.
The present disclosure is further described below with reference to the examples, but the examples are not intended to limit the present disclosure.
An MS culture medium used in the following Example or Comparative Example refers to a universal culture medium with a known formula in the art. Its components and preparation method may be found in Toshio Murashige, Folke Skoog. A Revised Medium For Rapid Growth
And Bio Assays With Tobacco Tissue Cultures. Physiollogia Plantarum, 15: 473-497(1962); 1/2
MS culture medium is an MS culture medium in which a large number of elements are halved while other components remain unchanged. The WPM culture medium used is a universal culture medium with a known formula in the art. Its components and preparation method may be found in Lloyd G, Mc Brown B. Commercially-feasible micropropagation of mountain laurel,
Kalmia latifolia, by use of shoot-tip culture Combined Proceedings International Plant
Propagators Society, 30, 421-427(1980).
Example 1
A genetic transformation method of E. urophyila x E. grandis DH3213 of the present example includes the following steps: (1) Preparation of an explant material
Sterile plants of a fine cultivated variety E. urophylla x E. grandis DH3213 were used as a propagation material and cultured on a propagation medium (the propagation medium: a modified MS culture medium + 0.3 mg.I" of BAP + 0.05 mg.I"! of NAA + 30 g.1 of sucrose + 6.0 g.I'" of agar, where the modified MS culture medium was obtained by reducing the content of an ammonium nitrate component in an MS culture medium by half) for 20-25 d, and the culture was performed at an illumination intensity of 100-300 ymol.m=2.s™ for 16 h/d at a culture room temperature of 2312°C; and then subjected to dark culture for 10 d to obtain an albino shoot. (2) Cutting and pre-culture of the explant
A robust albino shoot was taken, a terminal bud and a resting bud were removed, and a 0.5-1.0 cm-length stem segment with a removed lateral bud primordium was cut as an explant; and the stem segment explant was inoculated on an adventitious bud induction culture medium (the adventitious bud induction culture medium: WPM + 0.25 mg.’ of TDZ + 0.1 mg." of NAA + 0.1 g.I" of hydrolysed casein + 0.5 mg.I"* of AgNO: + 30 g.I"" of sucrose + 6 gr’ of agar, with a pH of 5.8.) and pre-cultured for 3 d, and the culture was performed at an illumination intensity of 100-300 umol.m2.s"t for 16 h/d at a culture room temperature of 23+2°C. (3) Preparation of Agrobacterium
A GV3101 Agrobacterium strain carrying a pBl121 plasmid containing a GUS gene (and a target gene) preserved in a -80°C refrigerator was taken out, and triple streaking was performed on a plate containing 20 mg.I"" of rifampicin and 50 mg.lt of kanamycin to activate the
Agrobacterium. A single colony was picked and transferred into an LB liquid culture medium, and the bacteria were cultured in a constant temperature shaker at 28°C and 200 rpm for 16-24 h until an ODsgo value of a bacterial solution was 0.4-0.7; the cultured bacterial solution was centrifuged at 4°C and 5,000 g for 10 min, a supernatant was removed, and an equal volume of a resuspension solution (resuspension solution: 1/2MS + 200 uM of AS + 30 g.l! of sucrose, with a pH of 5.4) was added in a clean bench to resuspend the bacteria precipitated into the bacterial solution with the ODsge = 0.4-0.7. {4} Infection and co-culture
The stem segment explant precultured for 3 d in step (2) was taken out and soaked into the resuspended bacterial solution prepared in step (3) for 10 min, and during the period, the solution was slightly shaken at intervals to fully contact the Agrobacterium cells with the explant.
Then the explant was transferred to a sterile dry filter paper, and the surface bacterial solution was absorbed. Then the explant was transferred to a co-culture medium (co-culture medium:
WPM + 0.25 mg.I'" of TDZ + 0.1 mg.I'* of NAA + 0.1 g.I" of hydrolysed casein + 200 uM of AS + 30 g.l! of sucrose + 6 gl’ of agar, with a pH of 5.8.) for dark culture for 72 h. (5) Screening culture
The co-cultured stem segment explant was washed once by using a 200 mg. cefotaxime sodium solution, then washed with sterile water, and transferred to a screening culture medium (an adventitious bud induction culture medium containing 100 mg.I"! of Cef + 50 mg.l! of Tmt + 15 mg.I"" of Kan) for a screening culture, the culture medium was replaced every other 2 weeks, and the browning/dead explant was removed when the culture medium was replaced. An obvious green tiny bud was observed after the culture medium was replaced for 2-3 times. (6) Screening of a resistant plant
The explant with the green tiny bud was transferred to the propagation medium containing 100 mg.l! of Cef + 50 mg." of Tmt + 15 mg.I"! of Kan for induction and elongation culture of the adventitious bud. 2 weeks later after the culture, a part of leaf blades were marked and cut for
DNA extraction when a part of resistant buds were elongated to 2-3 cm and had 4-6 leaves. (7) Detection of a transgenic plant
A leaf of the resistant bud was taken on the clean bench, DNA was extracted by using a
CTAB method, and a PCR amplification was performed by using gene specific primers NPT!! and GUS. A line amplifying each target band at the same time was proved to be a transgenic line stably integrated into a nuclear genome of E. urophylla x E. grandis DH3213. At the same time, the detected line was further determined by using a GUS staining to ensure the expression of a GUS gene. The line was further verified as a transgenic line. (8) Propagation expanding and rooting
The identified transgenic plant was subjected to a propagation culture on the propagation medium containing 100 mg.I"* of Cef + 50 mg.I"* of Tmt + 15 mg." of Kan. A robust bud seedling with a length of 2-3 cm after propagation to a certain number was cut, and transferred a rooting culture medium (the rooting culture medium: 1/2MS culture medium + 0.3 mg.I"" of IBA + 20.0 g.I'" of sucrose + 6.0 g.I'" of agar + 0.05 g.I"* of activated carbon) for culture, and induced to a rooting seedling. Then the seedling was exercised and transplanted in time.
The method of the example was obtained after a large number of screening and optimization. The establishment and optimization of an adventitious bud induction regeneration system and a genetic transformation system involved in the method are described as follows: . Development of the adventitious bud induction culture medium and establishment of the adventitious bud induction regeneration system (1) Proportion of a culture medium and selection of an explant
Sterile plants of a fine cultivated variety E. urophylla x E. grandis DH3213 were used as a propagation material and cultured on a propagation medium (the propagation medium: a modified MS culture medium + 0.3 mg.I"t of BAP + 0.05 mg." of NAA + 30 g.I"! of sucrose + 6.0 a.I'" of agar) for 20-25 d in a culture room at an illumination intensity of 100-300 umol.m2.s"* for 16 h at a culture room temperature of 23+2°C. A leaf and a stem segment were respectively taken as an explant, an MS was as a basic culture medium, a combination of different concentrations of TDZ (0.05, 0.10, and 0.25 mg.l*) and IBA (0, 0.05, and 0.10 mg.I"') was added, 20 explants were placed in each plate, at least 180 explants were treated each time, 45 days later after the culture, a callus induction rate, an adventitious bud induction rate, and regeneration rate of callus-induced adventitious bud were investigated.
The result in Table 1 shows that the leaf and the stem segment were significantly different to treatment at different hormone concentrations. When the TDZ concentration was 0.10 mg.I"! and 0.25 mg.I", the callus induction rate of the leaf exceeded 80%; moreover, the adventitious bud induction rate was the highest, being 9.45% only when the TDZ concentration was 0.10 mg. in the leaf,. Most other treatments failed to induce an adventitious bud. The stem segment may obtain an adventitious bud regeneration in all the treatments. When the TDZ concentration was 0.25 mg.I", the highest adventitious bud induction rate of 22.78% was obtained, and the regeneration rate of callus-induced adventitious bud reached 65.80%. When the stem segment was taken as an explant, a highest genetic transformation efficiency may be obtained by exogenously adding TDZ at a concentration of 0.25 mg.I"* into the culture medium, and a formula of the culture medium was further adjusted on the basis.
Table 1
Effect of different hormone proportions on regeneration of E. urophylla x E. grandis DH3213
Type of Average Average
Average explant Number adventitious regeneration rate
TDZ IBA callus
Treatment of bud of callus- induced (mg.F) (mg. induction explants induction adventitious bud rate (%) rate (%) (%)
Stem 1 0.05 0 342 73.68% 16.21% 23.93% segment
Stem 2 0.05 0.05 342 94.36% 6.52% 6.96% segment
Stem 3 0.05 0.10 360 95.83% 6.39% 6.78% segment
Stem 4 0.10 0 300 24.45% 12.22% 51.67% segment
Stem 5 0.10 0.05 360 96.67% 4.45% 4.65% segment
Stem 6 0.10 0.10 360 96.94% 6.81% 7.14% segment
Stem 7 0.25 0 360 37.78% 22.78% 65.80% segment
Stem 8 0.25 0.05 360 97.50% 16.11% 16.65% segment
Stem 9 0.25 0.10 360 95.55% 15.00% 15.79% segment 10 Leaf 4.05 0 310 36.09% 6.91% 16.98% 11 Leaf 0.05 0.05 360 93.05% 0.00% 0.00% 12 Leaf 405 0.10 300 96.00% 0.00% 0.00% 13 Leaf 449 0 360 70.28% 9.45% 17.29% 14 Leaf 419 0.05 300 89.72% 0.00% 0.00% 15 Leaf 0,10 0.10 240 93.75% 0.00% 0.00% 16 Leaf 425 0 360 85.83% 3.61% 4.27% 17 Leaf 405 0.05 180 90.00% 0.00% 0.00% 18 Leaf 425 0.10 340 93.33% 0.00% 0.00% (2) Selection of a basic culture medium
To further increase an adventitious bud induction rate, a test was performed by adjusting different basic culture mediums (MS, BS, WPM, 1/2MS, 1/2B5, and 1/2WPM). To obtain more explants, the sterile plants of E. urophylla x E. grandis DH3213 as a propagation material were continuously subjected to dark culture for 10 d. A robust albino shoot was taken, a terminal bud and a resting bud were removed from the stem, and a 0.5-1.0 cm-length stem segment with a removed lateral bud primordium was cut as an explant. The same explant material was used for all the following tests. Each test was repeated for 3 times and at least 180 explant materials were used each time. The results (Table 2, FIG. 1) show that the adventitious bud induction rate was improved to 38.39% on the WPM culture medium, and the regeneration rate of callus- induced adventitious bud was improved to 71.51%, which was significantly higher than those on the MS medium.
Table 2 Effect of different culture mediums on regeneration of E. urophylla x E. grandis DH3213
Type of Average Average regeneration
Average callus
Treatment culture adventitious bud rate of callus-induced , induction rate (%) / / m medium induction rate (%) adventitious bud (%) 1 MS 63.57% 38.45% 54.80% 1/2MS 46.65% 21.96% 46.56% 3 B5 57.14% 29.25% 47.86% 4 1/2B5 38.80% 13.18% 27.66% 5 WPM 42.72% 38.39% 71.51% 6 1/2WPM 32.50% 18.67% 42.98% (3) Treatment with the addition of hydrolysed casein
To further increase the adventitious bud induction rate, a test was performed by adding 5 different concentrations of hydrolysed casein (0, 100, 200, 300, 400, and 500 mg.I'!) to the culture medium, each test was repeated for 3 times, and at least 180 explant materials were used each time. The result (Table 3, FIG. 2) shows that although the hydrolysed casein may not significantly improve the callus induction rate, it may slightly improve the adventitious bud induction rate, at the same time, may greatly improve the regeneration rate of callus- induced adventitious bud, and may significantly improve the regeneration rate of callus induction adventitious bud to 84.32% when 100 mg.I"" of the hydrolysed casein was added.
Table 3 Effect of different concentrations of hydrolysed casein added on regeneration of
E. urophylla x E. grandis DH3213
Average Average regeneration
Type of culture Average callus
Treatment adventitious bud rate of callus-induced medium (mg.I"") induction rate (%) 3 induction rate (%) adventitious bud (%) 1 0 50.39% 23.38% 52.89% 5 100 45.82% 32.78% 84.32% 3 200 41.94% 31.22% 77.54% 4 300 48.89% 34.44% 70.80% 5 400 41.61% 26.78% 74.95% 6 500 41.94% 28.31% 62.81%
(4) Treatment with the addition of different concentrations of NAA
To further increase the adventitious bud induction rate, a test was performed by adding different concentrations of NAA (0, 0.05, 0.10, and 0.15 mg.I'!) to the culture medium, each test was repeated for 3 times, and at least 180 explant materials were used each time. The results (Table 4, FIG. 3) show that the NAA may greatly improve the callus regeneration rate and the adventitious bud regeneration rate. When the concentration of the NAA was 0.10 mg.lt, the callus induction rate reached 99.33% and the adventitious bud regeneration rate reached 64.15%. Nevertheless, the regeneration rate of callus-induced adventitious bud was reduced, and a small amount of explants may be still browned and dead.
Table 4 Effect of different concentrations of NAA added on regeneration of
E. urophylla x E. grandis DH3213
Type of /
Average Average regeneration culture Average callus
Treatment adventitious bud rate of callus-induced medium induction rate (%) induction rate (%) adventitious bud (%) (mg.I' 1 0 54.50% 26.78% 50.62% 9 0.05 89.14% 41.21% 46.21% 3 0.10 99.33% 64.55% 64.15% 4 0.15 96.67% 36.24% 41.33% (5) Addition of silver nitrate
To further increase the adventitious bud induction rate, a test was performed by adding different concentrations of silver nitrate (0, 0.1, 0.3, 0.5, 1.0, and 2.0 mg.I"") to the culture medium, each test was repeated for 3 times, and at least 180 explant materials were used each time. The results (Table 5, FIG. 4) show that the silver nitrate may significantly increase the adventitious bud regeneration rate while maintaining the callus induction rate. When the silver nitrate was at 0.5-2.0 mg.I"", the regeneration rate exceeded 90%, but when the silver nitrate was at 2.0 mg.I"', the adventitious bud was short and difficult to grow high. Therefore, the concentration of the silver nitrate was selected to be 0.5 mg.I"}, and the adventitious bud regeneration rate reached 92.88%.
Table 5 Effect of different concentrations of silver nitrate added on regeneration of
E. urophylfa x E. grandis DH3213
Type of
Average Average regeneration culture Average callus
Treatment adventitious bud rate of callus- induced medium induction rate (%) induction rate (%) adventitious bud (%) (mg. 1 0 99.58% 65.83% 66.12% 2 0.1 100.00% 80.06% 80.06% 3 0.3 100.00% 82.14% 82.14% 4 0.5 96.67% 87.89% 92.88% 1.0 96.25% 87.08% 90.48% 6 2.0 96.67% 90.42% 93.48% (6) Establishment of a regeneration system 5 Through the above optimization, a complete adventitious bud induction regeneration system was established. The stem segment explant was transferred to the adventitious bud induction culture medium (the adventitious bud induction culture medium: WPM + 0.25 mg.I"! of
TDZ + 0.1 mg.l of NAA + 0.1 g.I'* of hydrolysed casein + 0.5 mg." of AgNO; + 30 g.I"" of sucrose + 6 gl’ of agar, with a pH of 5.8) to be cultured, 3 d later, the stem segment explant began to expand, after 10 d of the culture on the adventitious bud induction culture medium, a red and green callus was formed, and after 25-30 d of the culture, a large number of small adventitious buds may be observed to form; and the adventitious buds were transferred to the propagation medium (propagation medium: a modified MS culture medium + 0.3 mg.l' of BAP + 0.05 mg.I'" of NAA + 30 g.I'! of sucrose + 6.0 g.l’ of agar) to be cultured for 15 d, the buds began to grow high and were continuously cultured on the propagation medium for 30 d to form green and robust buds, 2-3 cm bud seedlings were cut and transferred to a rooting culture medium (rooting culture medium: 1/2MS culture medium + 0.3 mg.I"* of IBA + 20.0 g.I"" of sucrose + 6.0 g.I'" of agar + 0.05 g.l’ of activated carbon) to be cultured for 20 d, and robust buds were formed (FIG. 5).
II. Optimization of the genetic transformation system
The explant material used in the process of genetic transformation optimization was stem segment, and each test treatment was repeated for 3 times, and the number of the explants used in each repetition exceeded 100.
(1) Type of bacterial strain
When the stem segment of the E. urophylla x E. grandis DH3213 was infected with different
Agrobacterium tumefaciens strains (GV3101, LBA4404, EHA105, and AGL1), it was found that a GUS staining rate (70.8%) of the stem segment of the GV3101 was significantly higher than that of the other three bacterial strains (FIG. 7); therefore a subsequent test was performed with the GV3101. (2) Concentration of bacterial solution
To determine an optimal growth stage of Agrobacterium tumefaciens to improve transformation efficiency, Agrobacterium at six different growth stages (ODso = 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7) was used to infect the explant. The result (FIG. 8) shows that the transformation efficiency increased with an increased concentration of the Agrobacterium tumefaciens. There was no significant difference between 0.4-0.7. Therefore, the bacterial solution with ODsgs = 0.4-0.7 was selected for the subsequent transformation test. (3) Infection time
Infection time plays an important role in a genetic transformation process of a plant. As in the result of the effect of different infection time on transformation efficiency (FIG. 9), the transformation efficiency of the explant increased with an increase of time during the infection time of 5-40 min. The transformation efficiency of a single leaf did not show a significant difference 10 min later after the infection, but the longer infection time caused larger damage to the explant, such that the infection time of 10 min was used. (4) Co-culture time
Co-culture time after the Agrobacterium infection affects the genetic transformation efficiency. The result (FIG. 10) shows that when the stem segment of the E. urophylla x E. grandis DH3213 was co-cultured with the Agrobacterium for 3 d (72 h), the transformation efficiency was highest and reached 83.0%. Prolonged co-culture time failed to improve the transformation efficiency, but rather the long co-culture caused bacterial overgrowth which led to an explant death. The co-culture time of 72 h was used for transformation, so as to allow sufficient time for T-DNA of the Agrobacterium to be integrated into a plant genome and avoid a difficulty of sterilization after the explant was transferred to a screening culture medium due to the long-term co-culture. (5) Pre-culture time
Pre-culture time of the explant affects the transformation efficiency. Pre-culture may promote generation of cells on a surface of a damaged part of an explant, promote metabolism of the cells to generate a vir region induced compound, and promote a fusion of bacteria and plant tissues. Before infected with the Agrobacterium, the stem segment explant was cultured on the adventitious bud induction culture medium for 0-9 d. The result (FIG. 11) shows that the stem explant precultured for 3 d had the highest transformation efficiency (80.6%), and the increase of the pre-culture time significantly decreased the transformation efficiency, such that the pre-culture time of 3 d was used. (6) Addition amount of acetosyringone
Phenolic substances of acetosyringone (AS), etc. may promote the activation and expression of genes in a vir region on a Ti plasmid, thereby promoting the transfer of T-DNA.
The effect of promoting gene genetic transformation was different due to the added concentration. When the explant was infected with the Agrobacterium, different concentrations of AS (0, 100, 200, 300, and 400 uM) were added to a resuspension to explore its effect on genetic transformation. The result (FIG. 12) shows that the addition of the AS may significantly improve the transformation efficiency, and the addition of 200 pM of AS during the infection may enable the GUS staining rate of the stem segment to reach 90.8%, and significantly promoted the transformation efficiency of the E. urophylla x E. grandis DH3213. Therefore, 200 uM of AS was also added to the co-culture medium in a subsequent genetic transformation test. (7) Kanamycin screening
In order to conduct screening of a transgenic plant, a test of sensitivity to kanamycin by adventitious bud induction needed to be performed by adding different concentrations of kanamycin (0, 4.0, 8.0, 12.0, 15.0, 30.0, 45.0, and 60.0 mg.I'") to the adventitious bud induction culture medium, each test was repeated for 3 times, and at least 100 explant materials were used each time. The result (FIG. 6) shows that the kanamycin may significantly inhibit adventitious bud regeneration. When the kanamycin concentration was 30.0 mg.lt, the regeneration was completely inhibited. When the kanamycin concentration was 15.0 mg.lt, the adventitious bud could hardly be regenerated. In order to ensure that a transformed plant may be screened, the kanamycin concentration was selected to be 15.0 mg.I"*. (8) Screening and detection of a transgenic plant
Under the optimized conditions described above, a batch of explant materials (more than 300 explants) was cut and pre-cultured on the adventitious bud induction culture medium for 3 d, Agrobacterium tumefaciens GV3101 carrying a pBI121 plasmid was cultured to ODggs = 0.4- 0.7, and after each explant was infected with the bacterial solution for 10 min, the explant was transferred to the adventitious bud induction culture medium for dark culture for 72 h. The explant was taken out, the bacterial solution on the surface of the explant was washed away with sterile water, the water on the surface was sucked dry with a sterile dry filter paper, and then the explant was transferred to the adventitious bud induction culture medium containing 100 mg.l! of Cef, 50 mg.l' of Tmt, and 15 mg.I"! of Kan to be cultured. A new culture medium was replaced every other 2 weeks, and resistant adventitious buds may be observed after replacement for two times.
To further verify whether the T-DNA in the plasmid carried by the Agrobacterium tumefaciens was inserted into a nuclear genome of a plant to be transformed, the nuclear genome DNA of the plant to be transformed was extracted and subjected to a PCR amplification with a specific primer pair npt!! and uidA genes (GUS gene). The result shows that uidA (1.0 k) gene has been successfully integrated into the nuclear genome of 8 transformation lines.
The identified transgenic lines were further analyzed by GUS histochemical staining, and regenerated resistant buds were blue (FIG. 13); and a final stable transformation efficiency may reach 2.67%. Thus, a high-efficiency and stable genetic transformation system of the fine cultivated variety E. urophyila x E. grandis DH3213 was established.
Claims (9)
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| US20060101536A1 (en) * | 2004-11-05 | 2006-05-11 | Arborgen, Llc | Eucalyptus urophylla transformation and regeneration |
| CN101897298A (en) * | 2010-08-10 | 2010-12-01 | 普罗米绿色能源(深圳)有限公司 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
| CN104285815A (en) * | 2014-10-29 | 2015-01-21 | 广西壮族自治区国有东门林场 | Tissue culture and rapid propagation method of E. urophylla*E. Grandis DH32-13 |
-
2023
- 2023-02-10 CN CN202310091854.4A patent/CN116286964A/en active Pending
- 2023-06-20 NL NL2035132A patent/NL2035132B1/en active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060101536A1 (en) * | 2004-11-05 | 2006-05-11 | Arborgen, Llc | Eucalyptus urophylla transformation and regeneration |
| CN101897298A (en) * | 2010-08-10 | 2010-12-01 | 普罗米绿色能源(深圳)有限公司 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
| CN104285815A (en) * | 2014-10-29 | 2015-01-21 | 广西壮族自治区国有东门林场 | Tissue culture and rapid propagation method of E. urophylla*E. Grandis DH32-13 |
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| LLOYD GMC BROWN B.: "Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture", COMBINED PROCEEDINGS INTERNATIONAL PLANT PROPAGATORS SOCIETY, vol. 30, 1980, pages 421 - 427, XP001525827 |
| TOSHIO MURASHIGEFOLKE SKOOG: "A Revised Medium For Rapid Growth And Bio Assays With Tobacco Tissue Cultures", PHYSIOLLOGIA PLANTARUM, vol. 15, 1962, pages 473 - 497, XP002577845, DOI: 10.1111/j.1399-3054.1962.tb08052.x |
| WANG XIAOPING ET AL: "Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla x Eucalyptus grandis DH32-29 in Southern China", FRONTIERS IN PLANT SCIENCE, vol. 13, 29 September 2022 (2022-09-29), CH, XP093108394, ISSN: 1664-462X, DOI: 10.3389/fpls.2022.1011245 * |
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| CN116286964A (en) | 2023-06-23 |
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