CN1309715A - Process for selection of transgenic cells in meristematic region of cotton, coffee, cocoa, banana or grape - Google Patents

Abstract

This invention relates to a method for selecting transgenic meristem cells from cotton, coffee, cocoa, banana, or grape plants and subsequently preparing transgenic plants. The method includes the steps of: introducing a foreign gene into cells of the apical meristem of the hypocotyl of a cotton, coffee, cocoa, banana, or grape plant, or into tissues or organs containing said plant meristems; inducing multiple shoots from the apical meristem cells altered in the previous step by culturing their hypocotyls or tissues containing meristems in a medium containing a multiple shoot inducing agent; and selecting transgenic meristem cells from the apical region, wherein said cells are transformed by further culturing the hypocotyls or tissues containing meristems in a medium containing a selecting agent or by applying the selecting agent to the multiple shoots from the cells of the meristem region.

Description

A method for selection of transgenic cells in the meristem region of cotton, coffee, cocoa, banana or grape

field of invention

The present invention relates to the application of biolistic method to introduce exogenous gene, the exogenous gene is related to a molecule capable of translocating and selecting transgenic meristem cells of cotton, coffee, cocoa, banana or grape plants, and obtain transgenic plants by regenerating transformed tissues .

Background of the invention

Applying genetic engineering technology to introduce genes responsible for agricultural traits of interest is conducive to the development of new varieties of cultivated crops. To obtain transgenic plants, it is necessary to introduce foreign DNA into plant tissues and regenerate whole plants from such genetically transformed tissues. Depending on the species to be transformed, various tissues have been used to introduce foreign DNA. Among the various methods of transformation, meristems are preferred primarily because of the ease with which this tissue regenerates plants. Many methods for introducing foreign genes into apical meristem cells of higher plants have been proposed, among which the following methods can be proposed: a) biolistic system and b) Agrobacterium system. The introduction and integration of exogenous DNA in higher plant cells has been demonstrated by many scientists and described in various publications, such as (McCabe D. Martinelli B.J. (1993) transformation of superior cotton cultivars by particle bombardment of meristems. Bio/Technology 11:596-598; Keller, G., Spatoa L., McCabe D., Martinelli B., SWain W., John M.E. (1997) Transgenic cotton resistant to the herbicide bialaphos, Transgenic Research, 6 :385-392).

However, the low frequency of obtaining genetically transformed tissue, the low ability of the transformed tissue to regenerate fertile plants, and the use of transformation methods whose efficiency depends on genotype all make it difficult to obtain transgenic plants for most cultivated crops.

In the late 1980s, with the development of the biolistic method of directly introducing genes into plant cells (Sanford J.C., Klein T.M., Wolf E.D. & Allen N. (1987), the method of particle bombardment was used to transport substances to cell tissues; particle science and technology Journal, 5:27-37), a number of transgenic plants have been obtained for several plant species, including those that have proven difficult to transform by other methods. This is because it has become possible to introduce and express foreign genes in any kind of plant tissues. Thus, any tissue that has the potential to regenerate whole fertile plants is suitable for transformation.

The biolistic method was proposed by Sanford to introduce genetic material into the nuclear genome of higher plants. Since then, its wide application has been affirmed. And according to the observations of Sanford J C, Smith F D & Russel J.A. (1993): Optimizing biolistic methods for use in different biological applications; Methods in Enzymology: 217:413-510. It has proven to be an efficient and simple method for introducing and expressing genes in bacteria, protozoa, fungi, algae, insects, plant and animal tissues, as well as isolated organelles like chloroplasts and mitochondria. There are some other examples of the application of biolistic methods to obtain genetically modified organisms in the specialized literature, for example, US Patent 5,565,346, US 5,489,520 and WO 96/04392, among others.

In the biolistic approach, high-speed microprojectiles are used to carry and transduce nucleic acids and other substances into cells and tissues in vivo. (Rech E.L. & Arago F.J.L. (1997) biolistic method. In Brasileiro A.C.M. & Cameiro V.T.C (Ed) Handbook of Plant Genetic Transformation - EMBRAPA/Cenargen. This method is also called microprojectile bombardment, "gene gun" method, particle acceleration method and others. Different methods have been developed and established to accelerate nucleic acid-sequenced particles (made of tungsten or gold) to more than 1500 km/h. The basis of all these methods is to generate a band A shock wave of sufficient energy to expel a transport membrane containing particles coated with DNA. Can be vaporized water droplets by chemical explosion (dry powder), high-pressure emission of helium, high-pressure low-volume or low-pressure high-capacity discharge Generate the shock wave.

Those using high pressure helium and electrical discharges have shown a wide range of applications. Accelerated particles penetrate cell walls and membranes in a non-lethal manner, localizing randomly to organelles. The DNA is then freed from the microparticles by movement of the cytosol, and the process of integrating the foreign DNA into the genome of the organism to be altered occurs. (Yamashita T.Iada, A. & Morikawa H. (1991) - proved that more than 90% of the cells expressing b-glucuronidase all directly received the exogenous gene into the nucleus after particle bombardment; Plant Physiology 97: 829-831). Although the biolistic approach has been effectively and widely used, it still relies on the optimization of various physical and biological parameters that are fundamental to the efficient introduction of xenogeneic genes into plant tissues.

In 1985, the use of a genetically altered strain of Agrobacterium tumefaciens to transfer foreign genes into plants was first described. (Horsch RB, Fry JE, Hoffman NL, EichholtzD, Rogers SG & Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229-1231) Since then, many species have been passed through the Agrobacterium method Transformed. The basis for obtaining transgenic plants by Agrobacterium-mediated gene transfer is that the bacteria are able to transfer specific sequences from their DNA into the plant genome. Agrobacterium infection of plants is caused by their penetration of plant wounds into plant tissue. Bacteria are attracted to plant cells by a positive tropism associated with the shedding of phenolic compounds, sugars and amino acids (signaling molecules) to the damaged tissue with the wound. In addition to attracting bacteria, the signaling molecule also activates genes located in the virulence region called the Ti (tumor-inducing) plasmid, which is present in all virulent strains of Agrobacterium. Activation of genes in the virulence region induces the transfer of another Ti plasmid region (termed the T-region) from bacterial to plant cell genomes (Jouanin L, Brasileiro ACM, Lepe Jc, Pilate G & Corn D. (1993) Genetic Transformation: A Brief Review Methods and their application, achievements and prospects in forest trees, Annales des Scienll Forestieres (Annual Forest Science) 50:325-336, Tinland B. (1996) T-DAN integration into plant genome. Trends in Plant Science 1:178 -184).

Once integrated into the plant genome, the T-region is called T-DNA (transfer DNA). In plant genomes, T-DNA essentially induces the synthesis of phytohormones and opines. Synthetic phytohormones (auxins and cytokinins) alter the hormonal balance, causing uncontrolled proliferation of transformed cells and peripheral cells. This random proliferation of cells leads to the formation of tumors. Opines are simple molecules produced by condensation of an amino acid and a sugar. Opines are secreted from cells and are specifically acted upon by infectious bacteria present in the intercellular spaces of tumor cells, resulting in catabolism. Several opine types have been identified (nopaline, octopine, agropine, and others), and only the tumor-inducing strain is able to decompose its own opines.

Although tumor formation is the result of the expression of T-DNA genes integrated into the plant cell genome, these genes are not involved in the process of metastasis and integration, only the toxic region and the T-DNA ends (repeated by the 25-base-pair control sequence composition) is necessary for the above process. Therefore, it is possible to delete all the genes present in the T-DNA by a double recombination method, thereby generating defective strains that do not affect the transformation process. Those strains maintained the functional virulence region, and the original T-DNA gene was replaced by the tagged gene of interest. The gene of interest for this marker is flanked by repetitive sequences necessary for the transfer process. (Zambryski, P, JoosH, Genetello. C, Leemans J, Van Montagu M & Schell JS. (1983) Use of Ti plasmid vectors to introduce DNA into plant cells without affecting their normal regenerative capacity. EMBO Journal. 2:2143-2150 ). Therefore, this strain containing the modified T-DNA does not induce tumors but retains transforming ability.

Gene transfer into plant cells using Agrobacterium tumefaciens remains one of the most commonly used methods. (De Block M. (1993) Cell biology of plant transformation: Current status, problems, prospects and implications of plant breeding. Euphytica. 71:1-14). The system is simple, effective and inexpensive in every situation. In general, the resulting plants exhibited a low copy number of the foreign gene, and the DNA confined between the two ends of the T-DNA was specifically transferred. (DeBlock M. (1993) Cell Biology of Plant Transformation: Current Status, Problems, Prospects and Significance of Plant Breeding. Euphytica 71:1-14)

Infected tissue should usually produce callus tissue that has the ability to reform embryos or meristems. However, direct infection of embryos taken from mature seeds is also possible. This method is very inefficient and was published in Arabidopsis thaliana. (Feldmann KA & Marks MD. (1987) "Agrobacterium-mediated transformation of germinated seeds of Arabidopsis thaliana: a non-tissue culture method. Molecular and General Genetics 208:1-9) Another publication published a Similar method (Cheep, Fober & SligntomJL (1989) transforms soybean (Glycine.max (L.) Merril) with the method for Agrobacterium tumefaciens infection germinated seeds. Plant Physiology 91:1212-1218), however, it does not show Any signs of stable conversion.

The use of the Agrobacterium system as a tool for obtaining transgenic plants relies on two factors: first, there must be a clear pathogen-host relationship. Some dicots and most monocots and gimnosperms are insensitive or show very low susceptibility to Agrobacterium. (Christou P. (1993) The principle and practice of transferring multiple independent genes into resistant plants. In Vitro Cell and Developmental Biology, 29P:119-124). Second, it is necessary to develop tissue culture methods to obtain explants that are organogenic and capable of being transformed.

Recently, a biolistic approach combined with the Agrobacterium system (agrobiolistic) has been used to obtain transgenic plants. Microparticles made of tungsten are accelerated into plant tissue at high speeds. The result is tiny wounds that allow the Agrobacterium to cause infection. This method has made it possible to obtain transgenic plants of tobacco, sunflower, banana and grape. (Bidney DL. Scelonge CJ, Martich J, Burrus M, Sims L & Huffman G (1992) Microprojectile bombardment of plant tissue increases the transformation frequency of Agrobacterium tumefaciens. Plant Molecular Biology 18:301-313), (Malone- Schoneberg J, Scelonge CJ, Martich J, Burrus M & Bidney DL (1994) Steady Plant Science 103:199-207), (May GD, Afza R, Mason HS, Wiecko A, Novak FJ, Arntzen CJ (1995) via Agrobacterium Mediated transformation produces transgenic banana strains (Musa acuminata). Bio/Technology 13:486-492), (Scorza R, Cordts JM, Ramming DN, Emers RL (1995) Sygotic-origin grape (Vitris Vinifera L.) Transformation of somatic embryos and regeneration of transgenic plants, Plant Cell Reports, 14:589-592).

In order to obtain transgenic plants from the apical region of the hypocotyl, there are two basic necessary conditions, namely: 1) introducing the exogenous gene into the apical region cells at a high frequency, and introducing the exogenous gene into the plant genome. 2) Regeneration and formation of reproductive-competent transgenic plants from the resulting transformed cells.

With the development of transformation systems, it is possible to alter meristem cells. However, the development and further formation of fertile transgenic plants requires regeneration of the transformed cells and formation of plants.

During the past decades, many attempts have been made to obtain regenerated fertile plants of commercially important plants. For example, methods for multiple germination of embryonic apical and lateral meristems have been developed in different plants such as soybean, bean, eucalyptus, cocoa, pineapple, etc.

Other regeneration methods have been developed for certain plants such as soybean, bean, cotton and peanut, including induction of somatic embryogenesis from cultured mature and immature embryos. Known methods for obtaining transgenic plants based on transformation of meristematic cells have the following disadvantages: it is impossible to select transformed cells; Cell masses or organs composed of non-transgenic cells).

Therefore, the object of the present invention is to provide a method for obtaining transgenic higher plants with a high production frequency and which enables the selection of transformed cells containing a gene pair translocated in plant tissues and enriched in meristems One molecule of the region exhibits resistance.

Summary of the invention

The present invention relates to a method for selecting transgenic meristem cells in cotton, coffee, cocoa, banana or grape plants. The method includes the following steps:

a) Introducing exogenous genes into hypocotyls of cotton, coffee, cocoa, banana or grape plants

cells of the meristems, in the tissues or organs comprising the meristems of these plants;

b) by culturing the meristem in a medium containing a multiple germination inducer

Or a tissue organ comprising a meristem, inducing the altered meristem in step a

The cells in the tissue area germinate more,

c) by adding a molecule in a culture medium that is translocated and enriched in the apical meristem

In further culturing the meristem or the tissue or organ containing the meristem, select

the transgenic meristem cells obtained in step b; or d) by applying a molecule that is translocated and enriched in the apical meristem region of the embryo to the apical

multiple shoots in the apical region to select the transgenic meristems in the apical region obtained in step b

Tissue cells.

Detailed description of the invention

It has now surprisingly been found that it is possible to influence the selection of transgenic meristem cells containing a gene that exhibits resistance to a molecule that is translocated in the tissue and enriched in the hypocotyl or meristem region of the tissue. sex. This selection involves multiple germination and further steps to produce fertile transgenic plants at a high frequency of 1%-20%. This value is approximately using currently known methods. A magnitude of about 2 to 100 times the frequency is obtained, the latter having a magnitude of about 0.02%-0.5%. In addition, the method of the present invention can obtain transgenic plants in a shorter time than the prior art.

The now patent-pending method is suitable for transformation, regeneration and selection of different higher plants.

According to the present invention, hypocotyls containing apical meristem cells to be transformed in cotton, coffee, cocoa, banana or grapes or organ tissues containing meristems are prepared in a laboratory using a conventional method for particle Biolistic and/or Agrobacterium inoculation. Of course, the genes used for particle bombardment will depend on the specific purpose of each production process in question, ie, the genes will be selected according to the new trait desired to be imparted to the transformed plants. For example, where the purpose of production is to obtain herbicide resistant plants, a gene that provides such herbicide resistance will be utilized.

Following particle bombardment, hypocotyls or meristem-containing tissues are contacted with a medium containing multiple germination-inducing and selective agents and should be kept in the medium for a sufficient period of time, preferably over 1 to 30 days, to ensure Desired selection and induction. In a preferred embodiment of the present invention, cytokinin, i.e. 6-benzylaminopurine (BAP), is used as multiple sprouting inducer, and herbicides, such as imazapyr (available from American Cyanamid) or basta (available from AgrEvo), was used as a selection agent. An additional advantage of the present invention is that the method now claimed enables multiple germination to be achieved in a relatively short time, thus avoiding the genetic variation common in other known methods.

At the stage of selection for transformed cells, a molecule enriched in the apical meristem zone, such as the herbicide cited above, is transported through the ductal system of the hypocotyl and then concentrated in the meristem zone to act as a selection agent. In this way selection of cells can be accomplished without deleterious effects on the hypocotyls.

The present invention can be better understood with the help of the examples given below. These examples are illustrative only, and the parameters and conditions described therein should not be considered as limitations of the invention.

Example

Preparation of cotton, coffee, cocoa and grape hypocotyls for particle bombardment (biolistic)

Mature cotton seeds were sterilized in 70% ethanol for 1 min and in 4.0% calcium hypochlorite for 7 min. The sterilized seeds were washed with sterile distilled water and incubated in sterile distilled water at room temperature for 16-18 hours. The water was then removed and the seeds were incubated for an additional 16-18 hours at room temperature in the dark. In the case of cocoa, coffee and grapes, the seeds were sterilized in 70% ethanol for 1 minute and in 1.0% sodium hypochlorite for 20-30 minutes. The sterilized seeds were washed with sterile distilled water and incubated in sterile distilled water at room temperature for 16-18 hours.

The seeds of cotton, cocoa and grapes are peeled and the hypocotyls are removed. Primary leaves were excised to expose the apical meristem region. In the case of coffee, hypocotyls were removed and cultured in WPM medium for 1 week. Following this, primary leaves are excised to expose the meristem region.

The hypocotyls were then placed in a culture dish (10-15 hypocotyls/dish) containing a particle bombardment medium (hereinafter referred to as BM) consisting of a Murashige and skoog (1962) medium , supplemented with 3% sucrose, 0.7% vegetable gel, pH 5.7. These hypocotyls are arranged in a circle at equal distances of 6-12 mm from the center of the dish, with the apical meristem zone directly upward.

Once the material to be transformed is placed on the plate containing particle bombardment medium BM, it is bombarded with the gene of interest. In this case, various vectors are used that contain genes for resistance to the herbicides imazapyr or basta.

Tissues containing apical meristems were prepared for particle bombardment.

Selected banana tissues were sterilized in 70% ethanol for 1 minute and in 4.0%-9.0% sodium hypochlorite for 20-30 minutes. Sterilized tissues were washed with sterile distilled water.

These tissues were then placed in dishes containing particle bombardment medium (10-15 tissues/dish). The particle bombardment medium (hereinafter referred to as BM) consisted of a Murashige and Skoog (1962) medium supplemented with 3% sucrose, 0.7% phytogel, pH 5.7. These tissues were arranged in a circle at equal distances of 6-12 mm from the center of the dish, with the apical meristem zone directly upward.

Once the material to be transformed is placed in the plate containing particle bombardment medium BM, it is bombarded with the gene of interest. In this case a variety of vectors are used that contain genes for resistance to the herbicides imazapyr or glufosinate.

Preparation of microparticles and bombardment of meristems

Microparticles responsible for carrying foreign DNA into cells are sterilized and washed. 60 mg of microparticles of tungsten M ₁₀ (Sylvania) or gold (Aldrich, 32,658-5) were weighed and transferred to a microcentrifuge tube to which 1 ml of 70% ethanol was added. The mixture was stirred vigorously and continued for 15 minutes at the lowest speed of the mixer. Centrifuge at 15,000 g for 5 minutes and remove with a 1,000 μl micropipette and discard the supernatant. Add 1 ml of sterile distilled water and mix vigorously in a stirrer, centrifuge as in the previous step, discard the supernatant, and repeat this washing operation two more times.

After the last wash, the supernatant was discarded and the microparticles were resuspended in 1 ml of 50% glycerol (v/v). Equal parts of glycerol and distilled water were mixed, and the mixture was autoclaved and kept at room temperature.

DNA is then precipitated onto microparticles. For this purpose, 50 μl aliquots of the microparticle suspension were transferred to a microcentrifuge tube and 5-8 ml DNA (1 mg/μl) was added. The mixture was mixed rapidly (3-5 seconds) by shaking the outside of the tube with a finger. Add 50 μl of 2.5M CaCl ₂ , stir quickly and add 20 μl of 0.1M spermidine (SigmaS-0266). Spermidine is a reagent that easily absorbs water and can be oxidized.

The resulting mixture was incubated for 10 minutes at room temperature with gentle agitation, centrifuged for 10 seconds and the supernatant was carefully removed. Add 150 μl of absolute ethanol, then shake the outside of the tube again with your finger. The resulting mixture was centrifuged at 15,000 g for 10 seconds, and the supernatant was removed. Repeat the previous steps, add 24 μl absolute ethanol, mix vigorously and sonicate for 1-2 seconds.

3.2 μl of lysed sample was dispensed into the center of each transport membrane, which had been pre-positioned on the support membrane. Each precipitation is sufficient to prepare 6 transport membranes containing microparticles coated with the DNA of interest. The discs containing the DNA-coated microparticles are quickly deposited onto a plate containing drying material (silica gel) and placed in a desiccator.

Particle bombardment of the hypocotyl meristem region of higher plants was achieved using a particle accelerator using high-pressure nitrogen gas, as described in the literature below. Aragao FJL, Barros LMG, Brasileiro ACM, Ribeiro SG, Smith FD, Sanford JC, Faria JC, Rech EL: Inheritance of exogenous genes in transgenic bean (phaseolus Vulgaris L.) transformed by particle bombardment. Genetics Theory Applied, 93:142-150 (1996).

Embodiment 1: Obtain transgenic cotton strain (Gossypium spp) by the selection of herbicide imazapyr or glufosinate

After bombardment of hypocotyls with microparticles coated with imazapyr-resistant exogenous DNA, hypocotyls were transferred from particle bombardment medium (BM) to culture plates containing multiple germination induction and selection medium (ISM) inside. (ISM refers to multiple sprouting MS medium supplemented with 22.2 μl of BAP, 3% glucose or sucrose, 500-1000 nM of 0.8% agar, pH 5.7). Particle-bombarded hypocotyls were kept submerged in ISM for 2-3 weeks and then transferred to plates containing elongation medium (EM) (SM medium, 3% sucrose, 0.8% agar, pH 5.7), and stored in a growth chamber with a temperature of 27°C and a photoperiod of 16 hours (50 μmols m ^-2 S ^-1 ). Hypocotyls can also be cultured in medium without selection agent and selected after induced multiple shoot development. Thus, the herbicides glufosinate-ammonium (0.5-1%) or imazapyr (0.2%) were applied directly to the multiple shoots.

When the shoots reached 2-4 cm long, they were transferred to an MSIS medium (MS, 1% sucrose, 0.8% agar, 0.1% carbon black, pH 5.7), 27°C, 16-hour photoperiod (50μmols m ^-2 S ^-1 ) to enable plantlets to grow and root. A 1 mm section was cut from the base of the stem and leaf for analysis of foreign gene expression. Shoots expressing exogenous DNA were individually recorded and transferred to a new culture flask. Once the plantlets had rooted, they were transferred to containers containing an autoclaved soil:vermiculite (1:1) mixture.

The plantlets were covered for 7 days with a plastic bag tied with an elastic band. After 6-7 days, remove the elastic and plastic bag. Plants were transferred to soil-containing containers for seeding.

Embodiment 2: Obtain transgenic coffee strain (Cofea.spp) with herbicide imazapyr or scifosinate

After bombardment of hypocotyls with microparticles coated with imazapyr-resistant exogenous DNA, hypocotyls were transferred from particle bombardment medium (BM) to multiple germination induction medium (IM). (MS medium supplemented with 11.1-44.4 μM BAP, 3% sucrose, 0.6% agar, pH 5.7) in culture plates. Then, hypocotyls were transferred to a plate containing herbicide-containing medium (CMH) (medium SM, 3% sucrose, 100-500 nM IMAZAPYR, 0.7% agar, pH 5.7) and kept in a room. A growth chamber with a temperature of 27°C and a photoperiod of 16 hours (50 μmolsm ^-2 S ^-1 ) until multiple seedlings are induced. Hypocotyls can also be cultured in medium without selection agent and then selected after induced multiple shoot development. Thus, the herbicides glufosinate-ammonium (0.5-1%) or imazapyr (0.2%) were applied directly to the multiple shoots.

When the shoots reached 2-4cm long, they were transferred to an MSIS medium (MS, 1% sucrose, 0.8% agar, pH 5.7), 27°C, 16 hours light period (50μmolsm ^-2 S ^{- 1} ), so that small plants can grow and take root. A portion of lmm was excised from the base of the stem and leaf for analysis of exogenous gene expression. Shoots expressing the exogenous gene were recorded separately and transferred to a new culture flask. Once the plantlets had rooted, they were transferred to containers containing an autoclaved soil:vermiculite (1:1) mixture.

The plantlets were covered for 7 days in a plastic bag tied with an elastic band. After 6-7 days, remove the elastic and plastic bag. Plants were transferred to soil-containing containers for seeding.

Example 3: Transgenic cocoa plants (Theobroma cacao L.) were obtained by selection with the herbicide imazapyr or glufosinate.

After bombardment of hypocotyls with microparticles coated with imazapr-resistant exogenous DNA, the hypocotyls were quickly transferred from particle bombardment medium (BM) to induction medium (IM) (MS medium supplemented with 11.1- 44.4μM BAP, 3% sucrose, 0.6% agar, pH 5.7) culture plate. Then, hypocotyls were transferred to a plate containing herbicide-containing medium (MCH) (MS medium, 3% sucrose, 500-1000 nM IMAZAPYR, 0.7% agar, pH 5.7) and stored in a room. A growth chamber with a temperature of 27°C and a photoperiod of 16 hours (50 μmols m ^-2 S ^-1 ) until multiple seedlings are induced. Hypocotyls can also be cultured in medium without selection agent and also selected after induced multiple shoot development. In this way, the herbicides glufosinate-ammonium (0.5-1%) or imazapyr (0.2%) were applied directly to the multiple shoots.

When the shoots reached 2-4cm long, they were transferred to an MSIS medium (MS, 1% sucrose, 0.8% agar, pH 5.7), 27°C, 16 hours light period (50μmolsm ^-2 S ^{- 1} ) so that the plantlets can grow and take root. 1 mm sections were excised from the base of stems and leaves for analysis of foreign gene expression. Shoots expressing the exogenous gene were recorded separately and transferred to a new flask. Once the plantlets had rooted, they were transferred to containers containing an autoclaved soil:vermiculite (1:1) mixture.

The plantlets were covered for 7 days in a plastic bag tied with an elastic band. After 6-7 days, remove the elastic and plastic bag. Plants were transferred to soil-containing containers for seeding.

Embodiment 4: Obtain transgenic banana plant (Musa spp) by the selection of herbicide imazapyr or glufosinate

Tissues containing meristems were bombarded with microparticles coated with imazapyr-resistant exogenous DNA, in the same medium (MB) at 27°C with a 16-hour light period (50 μmolsm ^-2 S ^-1 ) Cultured for 7 days to induce multiple shoots. Afterwards, the germinated hypocotyls were transferred to a "magenta"-type box containing MSBH medium (MS medium supplemented with 10-20 μm BAP, 3% sucrose, 50-300 nM IMAZAPYR, 0.8 % agar, pH 5.7), temperature 27°C, 16-hour photoperiod (50 μmols m ⁻² S ⁻¹ ) for 7 days to reduce the total number of multiple seedlings. Hypocotyls were then transferred again to "magenta"-type boxes containing MS3S medium (MS supplemented with 5-10 μM BAP, 3% sucrose, 0.8% agar, pH 5.7) at 27°C , under the condition of 16-hour photoperiod (50μmols m ^-2 S ^-1 ), multiple seedlings could elongate. After two weeks, the hypocotyls begin to germinate. Hypocotyls can also be cultured in media without selection agents and selected after induced multiple shoot development. In this way, the herbicides glufosinate-ammonium (0.5-1%) or imazapyr (0.2%) are directly applied to multiple shoots.

When the shoots reached 2-4 cm in length, they were transferred to an MSIS medium (MS, 1% sucrose, 0.8% agar, pH 5.7), 27°C, 16-hour light period (50 μmolsm ^-2 S ^-1 ) Enable the plantlets to grow and take root. 1 mm long sections were excised from the bases of stems and leaves for analysis of foreign gene expression. Shoots expressing the exogenous gene were recorded separately and transferred to a new culture flask. Once the plantlets had rooted, they were transferred to containers containing a sterilized soil:vermiculite (1:1) mixture.

The plantlets were covered with a plastic bag tied with an elastic band for 7 days, the elastic band and plastic bag were removed after 6-7 days. Plants were transferred to soil-containing containers for seeding.

Embodiment 5: by the selection of herbicide imazapyr or glufosinate, obtain transgenic grape strain (Vitis Vinifera L.)

After bombardment of hypocotyls with particles coated with imazapyr-resistant exogenous DNA, hypocotyls were rapidly transferred from particle bombardment medium (BM) to induction medium IM (MS medium supplemented with 22.2- 44.4 μM BAP, 3% sucrose, 0.6% agar, pH 5.7) culture plate. Then, the hypocotyls were transferred to a plate with herbicide-containing medium (MCH) (MS medium, 3% sucrose, 100-600nM IMAZAPYR, 0.7% agar, pH5.7), and incubated at 27°C. , stored in a growth chamber with a 16-hour photoperiod (50 μmols m ^-2 S ^-1 ) until multiple seedlings are induced. Hypocotyls can also be cultured in media without selection agents and selected after induced multiple shoot development. Thus, the herbicides glufosinate-ammonium (0.5-1%) or imazapyr (0.2%) were applied directly to the multiple shoots.

When the shoots reached 2-4cm in length, they were transferred to an MSIS medium (MS, 1% sucrose, 0.8% agar, pH 5.7) for 16 hours in the photoperiod (50 μmolsm ^-2 S ^-1 ) , Under the condition of 27 ℃, the small plants can grow and take root. 1 mm long sections were excised from the bases of stems and leaves for analysis of foreign gene expression. Shoots expressing exogenous DNA were individually recorded and transferred to a new flask. Once the plantlets had rooted, they were transferred to containers containing a sterilized soil:vermiculite (1:1) mixture.

The plantlets were covered for 7 days with a plastic bag tied with an elastic band, after 6-7 days the elastic band and plastic bag were removed and the plants were transferred to containers with soil for seeding.

Shoots reaching 2-4 cm in length were transferred to an MSIS medium (MS, 1% sucrose, 0.8% agar, pH 5.7) for 16 hours in the photoperiod (50 μmols m ^-2 S ^-1 ), 27 Under the condition of ℃, the small plants can grow and take root. 1 mm long sections were excised from the bases of stems and leaves for analysis of foreign gene expression. Shoots expressing exogenous DNA were individually recorded and transferred to a new flask. Once the plantlets had rooted, they were transferred to containers containing a sterilized soil:vermiculite (1:1) mixture.

The plantlets were covered with a plastic bag tied with an elastic band for 7 days, the elastic band and plastic bag were removed after 6-7 days. Plants were transferred to containers containing soil for seeding.

Claims (5)

1. select the method for cotton, coffee, cocoa, banana or grapevine seedling transgenosis meristematic cell, may further comprise the steps:

A) foreign gene is imported the top of cotton, coffee, cocoa, banana or grapevine seedling plumular axis

The end meristematic cell in or contain in the merismatic tissue;

B) by in the substratum that contains multiple bud inductor, cultivating this plumular axis or containing merismatic

Tissue is induced the multiple bud of the meristem zone's cell that changes among the step a.

C) by in substratum, further cultivating this meristematic tissue, select to obtain among the step b

The meristematic tissue transgenic cell, a kind of molecular energy that contains in the wherein above-mentioned substratum

Displacement and to be enriched to cotton, coffee, cocoa, banana or grape tip of the embryo axis mitogenetic

Tissue; Perhaps

D) by a kind of cotton that is shifted and is enriched in, coffee, cocoa, banana or grape plumular axis

The molecular application in apical meristem district is selected step b in the multiple bud of top zone

The meristematic cell of the middle top zone that obtains.

2. the process of claim 1 wherein that multiple bud inductor is a kind of phytokinin.

3. according to the method for claim 2, wherein phytokinin is the 6-benzylaminopurine.

4. according to the process of claim 1 wherein that selective agent in the step (c) is a kind ofly to be enriched to the apical meristem district of described embryo or to comprise molecule in the merismatic tissue.

5. according to the method for aforementioned each claim, wherein cotton, coffee, cocoa, banana or grapevine seedling are selected from different Cultivars.