MXPA01004202A - Method for the genetic transformation and regeneration of transgenic prickly pear plants (opuntia sp.) - Google Patents

Abstract

The present invention describes a methods system of tissues culture in vitro for the genetic transformation interceded by Agrobacterium tumefaciens, and for the regeneration of prickly pear plants (Opuntia sp.). The relevant factors used in this system are:a) the use of a modified mean containing Murashige-Skoog (MS) supplemented with benzylamino-purine, gibberellin A3, and an additional 20%of nitrogen in the mean MS;b) selection and use of areolas (apical and axillary meristems) as target tissue (also called targeted tissue) both, for the plants regeneration and for the genetic transformation, in view of, the stem nature of said targeted tissue;c) the co-culture procedure consists in the inoculation with the pGV2260(pB1121)strain of Agrobacterium tumefaciens directly used in the areolas with a hypodermic syringe;and d) obtaining transgenic plants of prickly pear (Opuntia sp.) by means of the selection in means NOP1 supplemented with kanamicine?. The system permits a transformati on efficiency of 26%of the infected explants. This method is useful for the routinary vegetal transformation and regeneration that involves the introduction of exogenous genes in the prickly pear plant (Opuntia sp.), in a short period of time in comparison with the traditional methods of phyto-enhancement.

Description

DENOMINATION OR TITLE OF THE INVENTION Method for the Genetic Transformation and Regeneration of Nopal Transgenic Plants. { Opuntia sp.).

FIELD OF THE INVENTION The present invention is part of the field of biotechnology applied to the improvement of crops of economic importance. It refers to the methods of molecular biology and genetic engineering used for the generation of transgenic plants by introducing exogenous genes to plants of economic interest, in order to improve their nutritional, functional, sensory, pre and post-harvest handling qualities, as well as with the purpose of introducing said genes so that the transgenic plant produces proteins or chemical compounds of food, pharmaceutical, industrial, and / or other interest.

More particularly, this invention relates to a method for the genetic transformation and regeneration of prickly pear plants. { Opuntia sp.).

SUMMARY The present invention describes a system of in vitro tissue culture methods for genetic transformation, mediated by Agrobacterium tumefaciens, and for the regeneration of cactus plants. { Opuntia sp.). The critical factors used in this system are: a) the use of a modified medium containing Murashige-Skoog (MS) basal medium supplemented with benzylaminopurine, gibberellin A3, and an additional 20% nitrogen in the MS medium; b) the selection and use of areolas (apical and axillary meristems) as objective tissue (also called white tissue) both for the regeneration of plants and for genetic transformation, in view of the totipotential nature of said white tissue; c) the co-culture procedure consisting of inoculation with the strain pGV2260 (pBI121) of Agrobacterium tumefaciens directly into the areolas using a hypodermic syringe; and d) obtaining transgenic cactus plants. { Opuntia sp.) By selection in NOP1 medium supplemented with kanamycin. The system allows a transformation efficiency of 26% of the infected explants. This method should be useful for routine plant transformation and regeneration involving the introduction of important exogenous genes of interest in the cactus plant. { Opuntia sp.), In a much shorter time compared to traditional methods of plant breeding.

BACKGROUND / PREVIOUS ART In Mexico, most of the territory is arid and semi-arid zones that occupy almost 60% (1, 450,000 km) of the total surface; They have characteristics of low precipitation, extreme temperatures and soil with little amount of organic matter, so it is very difficult to produce basic crops. The cultivation of cactus. { Opuntia sp.) Is considered a good option to produce in these regions because it has morphological and physiological characteristics that allow it to adapt to conditions of low precipitation. Cactus production has allowed marginalized groups to obtain employment, take root in the countryside, produce food and generate income for their families. For example, cactus Opuntia genus represent about 100 species that have been used for hundreds of years by man in different forms (such as forage for animals, in the production of natural dyes such as red grana, for direct consumption in food human in the form of nopalitos (stems or young cladodes) and of the fruits called tunas, and as a source of nutraceutical compounds (Bravo, 1978, Scheinvar, 1999) .The economic and social importance of the nopal in Mexico lies, above all, in the large area occupied by ^ -Wf * nopaleras, both wild and cultivated, in the type and number of producers involved, in the type of regions where they are grown, and in the diversity of the products generated. an area of 30 million hectares and those cultivated only 45 thousand hectares, however, some of the aspects for which the crop is problematic are: low production, and the quality and high number of seeds that present the fruits that are marketed, among other factors (Castellanos et al., 1999; De León et al., 1999; Gutiérrez, 1999). It is estimated that in Mexico there are about 50-60 thousand ha of prickly pear planted for prickly pear, which makes this country the main producer worldwide. There are yields of 3-15 ton / ha of fresh fruit. The commercialization of tuna is limited by the short production period (June-November), lack of technology for postharvest handling and rapid ripening of the fruit. In the central region of Mexico, the harvest of the prickly pear is made from June in Puebla, and ends at the beginning of October in Zacatecas, concentrating production during July and August, especially in the states of Mexico and Hidalgo. 20 It is called nopal to several species of the Opuntia genus of the Cactaceae family; This family is endemic to America. Mexico has a great genetic diversity of Opuntia, which probably places it as the center of origin of the genus; This allows for a large number of wild genotypes and with different degrees of domestication for the use of family and commercial gardens (Fernández et al., 1999a). This diversity has been found in reproductive attributes, vegetative growth habits and qualitative aspects of relevance in the tuna producing regions (Fernández et al., 1999b, Vázquez Alvarado et al., 1999). Although in Mexico you can find a large number of cactus species, Opuntia ficus-indica (L.) Miller is one of the most widely known (Pimienta-Barrios et al., 1993). In general, the nutritional value of cactus fruits compares favorably with that of apples, apricots, cherries and oranges (Cantwell, 1991). However, also in nutritional terms, the low protein content of cactus and prickly pear is a problem for people whose diet is based mainly on this plant. The biochemical processes and molecular biology that determine the yield, disease resistance, and quality characteristics of these plants are just beginning to be understood. On the other hand, it is known that the seed is a source of proteins of nutritional value, represents 5-10% of the weight of the fruit, and that it contains between 5-16% of protein. The tuna has, from the nutritional point of view, a composition similar to that of other fruits; however, the presence of polysaccharides of the mucilage type makes a big difference in it and makes it difficult to process. Nopales have a high water content and have dietary fiber, of great interest in the contemporary diet due to their ability to prevent some diseases of the colon, obesity and diabetes (Aguilar Zamora, 1999, Cantwell, 1991, Gallardo et al., 1997; Sáenz, 1998; Silos Espino et al., 1999). Additionally, there are several Opuntia species that are used to produce nopalitos; however, Opuntia ficus-indica (L) Miller is the most commercially cultivated species. This is a cultivar without thorns very useful for production. The harvest is carried out throughout the year although productivity is higher during the spring and it is reduced in the middle of autumn and during the winter (Pimienta Barrios, 1993).

As for its reproduction, it is reported that Opuntia sp. presents self-fertilization and cross-fertilization. In addition, it carries out clistogamy, a form of autogamy in which pollination occurs before the flower opens. The systems of crossing are the set of sexual expressions of organisms that affect the genetic composition of their offspring; they are also the link between generations and the main determinant of the structure of populations. The improvement of crops of agricultural interest by traditional systems requires long periods of time; Therefore, the development of efficient in vitro plant transformation and regeneration processes has been of great help in rapidly improving important crops in recent years. The production of morphologically normal plants that contain and express exogenous genes (also called external genes, foreign or heterologous) has been made possible by the use of different strategies, being the natural transfer of genes that Agrobacterium tumefaciens performs one of the most used (Horsch et al., 1985). This system of gene transformation was established using plants of the Solanaceae family for its easy manipulation in tissue culture; although a large group of dicotyledons can be transformed successfully (Kaneyoshi et al., 1994). Protocols have been adapted from existing meristem transformation and regeneration technology for rapid propagation (Morel, 1972; Murashige, 1974), and have been used with gene transfer mediated by Agrobacterium tumefaciens (Caplan et al., 1983 Chilton et al., 1977; Depicker et al., 1983). Recent advances in the transformation of plants have increased the possibility of making genetically planned changes and thus modify the processes, expanding the use of germplasm useful for plant improvement programs (McEIroy, 1996). This information is critical to define the future of strategies for improvement in crops that have adaptation limitations (drought, salinity, low temperature), productivity (pests, diseases), and fruit quality (Pimienta-Barrios, 1994). It is well known that the use of the transformation system mediated by Agrobacterium tumefaciens can lead to stable incorporation of exogenous genes of interest to the genome of a number of plant species (Weising et al., 1988). Positive results have already been reported in several economically important crops (Ishida et al., 1996, Li et al., 1992, Miguel and Oliveira, 1999). However, for nopal there are no reports on its genetic transformation using any of the available strategies, including the Agrobacterium tumefaciens system. In the present invention, a system of methods for the genetic transformation, regeneration and in vitro propagation of cactus plant tissues is presented. { Opuntia sp.).

OBJECTIVES OF THE INVENTION Bearing in mind that the improvement of crops of agricultural interest, by the natural systems of crossing or traditional methods of genetic improvement, requires of long periods of time, it is an object of the present invention to provide a highly efficient system. efficient methods that allow for genetic transformation and the regeneration of prickly pear plants. { Opuntia sp.) With better agronomic, alimentary, functional, nutritional characteristics, and / or with compositions and / or physicochemical principles of high added value, in a much shorter time than that used in the previous systems or methods. Also, given the capabilities of the cactus plant. { Opuntia sp.) To grow abundantly in adverse agricultural and climatological conditions for other plants, it is an object of the present invention to provide a highly efficient system of methods that allow the generation of transgenic cactus plants that have the capacity to produce the plant or parts of the plant with the best agronomic, alimentary, functional, nutritional characteristics, and / or with novel physicochemical compositions and / or principles of high added value, in agricultural and climatological conditions that are limiting or oppressive for many other plants. The above objects, as well as other objects and advantages of the invention, are achieved by a system of in vitro tissue culture methods to carry out the genetic transformation, mediated by the bacterium Agrobacterium tumefaciens, and for the regeneration of nopal plants. . { Opuntia sp.). The critical factors used in this system are: a) the use of a modified medium containing Murashige-Skoog (MS) basal medium supplemented with benzylaminopurine, gibberellin A3, and an additional 20% nitrogen in the MS medium; b) the selection and use of areolas (apical and axillary meristems) as objective tissue (also called white tissue) both for the regeneration of plants and for genetic transformation, in view of the totipotential nature of said tissue; c) the use of a co-culture procedure consisting of inoculation with strain pGV2260 (pBI121) of Agrobacterium tumefaciens directly in the areolas using a hypodermic syringe; and d) obtaining transgenic cactus plants. { Opuntia sp.) By selection in NOP1 medium supplemented with kanamycin.

DETAILED DESCRIPTION OF THE INVENTION Plant material Opuntia ficus-indica cv. "Villa Nueva" was obtained from germplasm resources of Villa Hidalgo, Zacatecas, Mexico. Modified stems (cladodes) were selected from 8-year-old plants with high yields of nopalitos and prickly pear (80-90 ton / ha) (Flores Valdez, 1995).

Generation of explants in aseptic culture In order to obtain explants in aseptic culture in vitro from cactus cladodes cultivated in the field, three treatments were tested. In treatment one, the surface of cladodes 7-10 cm in length was washed with commercial detergent and tap water, followed by immersion in Clorox (commercial sodium hypochlorite solution commonly used as a laundry bleach) at 10% (v / v) for 10 min and 70% ethanol (v / v) for 5 min (Escobar et al., 1986). Treatment two consisted of treatment one except that, before dives in Clorox and ethanol, the cladodes were also immersed in a solution containing a mixture of the following fungicides: Agrimycine (1 mg / L), Benlate (2 mg / L) and Ridomil (2 mg / L). Treatment three consisted of treatment two with the addition of cupric sulfate (300 mg / L) to the fungicide solution; then rinsed with 70% (v / v) ethanol for 1 min, 1.6% Clorox (v? /) for 10 min, and sterile distilled water. The cladodes were cut into fragments of a square centimeter, each fragment containing an areola. The initial experiments to determine the optimal culture conditions for the induction of shoots, from explants containing areolas, were not successful due to the high level of contamination in vitro with bacteria and endogenous fungi of cactus. To solve this problem, the effect of several fungicides including Agrimycine, Benlate, Ridomil and cupric sulfate was tested in terms of its surface disinfection capacity to obtain aseptic plant material. Cupric sulfate was the only effective treatment; resulted in total asepsis of 10% of the explants, while the other fungicides did not produce aseptic explants. This observation, on the useful effect of cupric sulfate for the inhibition of fungal growth in tissue culture, is not a surprise since said compound has been used for a long time in the treatment of fungal diseases in agriculture.

Regeneration of plants The areolas consist of an apical meristem that directs the growth and development processes of the whole plant. Therefore, these structures were chosen as objective tissue due to their high totipotentiality, which is a requirement for tissue culture and genetic transformation of plants. The explants were cultivated for induction and propagation of calluses in medium NOP1: macronutrients and micronutrients, iron, myo-inositol and vitamins from Murashige-Skoog medium (Murashige and Skoog, 1962) plus ammonium nitrate (190 mg / L), potassium nitrate (160 mg / L), benzylaminopurine (BAP) 2 mg / L), gibberellin A3 (1 mg / L), citric and ascorbic acids (150 mg / L each), 2% sucrose, pH adjusted to 5.8 and solidified with agar-agar (7 g / L; Bioxon). For the establishment of aseptic cultures, the washes with the fungicide solutions mentioned above were used. The explants were cultured with a 16 h photoperiod with fluorescent Sylvania lamps of cold and white daylight (35 μmol / m2 / s) at 26 ° C. From aseptic cladodes established under tissue culture conditions, explants containing areolas were dissected and cultured in NOP1 medium for bud proliferation. The development of an average of 30 shoots per explant was observed after 45 days of incubation; the shoots were subcultured every 60 days in fresh NOP1 medium. The number and quality of outbreaks obtained with the NOP1 medium were similar to those reported by Escobar et al. (1986), in whose work Opuntia amyclaea was used as a source of plant material. The high efficiency of our shoot proliferation protocol is due to the fact that a 7-10 cm cladode can be dissected to obtain up to 25 explants, each containing an areola that efficiently responds to shoot formation. This morphogenetic process is a prerequisite for the experiments of genetic transformation of the plant, mediated by the Agrobacterium tumefaciens system or other alternative techniques of genetic transformation. After three weeks of incubation under illumination, the differentiated outbreaks of Opuntia ficus-indica were rooted in basal MS medium containing BAP (2 mg / L). Later, complete plants were established in soil for further growth and development.

Selective media The tolerance tests were carried out using three different aminoglycoside antibiotics: kanamycin, geneticin (G418) and paromomycin at 25, 50 and 100 mg / L, respectively. 70 shoots were grown in each medium by treatment for 45 days of incubation, until the growth and development of the shoots were totally inhibited. Data were obtained at 15, 30 and 45 days.

Tolerance assays were carried out using kanamycin, geneticin (G418) or paromomycin at 25, 50 and 100 mg / L each. The most efficient selective agent was kanamycin at 100 mg / L, as it stopped growth and development in 100% of the treated explants, compared to 88.6% using G-418 and 42% with paromomycin, after 45 days of incubation. All the outbreaks affected by the antibiotics showed chlorosis in their base, after which necrosis occurred and finally the death of the entire explant. These results agree with previous reports on genetic transformation, in which kanamycin is considered the best selective agent for dicotyledonous species.

Bacterial strain Plasmid pBI121 (Jefferson, 1987) was introduced into Agrobacterium tumefaciens strain C58C1 Rif (pGV2260) (Deblaere et al., 1985) by electroporation. This plasmid contains the neomycin phosphotransferase gene (npt II gene) as a selection marker, under the control of a nopaline synthase promoter. { nos), as well as the ß-glucuronidase gene (uidA gene, also called GUS) as a reporter gene, controlled by the 35S promoter of the cauliflower mosaic virus. Agrobacterium tumefaciens was cultured overnight at 28 ° C in liquid Luria-Bertani (LB) medium (Gibco-BRL, Life Technologies, Inc., Gai le sburg, MD) supplemented with rifampicin (50 mg / L), carbenicillin (50 mg / L). mg ,.) and kanamycin (50 mg / L), until reaching an optical density of 1.1 to 600 nm.

Protocol of genetic transformation of plants and generation of transgenic plants The explants of one square centimeter were infected specifically in the areolas (Figure 1A). The Agrobacterium tumefaciens cells were harvested from fresh cultures of 3 days of incubation in Petri dishes. The process involves inoculation using a 29G x 13 mm needle in a sterile hypodermic syringe, injecting approximately 10 μL of Agrobacterium tumefaciens suspended in sterile distilled water (approximately 5 x 10 ^ cells) at each injection site. The infected explants were co-cultured for 3 days in NOP1 medium and incubated under illumination with a 16 h photoperiod with Sylvania fluorescent lamps of cold and white daylight (35 μmol / m2 / s) at 26 ° C. After co-culture, the infected explants were rinsed three times with sterile water containing Cefotaxime (Claforan, 500 mg / L), and then placed in NOP1 medium supplemented with carbenicillin (500 mg / L) to inhibit the growth of Agrobacterium and kanamycin. (200 mg / L) to select the transformants. The explants were incubated under selection pressure for three months. Kanamycin resistant shoots were dissected from the original explant and transferred to fresh NOP1 medium for propagation and growth (Figure 1 B).

Explants of Opuntia ficus-indica co-cultured with Agrobacterium tumefaciens were subcultured in NOP1 medium supplemented with kanamycin (100 mg / L). All the uninfected explants died after one month of incubation in the selective medium. The tissues of Opuntia ficus-indica resistant to or kanamycin began to develop from the infected areas (areolas) in the form of small buds after five weeks of incubation. These resistant structures were dissected from the original explant for further development in selective medium with kanamycin for an additional three weeks (Figure 1 B). The efficiency of genetic transformation was 26.3% of the total of the 5 co-cultivated explants. In other words, from 216 infected explants, 57 transgenic plants were obtained. Kanamycin resistant plants were individually graded and subcultivated for further biochemical and molecular analysis. The kanamycin resistance of these plants represented the first evidence of stable genetic transformation. 0 Enzymatic assay of β-glucuronidase (GUS) GUS activity was determined in regenerated shoots resistant to kanamycin, by the histochemical assay described by Jefferson (1987). GUS expression was tested in all kanamycin resistant shoots. 5 GUS activity was observed in only 21 of the 57 plants resistant to kanamycin. The expression occurred mainly around vascular tissue and in a few cases in the areolas (Figure 1C). Wild type nopal explants (non-infected) did not express GUS activity (Figure 1 D). The absence of expression of GUS activity in the rest of the plants resistant to kanamycin can be explained in terms of silencing of the gene expression, which has been demonstrated in other species (Lambe et al., 1995). The DNA methylation has also been associated with the gradual loss of GUS expression. To test the possibility of presence of chimeric plants due to the complex organization of the apical bud itself, explants were taken from shoots resistant to kanamycin but without GUS activity and subcultured on fresh selective medium every two weeks. All of them responded positively regarding growth and development under selection, and it was possible to obtain 15-20 shoots from each explant after two months of culture under illumination (Figure 1 E). The expression of the GUS reporter gene provided confirmatory evidence of stable genetic transformation mediated by the Agrobacterium tumefaciens in Opuntia ficus-indica. The transgenic plants were successfully established under soil conditions as shown in Figure 1 F.

Isolation of Opuntia DNA 20 Genomic DNA was isolated from young cladodes using a modification of the protocol described by Shure et al. (1983). This modified method consists of collecting approximately 250 mg of transgenic or non-transgenic tissue in 2 mL Eppendorf tubes, and grinding in liquid nitrogen to a fine powder using a glass pistil attached to a homogenizer (CAFRAMO, Stirrer type RZR). The powdered tissues were resuspended with 500 μL of extraction buffer (7.0 M urea, 0.35 M NaCl, 0.05 M Tris-HCl, pH 8.0, 0.02 M EDTA, 1% sarcosine) for at least 45 min. The tissue homogenate was extracted with a volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v). The aqueous phase was separated by centrifugation. To ensure minimum levels of protein and polysaccharides, the DNA was further purified by adding a half volume of 7.5 M ammonium acetate, pH 8.0, and then incubating at room temperature for 60 min, after which it was centrifuged at 18,000 xg for 15 min.; The aqueous phase was then precipitated using an equal volume of isopropyl alcohol. The precipitated DNA was washed once with 70% (v / v) ethanol and resuspended in TE buffer (0.01 M Tris-HCl, 0.01 M EDTA, pH 8.0).

Analysis by polymerase chain reaction (PCR) PCR analysis was carried out according to Williams et al. (1990). Each sample (150 ng of each of the seven putatively transgenic Opuntia ficus-indica genomic DNAs) was incubated in a mixture containing the four 2-deoxyribonucleoside triphosphates (dNTPs, 0.4 mM each), 2.5 mM magnesium chloride, both suitable primer oligonucleotides (0.4 mM each), 1 unit of Taq DNA polymerase (Gibco-BRL, Life Technologies, Inc., Gaithersburg, MD) and sterile deionized water for a total volume of 25 μL. The primer oligonucleotides used for the amplification of the nptll gene were 5'-TATTCGGCTATGACTGGGCA-3 'and 5'-GCCAACGCTATGTCCTGATA-3', with an expected amplification product of approximately 650 bp. Due to the small amount of plant tissue needed for PCR analysis, seven transformed clones were analyzed that showed the same levels of kanamycin resistance as well as GUS expression. PCR analysis yielded the expected 650 bp fragment of the inner region of the npt II gene (Figure 2, Lanes 1-7). No amplification was observed when using the DNA from non-transformed plants (Figure 2, Lane 8). This analysis represented a third line of evidence in favor of the genetic transformation carried out in cactus. { Opuntia sp.).

Transfer analysis and Southern hybridization The total plant DNAs (50 μg measured by A260 / 280) obtained from wild type and putatively transformed plants were digested overnight at 37 ° C, using 10 units of Pstl per microgram of DNA; then they were separated on 0.7% agarose gels and transferred to a Nylon membrane (Hybond N +, Amersham Pharmacia Biotech Inc., Piscataway, NJ, Sambrook et al., 1989). Each filter was prehybridized at 65 ° C in a buffer containing 7% sodium dodecylsulfate (SDS), 0.5 M sodium phosphate and 1% serum bovine albumin for 4 h, and hybridized with the probe overnight at 65 ° C. C in the same shock absorber. Two 32p labeled probes were used by random priming reactions from plasmid pBI121: a 1.5 kb Pstl-Pstl fragment from the nptll gene, or a 2.0 kb BamHI-EcoRI fragment from the GUS gene ( Feinberg and Vogelstein, 1983). The filters were washed in 2x SSC (Sambrook et al., 1989) with 0.1% SDS at room temperature for 5 min; then they were washed with 1x SSC with 0.1% SDS at 65 ° C for 15 min, and a final wash of 0.5x SSC with 0.1% SDS at 65 ° C for 5 min. The filters were visualized by autoradiography using X-ray film (Cronex, Dupont).

As an additional confirmation that the nopal buds obtained (resistant to kanamycin and with GUS activity) were actually transgenic, nine GUS- and PCR-positive clones and one nontransgenic plant tissue clone were analyzed by transfer and hybridization analysis Southern type. When using a fragment of the npt II gene as a probe, all putatively transgenic propagated clones showed the expected hybridization signal of 1.5 kb, and at least four different integration patterns of the npt II gene were observed, while the non-transformed clone did not showed hybridization (Figure 3, lanes 1-9). After extensive washes, the same membrane was hybridized with a 2.0 kb fragment of the GUS gene; the expected hybridization signals were found in the corresponding transgenic plants (Figure 4, lanes 1-9). The different integration patterns were observed with both the GUS probe and the npt II gene. The observation of different integration patterns can be explained in terms of stable genetic integration in different chromosomes and / or in terms of the number of copies that were integrated into the genome of the plant. The results of the Southern blot analysis and hybridization provided the most important confirmatory evidence of the stable genetic transformation of Opuntia sp.

Conclusions In this report we describe reproducible protocols for transformation, for plant regeneration in vitro and for the propagation of nopal. { Transgenic opuntia sp.). The system of Agrobacterium tumefaciens was applied successfully for the stable genetic transformation of cactus. { Opuntia sp.). The efficiency of gene transfer from Agrobacterium cells to plant cells was high according to the relatively high number of kanamycin-resistant plants obtained, the β-glucuronidase activity, and the PCR and transfer and Southern-hybridization assays . These results clearly show that in cultured Opuntia explants, the areola can be easily and rapidly transformed by Agrobacterium tumefaciens. These observations agree with those reported by Ulian et al. (1988), Grimsley et al. (1988), Dale et al. (1989), Hussey et al. (1989), and Seabra and Pais (1998), whose work reported that in buds of petunia, wheat, corn, pea and European chestnut in culture, the youngest tissue (the apical meristem) was the best tissue selected as a target for genetic transformation by Agrobacterium. Therefore, we conclude that it is possible to integrate genes of interest in Opuntia species. This represents the first report of stable genetic transformation of cactus. { Opuntia sp.).

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Nucleic Acids Research 18: 497-504.

LEGENDS OF THE FIGURES Figure 1. Protocol of genetic transformation and regeneration of transgenic cactus plants. { Opuntia sp.) Mediated by the Agrobacterium tumefaciens system. A) Infection of apical meristems (areolas) using a syringe needle with 10 μl of suspension of Agrobacterium tumefaciens cells. B) Kanamycin-resistant outbreaks after two months of incubation under selection. C) Expression of ß-glucuronidase in explants resistant to kanamycin. D) Wild type explants showing absence of β-glucuronidase expression. E) Propagation of shoots in medium containing kanamycin. F) Transgenic cactus plants established in soil under greenhouse conditions.

Figure 2. PCR analysis of seven plants resistant to kanamycin (lanes 1-7) and wild type tissue (non-infected, lane 8).

Figure 3. Southern blot analysis and hybridization using an npt II gene fragment as a probe.

Figure 4. Southern blot analysis and hybridization using a fragment of the GUS gene as a probe.

Claims (7)

1. CLAIMS 1. A method to transform a cactus plant. { Opuntia sp.) Using Agrobacterium comprising the steps of: a) contacting nopal explants with Agrobacterium; b) growing in NOP1 medium containing carbenicillin at concentrations capable of inhibiting the growth of Agrobacterium, and kanamycin as an agent to select the explants expressing the selection gene; and c) regenerating the plants that express the transgene.
2. 2. The method of claim 1 wherein the culture of the in vitro explants is carried out in modified medium containing Murashige-Skoog (MS) basal medium supplemented with benzylaminopurine, gibberellin A3 and 20% additional nitrogen in the MS medium.
3. 3. The method of claim 1 wherein the transformed plants are regenerated in NOP1 medium supplemented with kanamycin.
4. 4. The method of claim 1 wherein the Agrobacterium strain is introduced to the explant by hypodermic syringe.
5. 5. The method of claim 1 wherein the explants are selected from areolas or meristematic tissues of the cactus stems or cladodes.
6. 6. The method of claim 1 wherein the prickly pear plant is Opuntia sp.
7. 7. The method of claim 6 wherein the prickly pear plant is Opuntia ficus- indica. The method of claim 1 wherein the Agrobacterium species is Agrobacterium tumefaciens. The method of claim 1 wherein the nopal plants are genetically transformed stably. . The method of claim 1 wherein the transgene is expressed in subsequent generations.