JP2003143987A - Gene transformation method of zoysia grass - Google Patents

Abstract

PROBLEM TO BE SOLVED: To carry out a gene transformation of zoysia grass with the gene by vector infection through A. tumefaciens. SOLUTION: This gene transformation method of zoysia grass (the genus Zoysia) comprises the following processes: i) a process for inducing and growing callus of the zoysia grass on a modified MS medium containing various hormones, ii) a process for introducing bialaphos-resistant bar gene by infecting the callus of the zoysia grass with Agrobacterium cells, iii) a process for coculturing the callus of the zoysia grass and Agrobacterium cells in a culture medium containing acetosiringone without containing 2,4-dichlorophenoxyacetic acid and calcium, iv) a process for removing Agrobacterium cells from the coculturing medium and v) a process for regenerating transgenic zoysia grass.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to zoysia grass (Zoysia jap).
onica Steud. ) (Korean
(also referred to as glass)). The present invention may be applicable to other closely related turfgrass species. The present invention also provides transgenic turfgrass plants that are resistant to herbicides, as well as experimental methods and processes for the production of such herbicide resistant turf.

[0002] The present invention is based on Zoysia japo
nica Steud. ) (Also known as Korean turf), an efficient Agrobacterium
tumefaciens mediated gene transformation method, optimized medium composition and culture conditions that greatly influence this transformation efficiency, and transgenic turfgrass plants and bialaphos with herbicide resistance
hos) provides relevant experimental tools for developing such transgenic turfgrass having a resistance gene (bar).

[0003]

BACKGROUND OF THE INVENTION Shiva is one of the most important species of turfgrass and is widely cultivated in Far East Asia including Korea, Japan and eastern China and temperate regions around the world. The growing area of turf has expanded rapidly in the United States and other countries in recent years, primarily due to its extraordinary characteristics, such as drought resistance and the ability to rapidly recover from traffic damage. To do. In addition, turf grows well on thin soils in virtually all climates. Due to these useful traits, Shiva is widely used in golf courses, stadiums, roadsides, residential gardens, and riverbanks. As the market size grows rapidly, consumers demand new varieties with improved resistance to pathogens, herbicides and various environmental stresses. Until recently, such new traits in turfgrass have been developed, mainly using classical breeding methods. However, more and more laboratories and laboratories are striving to apply molecular biology methods to genetically engineer turfgrass (Inokuma).
Et al., 1998; Park and Ahn 1998). Because it is now possible to develop or modify useful traits in a predictable manner using these methods.

Many monocots are Agrobacte
Not easily infected by Rium tumefaciens, but efficient A. The tumefaciens-mediated transformation system is used in several species of the grass family (eg, rice (Hiei)).
Et al., 1994; Rashid et al., 1996), corn (Ishida et al., 1996) and wheat (Ch).
eng et al., 1997) have been successfully developed in recent years. However, unfortunately, such A. tu
The mefaciens-mediated transformation method is carried out by
entgrass) (Yu et al., 2000) has not yet been established for turfgrass (Chai and Sticklen, 1998). In particular, genetic transformation of turf is further hindered by some additional technical problems. For example, turfgrass seed germination rates are very slow, and the production of renewable callus is difficult (Asano 1989).

Callus morphology has been demonstrated in various plant species (Armstrong and Gre.
en 1985; Ke and Lee 1996; Luo.
And Jia 1998), closely related to plant renewal potential. In recent years, the present inventors have established an efficient callus induction and plant regeneration system for Shiba (Bae et al., 2
001), which was filed as Japanese Patent Application No. 2001-223278 on July 24, 2001.

[0006]

SUMMARY OF THE INVENTION In accordance with the present invention, A.
It is now possible to genetically transform turf with the gene of interest by tumefaciens-mediated infection.

Some potential target traits for genetic transformation of turf include improved surface planting capacity, resistance to traffic damage, rapid recovery after damage,
Resistance to biotic and abiotic stresses, manipulation of growth rate (acceleration or delay), and shade avoidance.
Of particular interest is that growth rate and / or shade avoidance can be manipulated, resulting in dramatically reduced maintenance costs for irrigation and mowing.

With the recent rapid accumulation of molecular biology techniques and the establishment of efficient tissue culture and gene transformation systems for plants, any gene of agronomic importance is now
It can be easily introduced into any desired plant for the purpose of enhancing crop yield and quality and environmental adaptability.
In the present invention, the inventors have found that efficient Agroba
Cerium tumefaciens mediated method for transformation of turf, and transgenic turf plants having herbicide resistance are provided.

As used herein, the term "gene transformation" refers to the means for introducing a gene or genetic material into a higher plant of interest in a predictable manner.
This gene or genetic material is stably integrated into the plant genome and is inherited through generations.

[0010]

(Summary of the Invention) The present invention provides a reliable Agrobacterium against grass.
Regarding the tumefaciens-mediated transformation method, this transformation method can be conventionally used for the genetic transformation of turf or closely related turfgrass species.

The present invention also provides a method for genetically transforming Shiva (genus Zoysia), which comprises the following steps: i) Inducing callus of Shiva on a modified MS medium containing various hormones. And the step of growing; ii)
The step of infecting Agrobacterium cells to the callus of this shiba and introducing the bialaphos resistance bar gene; iii) 2,4-dichlorophenoxyacetic acid and calcium in a coculture medium containing acetosyringone, Callus and Agrobacte
co-culturing rymium cells; iv) eliminating Agrobacterium cells from the co-cultivation medium;
And v) the step of regenerating the transgenic grass.

In addition, Agrobacterium cells carry the pGPT-HB transformation vector, and 3
Type callus (our previous application number 2001-22
3278) is co-cultured in the co-culture medium for 5-7 days.

The present invention also provides a transgenic turfgrass bacilli by the above-mentioned method, wherein the transgenic turfgrass bas is under the control of a plant-specific promoter.
The r gene has been stably transformed. A particular promoter is the ubiquitin promoter.

The important medium components or culture conditions evaluated in the present invention include callus type, coculture period,
2,4-D (2,4-dichlorophenoxyacetic acid), Ca
Cl ₂ , and acetosyringone. The highest transformation efficiency was obtained when type 3 callus was co-cultured on a medium not containing 2,4-D for 5 to 7 days. Furthermore, calcium removal during co-culture and 30-7
Inclusion of 0 mg / l acetosyringone greatly enhanced this transformation efficiency.

This optimized transformation protocol is
Up to 20.5% of shoots plated on selective medium showed herbicide resistance when used for the transformation of turf with the bar gene.

Further, the present invention provides a transgenic turfgrass plant having herbicide resistance. This transgenic turf contains 2 g of a 5 g / l herbicide solution.
During the week it could survive even after daily spraying and eventually grew to maturity. On the other hand, the control plant
Growth was arrested and death when treated under the same experimental conditions.

Accordingly, the present invention provides a herbicide-resistant transgenic Shiva callus (registration number KCTC-1007).
6BP) is further provided.

The present invention provides a method for genetically transforming Shiva (genus Zoysia), which comprises the following steps: i)
Inducing and growing the callus of the turf on modified MS medium containing various hormones; ii) Infecting the callus of the turf with Agrobacterium cells and introducing the bialaphos resistance bar gene; ii
i) a step of co-cultivating callus and Agrobacterium cells of Shiva in a co-culture medium containing acetosyringone without 2,4-dichlorophenoxyacetic acid and calcium; iv) Agr from the co-culture medium
providing a method comprising the steps of removing O. bacterium cells; and v) regenerating the transgenic turf.

In one embodiment, Agrobac
terium cells carry the pGPT-HB transformation vector.

In one embodiment, a type 3 callus is
Co-culture for 5-7 days in the co-culture medium.

[0021] The present invention also provides a transgenic turfgrass according to the above method, which is stably transformed with the bar gene under the control of a plant-specific promoter.

In one embodiment, the bar gene is expressed under the control of the ubiquitin promoter.

The invention further provides a biaraphos resistant ba.
r gene (KCTC-10076BP) was introduced,
Provide transgenic Shiva callus.

The above callus is based on the Budapest Treaty, dated September 21, 2001, and is the Korea Collection for Type Culture (Korea Collect).
ionfor Type Cultures) (# 52
Oun-Dong, Yuson-ku, Taejo
n, Seoul, 305-333, Republico
f Korea) with registration number KCTC-10076BP
Deposited at.

[0025]

DETAILED DESCRIPTION OF THE INVENTION Genetic transformation of agronomically important plants is a potential method for developing new varieties of plant species with novel or improved useful traits. Is. Target traits associated with crop plants include improved or delayed growth rates, enhanced resistance to biotic and abiotic stresses, increased yield, and flexible adaptability to environmental changes. Pre-requisites for successful genetic transformation of plants are:
A robust system for tissue culture and plant regeneration, and a robust delivery device for genetic material. With recent advances in plant molecular biology and methodology, any gene of interest can be easily delivered to any plant species of interest. However, some crop plants (especially monocotyledons) are awkward to genetically engineer and it is not easy to perform plant regeneration in common laboratories. Turfgrass is widely used for a variety of purposes, including golf courses, lawns, residential gardens, and recreational parks,
And it has emerged as a potential commercial target plant for plant biotechnology applications in recent years. However, efficient tissue culture and gene transformation systems have not been developed. As a result, biotechnological genetic manipulation of turfgrass has not yet advanced.

The present invention is directed to zoysiagras.
s) provides medium composition and culture conditions optimized for efficient gene transformation and plant regeneration from seed-derived callus. Shiba, a subtype of warm-type turfgrass, is widely distributed and cultivated in temperate regions including Far East Asia. Moreover, cultivated areas have been expanding rapidly in recent years due to several advantages, such as resistance to drought damage and traffic damage. In the present invention, the most efficient transformation was achieved when type 3 callus was co-cultured for 6 days in a co-culture medium containing 2,4-D but having 50 mg / l acetosyringone. . Calcium, which is known to improve transformation efficiency in other plants, has a negative effect. Plant tissue culture and regeneration experiments are routinely performed in well-established plant molecular laboratories and are well known in the art.

The present invention also relates to herbicide-resistant grass plants, which can survive for two weeks every day even after spraying with the bialaphos solution. Growing this transgenic turf on golf courses and arenas makes it easier to selectively remove weeds by simply spraying the herbicide and dramatically reduces maintenance costs. In addition, a small amount of fertilizer is expected to be needed to grow this transgenic grass. This is because weeds that consume nutrients are efficiently removed. The transgenic turf did not show any morphological distortion and grew well and was indistinguishable from the parent plant.

The present invention may also be applied to other closely related turfgrass species as well as other monocotyledonous species, which probably belong to the family Gramineae. Experimental procedures for the development of such genetically engineered plants are well known in the art. The present invention provides an efficient A. cerevisiae of Shiva by using a highly renewable callus line. tumefa
It is the first invention on ciens-mediated transformation.

[0029]

(Example) (Preparation of renewable callus) Approximately 100 grains of Shiba (Zoy
sia Japana Steud. The matured seeds in 1) were surface sterilized with 1 ml of 2% sodium hypochlorite in a 2 ml centrifuge tube for 15 minutes with stirring using a tube mixer. The seeds were then washed 3 times with sterile double distilled water. About 10 for callus induction
0 seeds, 3% sucrose (w / v), 100 mg
/ L α-ketoglutaric acid, 4 mg / l thiamine-HC
1, 2 mg / l 2,4-D, 0.2 mg / l BA,
MS loaded with filter paper (Murasige and Skoog 1 containing 0.2% gellite (w / v)).
962) Placed in medium Petri dish. This medium was added to 1.2
The pH was adjusted to 5.8 with HCl before autoclaving at ˜1.3 kg / cm ² pressure and 121 ° C. for 20 minutes. Callus induction 26 ± 1 for 3 months in complete darkness
It was carried out in a culture room at ° C. Petri dishes (diameter 9
0 mm) contains about 30 ml of medium and contains parafilm (American National Can,
USA). 30 provided by fluorescent tube
After a further 3 days of incubation under continuous white light with an intensity of μmolm ⁻² s ⁻¹ , only green tissue was treated with 1 mg / l 2,4-D and 0.2 mg / l.
Transferred to MS medium containing BA and subsequently cultured in the dark for further growth. After culturing in the dark for 5 weeks, callus from independent seeds with green color was transferred,
It was then further cultivated in the dark in filter media-loaded MS medium containing a combination of different hormones of 2,4-D and BA. Four types of callus were finally obtained. Four
After subculture at weekly intervals, callus types 1, 2, and 4 were developed in medium containing 1 mg / l 2,4-D and callus type 3 at 4 mg / l 2,4.
Generated in -D containing medium (Figure 1). Each Petri dish (90 mm diameter) contains 25 ml of medium and M
icropore Surgical Tape (3M
(Health Care, USA).

Characterization of four callus types In order to investigate the morphological changes that occur in subcultures of type 1 callus, type 1 callus was subjected to 2,4-D (1,2, 4,
And 8 mg / l) and BA (0, 0.01, and 0.1 mg / l) were transferred to MS medium containing various hormone combinations. After 5 weeks of subculture in the dark, 4 callus types were observed. Each callus type was weighed and the weight percentage was calculated. Type 2, 3 and 4 callus were also tested in the same manner as for type 1 callus.

(Optimization conditions for Agrobacterium infection) AIG containing pIG121Hm. tune
Faciens disarmed strain EHA
101 was used for this experiment. pIG121Hm T-
DNA includes hygromycin resistance gene (hpt), kanamycin resistance gene (npt), and intron-.
It contained the gus gene (uidA). Agroba
50 cells of C. tumefaciens
mg / l hygromycin, 100 mg / l kanamycin, and 100 mg / l spectinomycin (pGP
(TV-HB) was grown in 28 ml of 100 ml Erlenmeyer flask containing 20 ml of LB medium (Table 1) supplemented with shaking at 160 rpm overnight.

Table 1 shows the different media and their composition used in the transformation system of Shea.

[0033]

[Table 1] Cells in 10 ml suspension culture were transferred to 50 ml polypropylene tubes (Becton Dickinson).
Labware, USA) for 20 minutes 250
Collect by centrifuging at 0 rpm, and 1
Resuspended in 0 ml of liquid infection medium (LqMSAS in Table 1) by gentle vortexing.

Acetosyringone was prepared at a concentration of 100 mg / ml by dissolving the appropriate amount of powder in dimethylsulfoxide and stored in the dark at 4 ° C. This was added to sterile media to the appropriate final concentration when needed. Callus grown in MSCG4 medium was immersed in Agrobacterium cell suspension for 1 minute. After dehydration on sterilized filter paper, the callus was treated with a co-cultivation medium containing 4 mg / l 2,4-D (M
(SAS) in the dark for 15 days at 26 ± 1 ° C. After co-cultivation, the callus was treated with a surfactant (0.0
Thoroughly wash in sterile double-distilled water supplemented with 2% pluronic F68) by vortexing the wash solution until clear, and finally 1000 mg / l carbenicillin (cal).
benicillin) and 0.02% surfactant in sterile double distilled water. After washing, half of this callus was used for GUS activity assay by the method described by Schenk et al. (1998).

Several factors were tested to optimize transformation efficiency. (1) For comparison of different callus types for invasion, callus types 1, 2, and 3 were co-cultured in MSAS medium without 2,4-D for 6 days; (2) Co-existence For comparison of culture period,
Callus 3, 6, 9, 12, 15, 18, 21, 2
Co-cultured for 4, and 27 days; (3) different amounts of 2,4-D (0, 1, 2,
4, and 8 mg / l), CaCl ₂ (0, 11, 11
0, 220, and 440 mg / l), and acetosyringone (0, 10, 50, 100, and 200 mg)
/ L) was added to the MSAS medium.

(Co-culture of pGPTV-HB) A. Optimized conditions for tumefaciens infection included the use of type 3 callus as a donor, a coculture period of 9 days, exclusion of 2,4-D and CaCl ₂ from the medium, and the use of 100 mg / l acetosyringone. I was out. All media used in the experiments and their composition are summarized in Table 1. After co-culturing with pGPTV-HB, the callus was transferred to MSCG medium with filter paper,
Then, the cells were cultured in the dark for 2 to 4 weeks. The callus was then transferred to MSSI medium with filter paper for shoot induction. Various concentrations (125, 250, and 500 mg
/ L) carbenicillin was added to the shoot induction medium.
The induced shoots were subsequently transferred to MSRS medium for 4 weeks of rooting and selection. 5 mg / l bialaphos and 10 mg / l hygromycin were used for the selection of bialaphos- and hygromycin-resistant plantlets. Rooted plants on selective medium were transferred to MSPG medium without antibiotics and bialaphos and grown further. Then, the fully grown plants are transferred to a pot containing soil and
Grow in an environmentally controlled growth chamber set at 80% relative humidity, and an 18 hour photoperiod at Iradiance with an intensity of 30 μmol m ⁻² s ⁻¹ provided by a cold white fluorescent tube. When the roots were fully developed, the pots were transferred to the greenhouse.

(Analysis of Transgenic Sheep Plant) In order to test whether or not the putative transgenic grass plant is resistant to the herbicide, 5 g / l of the herbicide was added to the putative transgenic grass plant. Solution (1g / l
Biaraphos, Meiji Seika, Japan)
Was sprayed. In addition, to study whether the herbicide resistance gene was integrated into the plant genome, Ro
Genomic DNA was isolated from the leaves of transgenic plants according to the method of Ger and Bendich (1985). 20 μg of genomic DNA digested with HindIII or BamHI was separated on a 0.8% agarose gel using standard methods (Southern 19
75) nylon membrane (Hybond-N
⁺ ) And probed with ³² P-labeled hpt or bar gene sequences. Random priming method (Sambrook)
Et al., 1989).

(Results) (Callus morphology suitable for Agrobacterium infection) Cultivation of mature seed-derived type 1 callus in MS medium containing various combinations of 2,4-D and BA was carried out by mixing a mixture of type 1 to 4 callus. Occurred (Table 2).

Table 2 shows the different growth of Shiva type 1 callus in media containing different combinations of growth regulators.
10 mg of callus type 1 was grown in MS medium with various combinations of 2,4-D and BA, and 2 g / l gellite in the dark for 5 weeks at 28 ° C. Each value represents an average calculated from 10 calli. Callus morphology was evaluated by: 1 = slightly white to yellow, dense and non-friable, 2 = white, dense and friable, 3 = yellow, dense and very friable, 4 = translucent,
Soft and watery.

[0040]

[Table 2] The resulting four types of callus were morphologically characterized. Type 1 callus is slightly white to yellow or light green in color and has a dense and non-friable structure. Multiple shoot primordia were observed on the surface of some type 1 callus (Fig. 1
A). Type 2 callus is white and has a dense and friable structure (FIG. 1B). Type 3 callus was yellow and had a dense and very friable structure (Figure 1C). Type 4 callus is unique in that it has a soft, watery appearance and is translucent (FIG. 1D). Type 1 to 3 callus was reproducible, but type 4 callus was not reproducible. Best growth and incidence of type 1 and type 2 callus (67.4 ± 9.1 mg, 58%, and 3 respectively)
2.3 ± 5.2 mg, 28%) and best growth and incidence of type 3 callus (33.7 ± 11.3 mg, 59)
%) Were obtained in MS medium containing 0.01 mg / l BA in combination with 1 and 4 mg / l 2,4-D, respectively (Table 2). The incidence of non-renewable type 4 callus was reduced by 1 / 1.8 to 1 / 3.4 times by adding 0.01 mg / l BA in combination with 2,4-D. Therefore, 0.01 mg / l BA in combination with 1 or 4 mg / l 2,4-D was used for further growth of type 1-2 or type 3 callus, respectively.
The subculture of type 2 and type 3 callus was similar to that of type 1 callus subculture, and the type 1 type 2 type 3 callus
And a mixture of callus and type 4 callus. However, subculture of type 4 callus yielded only type 4 callus. Interestingly, callus types 1 to 3 showed morphological variations when using different combinations of hormones. Furthermore, when callus was subcultured in the light for 3 days before subculture, green callus appeared. From this green callus, small albino plants regenerated in shoot induction medium (Fig. 2C). By suppressing the development of type 4 callus, and by selecting green callus for each subculture step, the reproducibility of green plants from callus lines was over 2 years without any harmful effects. (FIG. 2C).

A close relationship between callus morphology and shoot renewal has been demonstrated in various plant species (Armstrong and Green 1985; K.
e and Lee 1996; Luo and Jia 19
98). Long-tailed squirrel (Kentucky bluegr)
ass) (Poa progressions L.)
Callus derived from coleoptile and embryo can be classified into four types based on morphology and friability (Ke and L
ee 1996). The callus type 4 of calliphora was soft, unstructured, and translucent, from which no shoots were regenerated. This result is similar to that obtained with Shiva. Induction of type 4 callus in stag beetles could only be regulated by auxin, whereas induction of turf could be regulated by decreasing cytokinin levels. Astrangalus a
In dsurgens, callus derived from hypocotyl was also classified into four types (Luo and Jia 1998),
None of the four callus types showed morphological changes, even after subculture for 8 months. On the other hand, with the exception of type 4, each subculture of type 1-3 Shiva callus types yielded 4 callus types. Shiva, Nagahaga, and Ast
These differences between rangalus adults are reflected in the differential reproducibility between different species.

(Factors Affecting Transformation Efficiency) (Effect of Callus Type) Type 1 and type 2 callus grown on MSCG1 medium and type 3 callus grown on MSCG4 medium were treated with A. tumefaciens EHA
101 (pIG121Hm) was co-cultured with 2,4-D-free co-culture medium for 6 days, and measurement of GUS expression was used to examine the relationship between different callus types and transformation efficiency. (Table 3, Figure 2A).

Table 3 shows the transformation efficiency of different callus types. MSA not including 2,4-D
26% on S medium (see Table 1) in the dark for 6 days
Co-culture was performed at ℃. Three replicates were measured and averaged.

[0044]

[Table 3] GUS expression was detected on type 1 and type 3 callus, but no locus on type 2 callus. Type 3 callus produced the highest GUS expression frequency of 721 ± 201 locus per gram fresh weight of callus. This was 34 times higher than that observed in type 1 callus. This result is better than any other callus type
It shows that type 3 callus is more easily transformed.

(Effect of 2,4-D Concentration) The transformation frequency of each callus type, which was determined by the number of blue spots per unit fresh weight of callus, was also 2,4 in the coculture medium shown in Table 2. Affected by -D concentration. The effect of 2,4-D in co-culture medium was examined by counting locus ceruleus on callus grown on MSCG4 medium (FIG. 3). After 9 days of co-cultivation, there were 793 ± 145 blue spots per 1 g fresh weight of callus on co-culture medium without 2,4-D. However, the number of blue spots is
It decreased as the 2,4-D concentration increased (Fig. 3). This observation shows that 2,4-D is Agrobacterium
It has a negative effect on the mediated transformation efficiency. This is in contrast to what was observed in other plants. The positive role for active cell division in Agrobacterium-mediated transformation has been discussed in various plant species. Flax (McHuge
n et al., 1989) and eggplant (Claudia et al., 20).
00), pre-culture of explants in regeneration medium containing 2,4-D prior to Agrobacterium infection was
It was essential for efficient gene transfer. Active cell division in wounded explants greatly improved the integration of T-DNA fragments into plant genomic DNA (McHughen et al., 1989; Muthukuma).
r et al., 1996; Claudia et al., 2000). Two
It is possible that co-culture medium without 4-D can activate cell division in Shiva callus or promote gene transfer of itself, although the exact molecular mechanism is not understood.

(Co-cultivation period) A co-cultivation period of 2 to 3 days is generally used in the transformation of Gramineae (Hiei et al., 1994; Rashid et al., 199).
6; Dong et al., 1996; Ishida et al., 199.
6, Cheng et al., 1997), while an extended co-culture period of up to 5 to 7 days resulted in
tissimum, citrange,
And increased Agrobacterium-mediated transformation efficiency in Agapanthus (Cervera et al., 199).
8; Suzuki et al., 2001). Prolonging the co-culture period results in extensive proliferation of bacterial cells and usually reduces regeneration frequency (Cervera et al., 1998). Although overgrowth of bacterial cells was observed during the 6-day co-culture period, thorough washing with vortex was able to protect the callus from bacterial contamination and did not reduce transformation efficiency (data. Not shown).

(CaCl ₂ concentration in co-culture medium)
A. The effect of CaCl ₂ on tumefaciens mediated transformation frequency was investigated. Co-culture medium without calcium significantly increased the number of GUS-expressing blue spots (Fig. 4). The number of blue spots was 1368 per 1 g of fresh callus on the co-culture medium containing no CaCl ₂ , but it decreased as the CaCl ₂ concentration increased. However, callus growth was slower in the calcium-free coculture medium than in the calcium-containing medium. Therefore, callus must be rapidly transferred to callus growth medium containing calcium after co-cultivation. A similar calcium effect is found in Hevea brasulien
Observed in sis (Montoro et al., 2000).

Calcium plays an important role in the plant response to pathogenic infections and has been discussed in recent physiology studies (Dierk 1998). This calcium-mediated plant defense mechanism is also associated with Agrobact
It can be induced in Shiva callus when infected with erial cells. This may explain why Shiva callus is more easily infected in calcium-free co-culture medium.

(Effect of Acetosyringone Concentration) Acetosyringone, which is a phenol compound, is
erium Ti plasmid is well known as an inducer that activates vir gene expression (Stache
l et al., 1985). Acetosyringone has been described by Japonikaine and Indicaine (Hiei et al., 1994; Ra.
shid et al., 1996), phalaenopsis (pha)
raenopsis), orchid and agapanthus (Su
In monocotyledons such as zuki et al., 2001),
It has been shown to be important for Agrobacterium-mediated transformation. Acetosyringone is a cucumber,
Transformation efficiency in dicotyledonous plants such as broccoli, soybean, and citrange is also greatly increased (Ni
shibayashi et al., 1996; Cervera.
Et al., 1998; Santarem et al., 1998; Hen.
zi et al., 2000).

When the coculture medium containing no acetosyringone was used, the transformation efficiency was very low, and 86.3 ± 24.0 blue spots per 1 g of fresh callus. Addition of 50 mg / liter of acetosyringone to the co-culture medium significantly increased the number of locus ceruleus by 9-fold (Fig. 5). However, concentrations higher than 100 mg / liter had a negative effect on transformation. In particular, callus co-cultured on a medium containing 200 mg / l acetosyringone did not grow on the callus culture medium after co-culture.

(Transformation of turfgrass with bar gene) In order to evaluate the optimal transformation system for turfgrass plants, pGPTV-HB containing herbicide resistance gene (bar)
Established protocols were applied for genetically transforming turf plants with transformation vectors. The main components of this protocol were the use of type 3 callus and a co-culture period of 9 days. This co-culture medium is 2,4
It did not contain -D and CaCl _2, but containing acetosyringone 50 mg / l. The co-cultured callus was grown on callus growth medium and subjected to shoot induction without hygromycin and bialaphos selection.
Occasionally, Agrobacterium cell contamination was
250 mg / liter carbenicillin was used in shoot induction medium as observed on medium containing mg / liter carbenicillin.

Selection was carried out only on rooting medium containing 10 mg / l hygromycin or 5 mg / l bialaphos. Rooted shoots appeared after 4 weeks. When the rooted shoots were subcultured again on a fresh selection medium for 2 to 4 weeks, the escaped root (escape) on the first selection medium finally died,
On the other hand, putative true transgenic plants rooted and actively expanded. The frequency of resistant plants is about 20.5%
Was (Table 4).

Table 4 shows 1 shows the survival rate of shoots regenerated from callus infected with tumefaciens. EHA101
(PGPTV-HB) shoots regenerated from infected callus,
Bialaphos (B) at 5 mg / liter and 10 mg
MSR containing 1 / liter of hygromycin (H)
Cultured for 4 weeks on S medium (see Table 1). Survival rate is 10 times the number of resistant plants produced / sown shoots.
It is multiplied by 0.

[0054]

[Table 4] Herbicide Resistance Assay for Transgenic Sheep Plants Transgenic and non-transgenic plants were established in soil and then 5 g /
Liter herbicide solution (Meiji Seika, Jap)
an) was applied daily for 2 weeks. After applying the herbicide solution for 2 weeks, the transgenic plants survived the bialaphos application and grew to maturity. However, control plants stopped growing and eventually died (Figure 3). This result indicates that the bar gene is normally expressed in transgenic plants.

DNA Gel Blot Analysis Bialaphos resistant plants were analyzed by DNA gel blot analysis to confirm that the herbicide resistance of the transgenic grass plants was derived from the bar gene integrated into the plant genome. Genomic DNA was isolated from transgenic plants, digested with HindIII, and b
Hybridized with ar or hpt specific probes (Fig. 6A). At the same time, genomic DNA from control plants was also analyzed in the same way. The control sample did not show any bands (Fig. 7B,
Lane c; Figure 7C, lane c). On the other hand, samples from transgenic plants showed bands that hybridized specifically with the bar and hpt specific probes (Figure 7B, lane 1; Figure 7C, lane 1). pGPTV
-The T-DNA fragment of HB contains a single HindI
Having an II site (FIG. 7A), the number of hybridized bands reflected the copy number of the gene copy integrated in the transgenic turf plant. The bands detected represented fragments larger than 1.6 kb or 1.8 kb for the bar or hpt specific probes, respectively, as expected from the map of pGPTV-HB (FIG. 7A). This indicates that the copy number of the integrated gene was 2 (FIG. 7B, lane 1; FIG. 7C, lane 1). Genomic DNA Southern blots with BamHI and bar probes also gave the same results. This indicates that the copy number is 2 (Fig. 7B, lane 2). These results indicate that there were two stable integration events, clearly demonstrating that the herbicide resistance observed in this transgenic plant was determined by the integrated bar gene.

A. of Shiba in the present invention tumefac
The iens-mediated transformation system speeds up genetic engineering of grass and closely related turfgrass species. The present invention provides optimized medium composition and culture conditions for genetic transformation of turf, as the gene of interest improves resistance to biotic and abiotic stress. , And is especially beneficial when introduced into turf to regulate growth rate.

(References)

[Table 5] (Summary) The present invention relates to a zoysia plant (Zoysia japo).
nica Steud. The present invention provides an efficient gene transformation system for Optimized medium composition and culture conditions for turf transformation are also provided. A reliable transformation system for this turf was developed by optimizing several factors that significantly affect callus growth and plant regeneration. Callus type and co-cultivation period absolutely affected the transformation efficiency. 2,4-D, CaCl ₂
The concentration of acetosyringone was also an important factor. The best results were achieved when type 3 callus was co-cultured on a co-culture medium without 2,4-D for 6 days. Calcium removal and 50 mg during co-culture
Addition of acetosyringone / l dramatically enhanced its efficiency. The present invention also provides a bialaphos-resistant turf plant, which is used in golf courses and arenas,
Maintenance costs can be saved.

[0058]

According to the present invention, A. tumefaci
It is now possible to genetically transform turf with the gene of interest by ens-mediated infection.

[Brief description of drawings]

FIG. 1 is a photograph showing four different types of callus grown on callus growth medium. (A) Type 1 callus has a slightly white to yellow or light green color, and exhibits a dense and non-friable structure. (B) Type 2 callus is white and exhibits a dense and friable structure. (C) Type 3 callus is yellow and exhibits a dense and very friable structure. (D) Type 4 callus has a translucent, soft, watery appearance. The four callus types were obtained on callus growth medium supplemented with different combinations of growth regulators as summarized in Table 2.

FIG. 2 shows that A.p. carrying the pGPTV-HB vector (see FIG. 7). transgenic GU in Shiva callus infected with tumefaciens
It is a photograph which shows S expression. (A) Transgenic GUS expression in Shiva callus. (B) A. tumefa
Rapid growth of callus on filter paper and MSCGCB medium without antibiotics after ciens infection.
(C) Shoot induction on filter paper and on antibiotic-free shoot induction medium. (D) Shoot elongation and rooting on Bialaphos-containing medium. A. tumefacier
Shoots derived from ns-infected callus were cultivated on a selective rooting medium MSRS containing 5 mg / l bialaphos.

FIG. 3 shows the effect of 2,4-D on transformation efficiency. Callus cultivated on callus growth medium containing different concentrations of 2,4-D were counted for blue spots per gram fresh weight.

FIG. 4 shows the effect of CaCl _{2 on} transformation efficiency. As described in the brief description of FIG. 3, except that the culture medium contained different concentrations of CaCl ₂ .
tumefaciens infected calli were cultured.

FIG. 5 shows the effect of acetosyringone on transformation efficiency. This experiment was performed as described in the brief description of Figure 3, except that it contained different concentrations of acetosyringone.

FIG. 6 is a photograph showing a transgenic turfgrass plant (right) and a parent turfgrass plant (left). These moss plants were sprayed with herbicide solution daily for 2 weeks (5
Bialaphos in g / l). (A) Plants that were not sprayed with the herbicide solution. (B) Plants sprayed daily with herbicide solution for 2 weeks.

FIG. 7 shows a schematic of the transformation vector and Southern blot analysis to confirm the presence of the herbicide resistance (bar) gene in the plant genome. (A) Vector map of the transformation vector pGPTV-HB. Pnos,
Nopaline synthase gene promoter; Hyg ^R ,
Hygromycin resistance gene (hpt); NOS, nopaline synthase gene terminator; Ubi-P,
Ubiquitin promoter from corn; Bar,
Bialafos resistance gene; B, BamHI; E, Ec
oRI; H, HindIII; S, SacI. 3 is a photograph showing Southern blot analysis of (B) and (C) transgenic turf plants. Genomic DNA is transferred to HindI
Digested with either II (lane 1) or BamHI (lane 2). Membranes were hybridized with either bar (B) or hpt (C) probes. Genomic DNA of untransformed plants
Was included as a negative control (lane c).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Zhang Fubae             South Korea, Chungnam, Sanchon-             Si, Maegok-dong, Sanjeongyu             Niversity (72) Inventor Jeon Gukan             Republic of Korea, 500-480 Kwangju, Bu             Kugu, O'Yang-Don, number 1 (72) Inventor Pill-Soonson             Republic of Korea, 500-480 Kwangju, Bu             Kugu, O'Yang-Don, number 1 (72) Inventor Chun Mo Park             Republic of Korea, 500-480 Kwangju, Bu             Kugu, O'Yang-Don, number 1 (72) Inventor Hyo Yeong Lee             South Korea, Chungnam, Sanchon-             Si, Maegok-dong, Sanjeongyu             Niversity F term (reference) 2B030 AA02 AB03 AD05 CA06 CA17                       CA19 CD03 CD06 CD09 CD13                       CD17                 4B024 AA08 BA79 DA01 GA11 GA17

Claims (6)

[Claims] 1. A method for genetically transforming Shiva (genus Zoysia), which comprises the following steps: i) Inducing and growing callus of Shiva on a modified MS medium containing various hormones. Ii) a step of infecting the callus of the shiba with Agrobacterium cells and introducing a bialaphos-resistant bar gene; iii) in a coculture medium containing 2,4-dichlorophenoxyacetic acid and calcium but not acetosyringone , Shiva Callus and Agrobacterium
A method comprising co-culturing m cells; iv) removing Agrobacterium cells from the co-cultivation medium; and v) regenerating the transgenic shiva. 2. The Agrobacterium cell is p
The method according to claim 1, which comprises a GPT-HB transformation vector. 3. A callus of type 3 in a co-cultivation medium of 5 to 7
The method according to claim 1, wherein co-culturing is carried out for a day. 4. The bar gene is stably transformed under the control of a plant-specific promoter.
Transgenic turf by the method described in. 5. The transgenic shiba of claim 4, wherein the bar gene is expressed under the control of a ubiquitin promoter. 6. A bialaphos resistance bar gene (KC
TC-10076BP) introduced transgenic Shiba callus.