JPH07203790A - Production method of transformed eucalyptus plant - Google Patents

Abstract

PURPOSE:To obtain a transformed Eucalyptus plant steadily in a short period of time, by infecting a tissue piece of Eucalyptus pant with bacteria of Agrobacterium and by forming transformed cells under a specific condition. CONSTITUTION:A tissue piece of Eucalyptus plant is infected and cultured in a infection medium to be infected with bacteria of Agrobacterium. The bacteria are removed by vertical rotation ceelture using a sterile medium added with antibiotics, and then lumps of transformed cells are selected out by the vertical rotation culture in a selection medium added with antibiotics. The transformed cell lumps are cultured by this culturing method under irradiation of light to convert them to a primordium of nuresry stock to produce a transformed Eucalyptus plant. By the introduction of a useful gene, an excellent new variant of Eucalyptus plant is produced and it is possible to obtain a nursery stock having the useful gene in a short period of time.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a Eucalyptus plant transformed with Agrobacterium, more specifically, a transformed plant depending on a conventional transformation method with Agrobacterium. It is intended to provide an effective method for producing a transformed plant for a plant of the genus Eucalyptus for which the above-mentioned was not obtained.

[0002]

2. Description of the Related Art As a plant breeding technique, methods such as selection and crossing have been widely used. These methods can be considered to be gene transfer in a broad sense in which plant genes are treated as a large unit. On the other hand, advances in gene manipulation techniques have made it possible to isolate specific genes, modify the genes, and introduce them into plants in some plant species. By applying these gene manipulation techniques, highly practical breeding is being achieved in herbaceous plants such as tobacco, rice and tomato. However, on the other hand, in many plants, plant breeding by genetic engineering is difficult, and a major reason for this is considered to be the incomplete transformation technique.

Plant transformation techniques include (1) a biological method of introducing a gene via Agrobacterium.
(2) It is roughly classified into a method of directly introducing a gene by subjecting a plant cell to a physical or chemical treatment. Both methods need to be used properly depending on the regeneration system of the target plant. That is, when using the biological method, it is necessary to regenerate the transformed plant from the tissue, and when using the direct method, it is necessary to regenerate from the protoplast. In such a situation, in the case of a plant in which the redifferentiation system is not established, considering that it is easier to establish a redifferentiation system from a tissue than to establish a redifferentiation system from protoplasts, It is considered that it is industrially beneficial because the transformation method has a wider application range.

Even in woody plants, which have been considered to be difficult to cultivate tissues, loblolly pine (Sederoff) has been detected by a biological method using Agrobacterium.
et al. Bio / Technology 4: 647-649 (1986)), poplar (Fillatti et al. Mol.Gen.Genet. 206: 192-199 (198)
7)), walnut (McGranahan et al. Bio / Techno
logy 6: 800-804 (1988)), apple (James et al. Pla
nt Cell Rep. 7: 658-661 (1989)), Plum (Mante et.
al. Bio / Technology 9: 853-857 (1991)) and the like have been reported to have succeeded in producing transformed plants. Factors that succeeded in producing transformed plants with these woody plants are:
It is considered that this is due to the establishment of a redifferentiation system from tissues, but it is still difficult to establish a redifferentiation system with new plant species other than these.

On the other hand, the present inventors have already proposed a method for regenerating a plant from a protoplast of a eucalyptus plant through colonies (Japanese Patent Laid-Open No. 2-129631). Further, a method for producing a transformed plant in which a gene has been introduced into the protoplasts by an electroporation method has been proposed (JP-A-4-53429). However, even with these new and effective methods, if the plant species are different, a redifferentiation system from protoplasts must be newly established. Furthermore, there is room for improvement in that not only it takes a long time to obtain a transformed plant, but also the frequency of obtaining a transformed plant is low.

The present inventors have also proposed a transformation method of infecting shoot primordia with Agrobacterium (Japanese Patent Laid-Open No. 2-138966). However, since Eucalyptus plants have low infectivity to the shoot primordia of Agrobacterium, it has been necessary to examine excellent tissues as an infectious material for Agrobacterium or to discover factors that improve the infectivity. Furthermore, there is room for improvement in that it is difficult to regenerate from callus of the genus Eucalyptus on a solid medium.

[0007] Therefore, the present inventors infect various tissues of Eucalyptus plants with Agrobacterium, investigate the transformation rate between tissues, and improve the transformation rate in optimal tissues. We examined the factors. Furthermore, tissue pieces transformed by infection with Agrobacterium are subjected to vertical rotary culture in a medium containing antibiotics after the infection treatment to distinguish them from non-transformed cells and to efficiently transform transformed shoot shoots. This method of forming a group was completed. Transformed plants are easily regenerated from the transformed shoot primordium thus obtained.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a method for efficiently transforming a Eucalyptus plant having low infectivity of Agrobacterium and efficiently producing a transformed plant from the transformed cell. With the goal.

[0009]

Means for Solving the Problems The present invention is directed to eradication of Eucalyptus plant tissue pieces by infection culture in an infection medium and infection with Agrobacterium, followed by addition of antibiotics to the infected tissue pieces. The transformed cell clumps were selected by sterilizing the cells by vertical rotation culture in the medium, and then by performing vertical rotation culture on the infected tissue pieces in the selection medium containing antibiotics. Characterized by producing transformed shoot primordia by further culturing the cell clumps in vertical rotation under light irradiation, and producing transformed Eucalyptus plants via the obtained shoot primordia And a method for producing a transformed Eucalyptus plant.

The present invention also provides a method characterized by using a cotyledon or a hypocotyl as a tissue piece of a Eucalyptus plant, a method characterized by adding galactose and acetosyringone to the infection medium thereof, There is also a method characterized in that the infection culture is liquid stationary culture or solid culture, and a method characterized in that the sterilization culture is liquid rotary culture. Hereinafter, the method for producing the transformed Eucalyptus plant of the present invention will be described in detail.

(Cotyledons and hypocotyls for infecting Agrobacterium): Cotyledons and hypocotyls of Eucalyptus plants to be transformed are sterilized by seeds, and then plant tissue culture media such as B5. It is placed on an agar medium such as a medium or MS medium, germinated, and aseptically excised with tweezers and a knife.

(Preparation of Agrobacterium): Agrobacterium is prepared by detoxifying the Ti plasmid or a non-detoxifying strain. The gene to be introduced is modified so as to be expressed in plant cells, then ligated to a Ti plasmid vector or a binary vector, and transformed into Agrobacterium for use (MD, Chilton et al. Proc. Natl. .Acad.Sci.US
A 77: 4060-4064 (1980)), (L. Herrera-Estrella et
al.Nature 303: 209-213 (1983)). The Agrobacterium obtained by the above method was added to an L-liquid medium (Miller Experiments in
Molecular Genetics (1972) 10g / 1 Bact-tryptone, 5g /
1 Bact-yeast extract, 5 g / 1 NaCl) at 30 ° C. overnight. D. Incubate until ₆₀₀ is over 0.8.

(Agrobacterium infection): cotyledon,
The plant tissue of the hypocotyl was treated with 0.1% surfactant (Tween-20)
After washing with, cut into 2-10mm size with a knife, and infect Agrobacterium infection medium, eg WPM
(Loyd & McCown Proc.Int.Plant Prop.Soc. 30: 421-
427 (1980)), B5 (Gamborg et al. Exp. Cell Res. 50).
: 151-158 (1968)), MS (Murashige & Skoog Physio
l.Plant 15: 473-497 (1962)) 1- (2-chloro-4-) as a plant hormone cytokinins in a medium or the like.
Pyridyl) -3-phenylurea (4-PU), benzyladenine (BA) or kinetin, and auxins such as naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) or indoleacetic acid. (IAA), etc., and sucrose, galactose, acetosyringone, and Agrobacterium are inoculated in a liquid medium. More preferably, the plant tissue pieces are soaked in a culture solution of Agrobacterium, and then they are implanted in a solid medium containing a substance other than Agrobacterium. 20 ~
It is allowed to infect with Agrobacterium by static culture at a temperature of 30 ° C. under light-shielding conditions for 1 to 2 days.

(Disinfection of Agrobacterium): A plant tissue piece infected with Agrobacterium is disinfected in a disinfection medium such as WPM, B5, MS medium or the like, and 4-PU as cytokinins which are plant hormones. BA or kinetin, and NAA, 2,4-D as auxins
Alternatively, it is planted in a liquid medium containing sucrose and IAA or the like and an antibiotic for sterilizing Agrobacterium, such as carbenicillin, vancomycin, claforan or the like. This is subjected to vertical rotary culture for 3 to 14 days at a temperature of 25 ° C. and an illuminance of 0 to 2000 lux to remove Agrobacterium.

(Formation of transformed shoot primordia): A plant tissue piece from which Agrobacterium was completely sterilized was used as a basic medium, for example, WPM, B5, MS medium, etc., and cytokinins which are plant hormones. As 4PU, BA or kinetin, and as auxins NA
A, 2,4-D, IAA, etc., a carbon source, and a liquid medium containing an antibiotic corresponding to the selection gene that is introduced at the same time as the target transgene in order to select the transformed cell clumps Vertical rotation culture is carried out in it. The culture conditions are a rotation speed of 1 to 10 rpm, a maximum of 2,000 to 20,
The illuminance is 000 lux and the temperature is 20 to 30 ° C. 14
When the culture is continued by subculturing in a fresh medium at intervals of ~ 40 days,
About a month after the selection of transformed cells started,
Yellow-white transformed callus formation is observed in the plant tissue pieces. The obtained callus was separated from the plant tissue piece with a knife, and the vertical rotary culture was continued to give 40 to 12 from the start of the vertical rotary culture under light irradiation.
Transformed shoot primordia are obtained on day 0.

(Regeneration of transformed plant): Transformed shoot primordium obtained by rotary culture is used as a plant hormone, for example, in a medium for regenerating shoots, such as B5 medium and MS medium. Addition of auxins such as NAA, 2,4-D and IAA, cytokinins such as BA, 4-PU, kinetin, zeatin and thidiazuron and carbon source, and antibiotics for selecting transformed cell clumps. Incubate in the specified medium. Cultivation is performed at a temperature of 20 to 30 ° C.
The shoots can be regenerated by carrying out the irradiation at an illuminance of 000 to 3,000 lux for about 60 days, and further rooted to obtain a completely transformed plant.

[0017]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Test plant Eucalyptus camaldrensis (Eu)
calyptus camaldulensis) was used. (Production of cotyledons and hypocotyls): Eucalyptus camaldrensis seeds were sterilized with 10-fold diluted antiformin for 1 hour, and aseptically added to 70% ethanol for 15 hours.
It was sterilized by dipping it in antiformin diluted for 5 seconds and 5 times for 20 minutes. B5 which is a plant tissue culture medium
Germinated aseptically on agar medium. From the germinated plant, cotyledons and hypocotyls were aseptically removed using tweezers and a knife, and used as experimental materials.

(Preparation of Agrobacterium): Agrobacterium is a non-toxic L plasmid in which its Ti plasmid is detoxified.
BA4404 strain (MW Bevan et al. Nucleic Acids Res.
12: 8711-8721 (1984)) was used. Any gene as a transgene can be expressed by replacing the promoter with a plant promoter. Here, the commercially available β-glucuronidase (GUS) gene is used as the binary vector pBI.
It was ligated to 121 and transformed into Agrobacterium for use (RA Jefferson et al. EMBO J. 6: 3901-3907.
(1987)). Agrobacterium LBA4404 / carrying the above-mentioned β-glucuronidase (GUS) gene
pBI121 was added to the kanamycin antibiotic 100
3 times overnight in L-liquid medium containing at a concentration of μg / ml
Cultured at 0 ° C.

(Infection of Eucalyptus plant tissue with Agrobacterium): 3% sucrose was added to B5 basal medium,
0.02mg / l NAA and 0.2m as a plant hormone
Infection medium to which g / l 4-PU was further added with 10 mM galactose and 1 μM acetosyringone, and a culture solution of Agrobacterium LBA4404 / pBI121 adjusted to a concentration of 1% was added. Was produced. Cotyledon of Eucalyptus plant,
Each piece of hypocotyl tissue was washed with 0.1% surfactant (Tween-20), cut with a knife to a size of 2-10 mm, and transplanted. After static culture for 2 days, the cells were infected with Agrobacterium.

(Elimination of Agrobacterium): In order to remove Agrobacterium from the tissue piece infected with Agrobacterium, 3% sucrose was added to the B5 basic medium to obtain 0 as a plant hormone. 0.02 mg / l NAA and 0.2 mg / l 4-PU as 250 μg / m 2 antibiotics for further sterilizing Agrobacterium.
l Carbenicillin and 100 μg / ml vancomycin were added to the sterilized medium. This was subjected to vertical rotary culture at a temperature of 25 ° C. under a light-shielding condition at a rotation speed of 2 rpm for 7 days to remove Agrobacterium.

(Formation of transformed shoot primordia): A tissue piece from which Agrobacterium was completely eradicated was added to 0.05 mg / l NAA and 0.2 mg / l 4 of B5 basal medium.
-PU, 3% sucrose, and the antibiotic Geneticin (G418) in a liquid medium added at 30 μg / ml,
Three test pieces were planted per one test tube. By culturing for 7 days under light-shielding conditions at a temperature of 25 ° C., under irradiation with light of 200 lux on the lower side and 20,000 lux on the upper side, and by performing vertical rotary culture for 1 month at a rotation speed of 2 rpm, 2 -3 mm of transformed callus formation was observed. The obtained callus was subcultured to a fresh medium at intervals of 14 to 40 days, and the culture was continued. Transformed shoot primordia were obtained 40 to 120 days after the start of vertical rotary culture under light irradiation.

(Regeneration of transformed plant): 0.02 mg / l N was added to B5 medium to regenerate the transformed shoot from the transformed shoot primordium obtained by rotary culture.
AA, 0.2 mg / l BA, 1% sucrose, 0.2%
Gellan gum (Wako Pure Chemical Industries) and 30 μg / ml G41
The shoot primordia adjusted to a size of about 5 mm was transplanted to the solid medium containing 8 added. And the temperature of 25 ℃, illuminance 40
As a result of culturing under light irradiation at 00 lux for 16 hours, regeneration of shoots was recognized one month after transplantation. The obtained shoots were added to B5 medium with 0.01 mg / l NAA, 1% sucrose,
0.15% gellan gum and 30 μg / ml G418
By transplanting to a rooting medium supplemented with, rooting was recognized after 1 month and a complete plant body was obtained. That is,
In the case of this method, it took 5 to 12 months from the infection of the plant tissue piece to the production of the transformed plant.

(Confirmation of the presence of the transgene in the transformed plant): The presence of the transgene was confirmed by Southern hybridization in four individuals obtained by selection with an antibiotic. As described above, it was confirmed that several genes were introduced in the case of large numbers. From this, it was proved that the transformation of Eucalyptus plants by this method was effective.

(Confirmation of expression of transgene in transformed plant): In an individual whose presence of the transgene was confirmed by Southern hybridization, expression of GUS gene was subjected to tissue staining (S. Kosugi et al. Plant Science 70:
133-140 (1990)), strong GUS gene expression was confirmed around the leaves, root tips and root hairs.

Example 2 After aseptically sowing Eucalyptus camaldrensis, Example 1 was applied to cotyledons, hypocotyls, roots, adult leaves, and adult leaves of field plants.
After infecting Agrobacterium with the same infection medium as above, transformed callus was produced by the same method as in Example 1. Then, the infection rate of Agrobacterium due to the difference in material was compared by the transformed callus formation rate. As a result, the formation of transformed callus was observed in 9.9% of the cotyledon fragments and 11.3% of the hypocotyl fragments used in the experiment, whereas it was 0% in the root fragments. Also, when adult leaves that are commonly used for transformation with Agrobacterium are used as infectious materials, coculture with Agrobacterium causes browning of the leaf color, or non-transformed cells tend to grow. , It became difficult to obtain transformed callus.

Example 3 In the same manner as in Example 1 except that galactose and acetosyringone were not added to the infection medium when infection culture of Eucalyptus camaldrensis cotyledon and Agrobacterium was carried out. Transformation was performed and the effect of the addition of galactose and acetosyringone on the transformation rate was examined by the rate of formation of transformed callus. As a result, transformed callus was obtained in one of the 20 cotyledon pieces tested without addition. On the other hand, when added, transformed calli were obtained in 3 pieces out of 20 cotyledons, and it was revealed that the addition of galactose and acetosyringone was effective in improving the transformation rate of Eucalyptus plants.

Example 4 The effects of the Agrobacterium infection culture method and the eradication culture method on the transformation rate were examined. Specifically, when a cotyledon of Eucalyptus camaldrensis prepared by the same method as in Example 1 was infected with Agrobacterium, in the infection culture and sterilization culture, the presence or absence of agar was used. The number of transformed calli was determined by performing stationary culture or rotary culture of solid medium or liquid medium having the same components. As a result, as shown in Table 1, the infection culture of Agrobacterium can be efficiently carried out by liquid stationary culture, more preferably solid culture, and sterilization culture by liquid rotary culture. It was revealed.

[0028]

[Table 1]

Example 5 Eucalyptus camaldrensis cotyledon pieces infected with Agrobacterium in the same manner as in Example 1 were transformed in the same manner as in Example 1 except for the period of eradication, and the eradication was carried out. The effect of the period on the transformation rate was investigated. When the bacteria were eradicated immediately after infection with Agrobacterium, the formation rate of transformed callus was 0%, whereas it was 30% in 2 days.
Improved to 43% in 7 days and 40% in 14 days
The formation of callus was observed in the cotyledon of Cotyledon. From this,
It has been clarified that the period required for eradication of Agrobacterium is preferably 7 days.

Comparative Example 1 Method proposed by Ota et al. (Japanese Patent Laid-Open No. 4-53429)
According to the above, the gene prepared in the same manner as in Example 1 was introduced into the protoplasts of Eucalyptus camaldrensis by the electroporation method to produce a transformed plant, which required about 2 years. On the other hand, the transformed plant can be produced by the Agrobacterium method about 5 to 12 months after the infection. From this, it became clear that the Agrobacterium method can produce transformed plants in a shorter period of time than the electroporation method.

Comparative Example 2 A shoot primordium of a eucalyptus plant was infected with Agrobacterium using the same infection medium as in Example 1, and then transformed callus was transformed by the same method as in Example 1. I made it. As a result, no transformed callus was formed.

Comparative Example 3 The same method as in Example 1 except that Eucalyptus camaldrensis cotyledons infected with Agrobacterium in the same manner as in Example 1 were statically cultured in the process after the sterilization. Transformation was performed by. As a result, callus transformed in the same manner as in Example 1 was obtained, but no shoots were obtained even when placed in a seedling medium, the callus proliferated, and eventually died.

Example 6 (Test plant): Eucalyptus globulus was used as a Eucalyptus plant. (Production of cotyledons, hypocotyls and preparation of Agrobacterium): For Eucalyptus globulus, the same method as in Example 1 was used. (Infection of Eucalyptus plant tissue with Agrobacterium): In the same manner as in Example 1, 3% sucrose was added to B5 basal medium to give 0.02 mg of a plant hormone.
/ L NAA and 0.2 mg / l 4-PU, 1 more
Add 0 mM galactose and 1 μM acetosyringone, and further add 1% Agrobacterium L
An infection medium for Agrobacterium was prepared by adding a culture solution of BA4404 / pBI121. Into this medium, tissue pieces of the cotyledon and the hypocotyl of a Eucalyptus plant were added to 0.
After washing with 1% surfactant (Tween-20), implant a piece of tissue cut with a knife to a size of 2-10 mm,
The cells were statically cultured for 2 days at a temperature of 26 ° C. under light-shielding conditions to infect Agrobacterium.

(Disinfection of Agrobacterium): From a Eucalyptus plant tissue infected with Agrobacterium,
In order to remove Agrobacterium, the plant tissue pieces were sterilized and B5 basal medium was supplemented with 3% sucrose to obtain 0.02 mg / l NAA and 0.2 mg / l as plant hormones.
4-PU, antibiotics for sterilizing Agrobacterium, 250 μg / ml carbenicillin, 10
It was transplanted to a sterilization medium supplemented with 0 μg / ml vancomycin. This was subjected to vertical rotary culture at a temperature of 25 ° C. under a light-shielding condition at a rotation speed of 2 rpm for 7 days to remove Agrobacterium.

(Formation of transformed shoot primordia): A tissue piece from which Agrobacterium was completely sterilized was added to 0.02 mg / l NAA, 0.2 mg / l 4 of B5 basal medium.
-PU, 3% sucrose, and 30 μg / ml G418
Was added to the selective medium containing 3 parts per one test tube. By culturing for 7 days under light-shielding conditions at a temperature of 25 ° C., under irradiation with light of 2000 lux on the lower side and 20000 lux on the upper side, and rotating vertically for 1 month at a rotation speed of 2 rpm, 2 to give a yellowish white color in tissue. Callus formation of 3 mm was observed. The obtained callus was subcultured to a fresh medium at intervals of 14 to 40 days, and the culture was continued. Transformed shoot primordia were obtained 40 to 120 days after the start of vertical rotary culture under light irradiation.

(Regeneration of transformed plant): 0.02 mg / l N was added to B5 medium to regenerate the transformed shoot from the transformed shoot primordium obtained by rotary culture.
AA, 0.2 mg / l BA, 1% sucrose, 0.2%
A shoot primordia adjusted to a size of about 5 mm was transplanted to a solid medium containing gellan gum and 30 μg / ml G418. And the temperature of 25 ℃, illuminance 4000 lux,
As a result of culturing under light irradiation for 16 hours, regeneration of shoots was recognized one month after transplantation. The obtained shoots were added to B5 medium at 0.01 mg / l NAA, 1% sucrose, 0.15%.
When transplanted to a rooting medium supplemented with gellan gum and 30 μg / ml G418, rooting was recognized after 1 month, and the plant became a complete plant.

(Confirmation of Existence and Expression of Transgene in Transformed Plant): Regarding the individual obtained by transformation, the presence of the transgene was confirmed by Southern hybridization in the same manner as in Example 1. It was confirmed that at least one gene was introduced in an individual, and in the case of a large number, several genes were introduced. Furthermore, GU
Expression of the S gene was confirmed by tissue staining.

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to efficiently and stably produce a transformed plant even in a plant of the genus Eucalyptus, which has been difficult to produce a transformed plant. Furthermore, by introducing a useful gene, a superior new variety of Eucalyptus plant is produced, and by using the shoot primordia obtained in the transformation process of the new variety, a seedling having a useful genetic trait can be produced in a short period of time and at a low cost. It has become possible to supply.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Mariko Tsuji 24-9 Nozono-cho, Kameyama City, Mie Prefecture Shinji Paper Co., Ltd. 24-9 Nomachi Shin-Oji Paper Co., Ltd. Kameyama Laboratory, Forest Tree Breeding Research Institute

Claims (4)

[Claims] 1. A eucalyptus plant tissue piece is cultivated in an infection medium and infected with Agrobacterium.
The infected tissue pieces were sterilized by vertical rotation culture in a sterile culture medium containing antibiotics, and the infected tissue pieces were then cultivated by vertical culture culture in a selective culture medium containing antibiotics. The transformed cell clumps were selected, and the cell clumps obtained by the selection were further subjected to vertical rotation culture under light irradiation to produce a transformed shoot primordia, and the transformed shoot primordia were used for trait transformation. A method for producing a transformed Eucalyptus plant, which comprises producing a transformed Eucalyptus plant. 2. The method according to claim 1, wherein the tissue piece of a Eucalyptus plant is a cotyledon or a hypocotyl. 3. The method according to claim 1, wherein galactose and acetosyringone are added to the infection medium. 4. The method according to claim 1, 2 or 3, wherein the infection culture is liquid static culture or solid culture, and the sterilization culture is liquid rotary culture.