MXPA06007412A - A tissue culture process for producing cotton plants - Google Patents

Abstract

A method for plant regeneration in cotton via developmentally synchronized somatic embryogenesis is disclosed. The invention is simple, fast, reproducible and convenient for applications in plant genetic engineering and results in the achievement of synchronised somatic embryogenesis by a step of inositol starvation.

Description

PROCESS FOR THE TISSUE CULTIVATION FOR THE PRODUCTION OF COTTON PLANTS FIELD OF THE INVENTION The present invention relates to a tissue culture process for producing a large number of viable cotton plants in vitro from a specific tissue of the plant. The invention provides a method for synchronized somatic embryogenesis and opens up new possibilities for obtaining an agronomically improved uniform population of cotton plants by modern methods of agrobiotechnology and genetic engineering. The protocol provides an important step in the success of the cotton improvement program using tissue culture technology.

BACKGROUND OF THE INVENTION Cotton is a globally important crop, which is grown mainly for fiber. The seeds provide an important source of feed for livestock. Cotton has influenced the economic development of many nations around the world. Therefore, cotton improvement programs using modern methods of agrobiotechnology are of global interest. This has increased the importance of developing tissue culture methods to facilitate the application of modern genetic engineering techniques to the cotton plant. Despite the much talked about economic value of cotton, the improvement of cotton through genetic engineering has taken place at a relatively slow speed, due to the absence of reproducible methods, which consume less time and efficient to regenerate organized tissues and plants. cotton at high frequency. Regeneration by tissue culture techniques is well established. Although the totipotency of a plant cell is a well-known phenomenon, each plant or part of the plant requires specialized studies to invent the conditions that allow such regeneration at a high efficiency and frequency. There seems to be a consensus that the success in inducing differentiation depends on the type of explant, the physiological condition of the explant and the physical and chemical environment of the explant during culture. Thus, the science of tissue culture has been aimed at optimizing the physiological condition of the plant of origin, the type of explant, the growing conditions and the plant growth regulators or other medium supplements used to initiate the response of the plant. tissue. A wide variety of plant species has been regenerated successfully in in vitro organogenesis or via somatic embryogenesis. The selected mode of regeneration is often based on the relative case, efficiency and applicability of the method for the genetic transformation of a plant species. A method not based on meristem, that is, somatic embryogenesis is always a preferred mode, since it eliminates the possibility of obtaining a false positive or chimeric transformants. The regeneration via somatic embryogenesis of an explant may involve several stages of growth. More frequently, an explant of a part or organ of a mature plant or germinated seedlings is given a chemically defined nutrient medium under sterile conditions. After incubation, the part of the excised plant, under artificially controlled conditions of light, temperature and photoperiod gives rise to a mass of undifferentiated cells, referred to as a callus. As a result of growing the callus under a set of physical and chemical means, ie, nutrients, light, temperature, photoperiod and adding an appropriate combination and concentration of plant growth regulators, or by suddenly eliminating these, it has been reported that the calluses of some plant species generate an embryogenic callus, which, in turn, undergoes to form a somatic embryo, a process called somatic embryogenesis. Somatic embryos develop outside of somatic cells. Each somatic embryo is a mass of organized tissue, able to develop in a complete plant. Somatic embryos are very similar to zygotic embryos developed in the seed, except that they develop without involving a reduction in cell division (meiosis) and are often larger in size. Changes in the concentration of one or more constituents of the medium can lead to changes in in vitro development and tissue differentiation of the plant. It has been reported that signaling pathways mediated by phosphoinositols influence the development and embryogenesis in plants. The deprivation of the tissues in a particular stage of inositol, during a defined period of time can produce the synchronization of the development of the tissues without deteriorating their viability. However, the implication of inositol in achieving the synchronization of the development of the plant or the in vitro embryo has not been reported before and is the most critical aspect of this invention. Now, several reports on the techniques of somatic embryogenesis of cotton have been published / patented. The frequency of somatic embryogenesis between explants during a defined treatment is typically low. In addition, the number of embryos obtained per explant is not high. The long period of time required for in vitro embryogenesis and the dependence of the genotype process is further added to the problem of somatic embryogenesis in cotton. It would be advantageous if a protocol could be developed, even for a cultivated cotton model plant, to provide a set of formulations and conditions, whereby the frequency of somatic embryogenesis can be improved and if such a process needs less time. Synchronized development can be beneficial to collect a large number of regenerated plants. Synchronized development provides not only a large number of plants, but also a uniform population, with all the plants in the same growth phase. The simplification of the steps and the formulations can also improve the applicability of the protocol. The use of a liquid medium can facilitate more efficient selection during genetic transformation (development of the transgenic plant) since the selection pressure (for example, resistance to antibiotics) can penetrate the cells more uniformly in the liquid culture. All these aspects have been achieved by the present invention. The synchronized development of embryos in cotton has not been mentioned in the prior art and is an important aspect of this invention.

DESCRIPTION OF THE PREVIOUS TECHNIQUE There are several published reports that deal with tissue culture conditions, which lead to embryogenesis and to the regeneration of the plant in cotton. Davidonis and Hamilton, in Plant Sci. Letter (1983) 32: 89-93 and U.S. Patent 4,672,035 (1987) report somatic embryogenesis in 2-year callus of G. hirsutum. Shoemaker et al. , 1986 characterized somatic embryogenesis and regeneration of the plant in 17 cotton cultivated plants (Plant Cell Rep. 3: 178-181). Trolinder and Goodin (1987) and Finer (1988) reported somatic embryogenesis in a cotton suspension culture (Plant Cell Rep 6: 231-234; Plant Cell Rep. 7.- 399-402). These protocols took several months to develop regenerated plants. The methods are highly dependent on genotypes (Trolinder and Xhixian, 1989 Plant Cell Rep. 8: 133-136) and therefore, are applicable to only a few cultivated cotton plants. Due to the length of culture time, plants developed via these protocols are often reported as being sterile or having cytogenetic abnormalities (Trolinder and Goodin, 1987 Plant Cell Rep. 6: 231-234). Pangan (1993) in U.S. Patent No. 5,255,902 and Rangan and Rajasekaran (1997) in U.S. Patent No. 5,695,999 reported the embryogenesis of seedling explants in some cotton varieties. Gawel and Robacker (1990) in Plant Cell Tiss. Organ Cult. 23: 201-204 compared somatic embryogenesis in cotton in semi-solid versus liquid proliferation medium. Kumar et al., 1998 reported somatic embryogenesis in Coker 310 Fl hybrids with Hindus cotton varieties, using the modified protocol of Trolinder and Goodin. Zhang et al., 2000 in Plant Cell Tiss. Organ Cult. 60: 89-94 describe the somatic embryogenesis of an abnormal somatic embryo derived from explants. Several of these protocols or modifications thereof have been used in the transformation of cotton mediated by Agrobacterium (Umbeck et al., 1987 Bio / Technology 5: 263-266; Firoozabady et al., 1987 Plant Mol. Biol. 10: 105 -116) or the transformation of cotton mediated by particle bombardment (Finer and McMullen, 1990 Plant Cell Rep. 8: 586-589). Certain bacteria genes, such as those that code for herbicide resistance (Bayley et al., 1992 Theo, Appl. Genet, 83: 645-649) and Bacillus thuringiensis endotoxin genes (Perlak et al., 1990 Bio / Technology 8: 939-943) have been successfully expressed in the transgenic plants. Strickland (1998) in U.S. Patent No. 5,846,797 reported the regeneration of transformed explants in growth regulator free medium. Once an efficient method for regeneration, especially somatic embryogenesis becomes available, it can be used conveniently for the transformation or genetic engineering of cotton. A comparative graph between the various prior techniques is presented below in Table 1: Table 1: Revision of the prior art on regeneration in cotton in order to induce somatic embryogenesis. Explanatory Report Phytohormones Synchrony Notes developed Davidonis GH and Cotyledon NAA, Kin. Calluses of 2 Hamilton RH (1983) years of G: hlrsutum L. cv. Plant regeneration Coker 310 cultivated in from callus of G. medium LS containing 30 hirsutu L. Plant gm / L glucose in absence Sci. Lett. 32: 89-93 of NAA and kin, 30% of the cultures gave rise to somatic embryos. Davidonis GH, Mumma -do- -do- -do- RO, Hamilton RH (1987) Controlled regeneration of cotton plants from tissue culture U.S. Pat. No. 4672035 Shoemaker RC, Couche Hipocotile NAA, Kin. Seventeen LS plants and Galbraith D.W. cultivated cotton G. (1986) hirsutum L. for the Characterization of somatic embryogenesis. somatic embryogenesis After a series of and regeneration plant transfers of the cotton Gossypium callus through the medium hirsutum L. Plant Cell containing MS salts, NAA Rep. 3: 178-181. and Kin., for several weeks, calluses were observed for the presence of somatic embryos. The cultivated plants Coker 201 and 52-367 Explanatory Report Phytohormones Synchrony Notes developed Coker 315 were identified as embryogenic. The embryos were isolated and developed in plants.

Trolinder NL and Goodin Hipocotilo 2, 4D, Kin. The globular embryos were JR (1987) Somatic observed in the culture of embryogenesis and calluses of 6 weeks. In plant regeneration in this stage, the cotton Gossypium calluses were subcultured with a Hrsutum L. Plant Cell liquid suspension medium Rep. 6: 231-234. free of growth regulator. After 3-4 weeks, the suspensions were sifted to collect the globular embryos and in the bud stage. The collected embryos developed in solidified medium until maturity. Mature embryos germinated in plants. Most of the plants developed by this method were sterile. (Only 15% of the plants were fertile).

Trolinder NL and Xhixian Hipocotilo 2, 4D, Kin. 38 cultivated plants, C (1989) Genotype strains and races of Gossypium specificity of somatic were selected for the embryogenesis response somatic embryogenesis with in cotton. Plant Cell the method developed for Rep. 8: 133-136 Coker 312. The selection indicated that there was variation for the genotype 52-367 - . 5 - . 5 -367 52-367 52-367 52-367 -367 52-367 Report 4 of Table 1 claims the development of plants that were sterile, while the present process described in this application provides healthy plants, which are fertile. The present method provides a high frequency of somatic embryogenesis over a randomly selected collection of seedling explants collected from plants growing in the field. This is a significant improvement over the previous relics, where the explants were taken from samples already selected for somatic embryogenesis (reports 11 and 12 of table 1). In addition, as now there is no published report describing synchronization in somatic embryogenesis of cotton. There is no report for any other plant, which describes inositol deprivation as a tool to induce synchrony in the development of somatic embryos. In the case of cotton, the synchronized development of the somatic embryo has not been possible in the prior art and is a very important achievement of this invention. The success of the present invention depends on the concentration and combination of the plant growth regulators used to induce callus on the explants, and the manner in which these calluses were cultured by the method of the present invention involving a short inositol deprivation phase 52-367 term. Once the calluses have been induced, the process does not need any regulator of the growth of the exogenous plant or other additives of the medium. (For example, additional KN03 in reports 4 and 6 and activated carbon in reports 11 and 12) at no later stage. The somatic embryo germinated and rooted in a liquid germination medium simplified on a > non-gelling agent, based on an expensive and simple support (for example, vermiculite). In this regard, the present invention describes a simple and less expensive protocol, suitable for commercialization. The present invention describes for the first time that inositol influences the development and differentiation of cells of plants grown in vi tro. The method details the use of inositol deprivation of cultures at and during a particular time for the synchronized development of the somatic embryo. When clusters of selected embryogenic cells were subjected to a period of 8-12 days of inositol deprivation and the cultures subsequently returned to the basal medium containing inositol, it is found that almost all embryos are in the globular stage. After 10 days of additional growth, most embryos (92%) were in the bud stage. And in a subsequent subculture, approximately 82% of the embryos were in the torpedo stage. When the embryogenic clusters 52-367 underwent 2 cycles of 8-12 days of inositol deprivation, development synchronization was not observed after the globular stage. In addition, this short-term deprivation of inositol not only synchronized the development of the embryos, the number of embryos recovered finally increased surprisingly to 4-5 times more than the highest value. Prior patents in this area on cotton are U.S. Patent No. 4,672,035; the Patent of the States No. 5,244,802; U.S. Patent No. 5,695,999; EP 344302 and U.S. Patent No. 5,846,797, wherein the inventors describe processes for the regeneration of cotton callus plants via somatic embryogenesis; U.S. Patent No. 5,846,797; U.S. Patent No. 5,004,863; U.S. Patent No. 5,159,135 and EP 344302, wherein the inventors describe a method for the transformation of cotton that involves somatic embryogenesis based on a regeneration process and WO Al 9215675, wherein the inventor describes a method for the Transformation mediated by bombardment of the embryonic axes from where the transgenic plants were developed. The process described in the present invention is very simple to adopt commercially, it is fast, 52-367 reproducible and suitable for applications in plant genetic engineering. Unlike the technologies of the state of the art, this process does not lead to the formation of plants with morphological and cytogenetic abnormalities unlike the case of Stelly et al., 1989, and does not produce false positive transformants in the transformation experiments, difference from the case of Sunil Umar and Rathore, 2001.

OBJECTS OF THE INVENTION It is an important object of the present invention to provide a method for somatic embryogenesis synchronized in a developed manner in cotton and the sustained regeneration of a large number of plants from specific tissues of plants. Another object of the present invention is to provide an improved process for the regeneration of cotton via somatic embryogenesis, in a way that the regenerated plants grow, mature and form fertile plants. Still another object of the present invention is to provide a simple and reproducible method for somatic embryogenesis, with the least involvement of plant growth regulators, and any other additive of the additional medium, supplied in a manner 52-367 exogenous.

SUMMARY OF THE INVENTION The present invention provides for the first time, an efficient method for the regeneration of the plant in cotton via somatic embryogenesis synchronized in a developed manner. The process of this invention is simple, rapid, reproducible and convenient for plant genetic engineering applications. The most critical novel aspect is the achievement of synchronized somatic embryogenesis through a step of inositol deprivation. This critical aspect is not mentioned or suggested in any prior art known to applicants. The process of the present invention employs a combination of a growth regulator (2,4-dichlorophenoxy acetic acid and Benzyl adenine), different from those used in previous reports, to induce callus from seedling explants. The process of the present invention achieves the production of callus-mediated somatic embryogenesis that takes a shorter time and provides a higher number of normal and fertile plants. Thus, according to the present invention, a method is provided for regenerating a large number of viable and fertile cotton plants, via embryogenesis 52-367 synchronized somatic of a hypocotyl segment or a mesocotyl segment or a cotyledon piece, the method comprises (i) treating the seeds of the cotton plant with a sterilant to remove any unwanted contaminants. (ii) cultivating the treated seeds of step (i) for germination in a first medium consisting of: (a) salts of any conventional medium, (b) vitamins of any conventional medium, (c) inositol, and (d) a source of carbon at a pH in the range of 5.2-6.0 and sterilize the medium by autoclaving, incubating the cultures at the temperature of 23-33 ° C in light (at 30-60 μmol / m2 / second intensity) or in darkness for a period of 6-12 days. (iii) cultivate the explants of the seedlings obtained in step (ii). (iv) culturing the explants obtained from step (iii) for the purpose of inducing a callus in a second solid medium, consisting of (a) salts of any conventional medium, (b) vitamins of any conventional medium, (c) ) inositol, (d) a carbon source, (e) plant growth regulators in combination of 2, 4D and BA and (f) a gelling agent, at a pH in the range of 5.4 to 6.2 and sterilize the medium by autoclaving, incubating the cultures in the range of 23-33 ° C in light of at least 90 μmol / m2 / second intensity with 16 hours of photoperiod; (v) continuing the cultivation for a period of 3-5 weeks until the calluses are formed on the cutting edges; (vi) transferring the calluses generated from step 2 to a third liquid medium, at a packing density of 600-1000 mg of callus / 50 ml of medium, the medium is comprised of: (a) salts of any conventional medium, ( b) vitamins of any conventional medium, (c) inositol and (d) a carbon source at a pH in the range of 5.2-6.0 and sterilize the medium by autoclaving, and incubate the cultures at a temperature between 23-33 ° C at a light intensity of 20-40 μmol / m2 / second, with a photoperiod of 16 hours for a period of 12-32 days, sufficient to form the embryogenic clusters. 52-367 '(vii) select the cell suspension through metal sieves of different mesh sizes and select the cells / clusters collected on the 40 mesh size and also subculture the selected clusters with the basal liquid medium, as in step (saw) . (viii) subculturing the embryogenic cells / clusters for a short period (8-12 days) with the basal liquid medium of step (vi) but without inositol, that is, the fourth medium. (ix) further subculturing the embryogenic cells / clusters with the basal liquid medium of step (vi) at a regular interval of 8-12 days. (x) incubating the cultures in steps (viii) and (ix) to the same condition of temperature, light, photoperiod as in step (vi). (xi) shake the cultures in step (vi), (viii) and (ix) at 110-130 rpm on a rotary shaker. (xii) transfer somatic, bipolar embryos in a fifth germination medium comprising: (a) salts of any conventional medium, (b) vitamins of any conventional medium, (c) inositol reduced to a quarter of the normal concentration and ( d) a carbon source, at a pH in the range of 5.2-6.0 and sterilize the medium by autoclaving and incubate the cultures at a temperature between 23-33 ° C at a light intensity of at least 60 μmol / m2 / second , with a photoperiod of 16 hours until the seedlings develop. In the present invention, the term "explants" refers to cotyledon pieces or segments of hypocotyl or mesocotyls. In a preferred embodiment of the present invention, the first and fifth media comprise salts of MS medium, vitamins of Gamborg B5 medium and a carbon source. The first and fifth media comprise salts of MS medium and vitamins of Gamborg B5 medium at half their standard concentration, while the second, third and fourth media comprise them at their standard concentration. The most preferred salts of the MS medium and its standard concentration comprise the following, as shown in Table 2: TABLE 2 Salt medium of Murashige and Skoog (1962) Component Concentration (mg / L) NH4N03 1650 N03 1900 CaCl2.2H20 440 2-367 Component Concentration (mg / L) MgS04.7H20 370 KH2P0 170 Kl 0.83 H3B03 6.2 MnS04.4H20 22.3 ZnS0 .7H20 8.6 Na2M? 04.2H20 0.25 CuS04.5H20 0.025 CoCl2.6H20 0.025 Na2.EDTA 37.3 FeS04.7H20 27.8 The preferred Gamborg B5 medium vitamins comprise the following, as shown in Table 3: TABLE 3 Component Concentration (mg / L) Nicotinic Acid 1.0 Pyridoxine HCl 1.0 Thiamin HCl 10 The preferred source of carbon in the first medium is selected from the group consisting of sucrose and glucose and such a carbon source employed is in a range of 1-3% w / v. The preferred carbon source in the second, third and fourth media is essentially glucose and such a carbon source employed is in a range of 1.5-45% w / v. The preferred carbon source in the fifth medium is essentially sucrose and such carbon is employed at a 52-367 range of 1-3% weight / volume. The preferred gelling agent in the second medium is selected from a group consisting of agar (used at a range of 0.6-0.8% w / v) and fitagel (used at a range of 0.15-0.29% w / v). The preferred organics in the first, second, third and fifth media is essentially myo-inositol, used at 100 mg / L in the first to the third medium and at 25 mg / L in the fifth medium. The plant growth regulators employed in the second medium are selected from the group consisting of combinations of 2,4D as axin and BA as cytokinin. In another preferred embodiment, the method of the present invention comprises: (i) sterilizing the seeds of the cotton plant to remove contaminants such as bacteria / fungi by conventional methods; (ii) cultivate seeds sterilized for germination in a medium shown in Table 4 (iii) cut the explants of the seedlings obtained in step (ii) (iv) to culture the explant obtained in step (iii) in a medium as shown in Table 5 at a pH in the range of 5.4 to 6.2 and sterilizing the medium by autoclaving 52-367 (v) cultivate the explants at a temperature of 23-33 degrees C, light at least 90 μmol / m2 / second under a photoperiod of 16 hours for a period of 3-5 weeks, until sufficient callus is formed (vi) transferring the calli to the embryogenesis induction medium shown in Table 6, with the following composition, at a pH in the range of 5.2-6.0 and sterilizing the medium by autoclaving (vii) maintaining the culture at the temperature of 23-33 ° C, light intensity in the range of 20-40 μmol / m2 / second under a photoperiod of 16 hours, for a period of 2-5 weeks, until the embryogenic clusters are formed (viii) sieving the cluster embryogenic and transfer to a private inositol medium having the composition shown in Table 7 at a pH in the range of 5.2-6.0 and sterilize the medium by autoclaving and cultivate at the temperature of 20-40 μmol / m2 / second under a 16-hour photoperiod for a short period of 8-12 days (ix) transfer go the culture with osmotic shock to the basal liquid medium of step (vi) and continue the culture in this medium where the somatic embryos are synchronized (x) transfer the mature bipolar somatic embryos to the germination medium of the embryo, as - . 5-367 shows in Table 8 a pH in the range of 5.2-6.0 and sterilize the medium by autoclaving. (xi) maintain the culture at the temperature of 23-33 degrees C in light of at least 60 μmol / m2 / sec under a photoperiod of 16 hours until the seedlings are sufficiently developed to be removed from acclimation. (xii) acclimatize the regenerated plants in a potting mix comprising garden soil: sand: vermiculite: moss in a 2: 1: 1: 1 ratio.

TABLE 4 Germination medium of the seed: a. Main salts Component Concentration (mg / L) NH4NO3 825 KNO3 950 CaCl2. 2H20 220 MgSO4. 7H20 185 KH2P04 85 b. Secondary salts Component Concentration (mg / L) Kl 0. 425 H3B03 3. 6 MnS04. 4H20 11. fifteen 2-367 ZnS04.7H20 4.3 Na2Mo04.2H20 0.125 CuS04.5H20 0.0125 CoCl2.6H20 0.0125 Na2.EDTA 18.65 FeS04.7H20 13.9 c. Organic Myo-inositol 100 d. Vitamins Component Concentration (mg / L) Nicotinic Acid 0.5 Pyridoxine HCl 0.5 Thiamine HCl 5 e. Source of Sucrose Carbon or Glucose 20 g / 1 at a pH in the range of 5.2-6.0 and sterilize the medium by autoclaving.

TABLE 5 Induction medium of callus a. Main salts Component Concentration (mg / L) NH4NO3 1650 KNO3 1900 CaCl2.2H20 440 2-361 MgS04.7H20 370 KH2PO4 170 b. Secondary salts Component Concentration (mg / L) Kl 0.83 H3BO3 6.2 MnS04.4H20 22.3 ZnS04.7H20 8.6 Na2Mo04.2H20 0.25 CuS04.5H20 0.025 CoCl2.6H20 0.025 Na2. EDTA 37.3 FeS04.7H20 27.8 c. Organic Myo-inositol 100 d. Vitamins Component Concentration (mg / L) Nicotinic Acid 1.0 Pyridoxine HCl 1.0 Thiamin HCl 10 e. Carbon source Glucose 30 g / 1 f. Agaric Geling Agent 0 Fitogel 8 g / 12.2 g / 1 Plant Growth Regulators Auxinas 0.44 to 4.4 μM Cytokinins 0.22 μM to 2.2 μM TABLE 6 Induction medium of embryogenesis a. Main salts Component Concentration (mg / L) NH4NO3 1650 KNO3 1900 CaCl2.2H20 440 MgS04.7H20 370 KH2PO4 170 s. Secondary salts Component Concentration (mg / L) Kl 0.83 H3BO3 6.2 MnS04.4H20 22.3 ZnS04.7H20 8.6 Na2Mo04.2H20 0.25 CuS04.5H20 0.025 CoCl2.6H20 0.025 Na2.EDTA 37.3 FeS04.7H20 27.8 2-367 c. Organic Myo-inositol 100 d. Vitamins Component Concentration (mg / L) Nicotinic Acid 1.0 Pyridoxine HCl 1.0 Thiamin HCl 10 e. Carbon source Glucose 30 g / 1 TABLE 7 Inositol deprivation medium a. Main salts Component Concentration (mg / L) NH4N03 1650 KN03 1900 CaCl2. 2H20 440 MgSO4. 7H20 370 KH2PO4 170 b. Secondary salts Component Concentration (mg / L) Kl 0.83 H3BO3 6.2 MnS04. 4H20 22.3 ZnS04. 7H20 8.6 Na2Mo04. 2H20 0.25 -367 CuS04.5H20 0.025 CoCl2.6H20 0.025 Na2.EDTA 37.3 FeS04.7H20 27.8 c. Vitamins Component Concentration (mg / L) Nicotinic Acid 1.0 Pyridoxine HCl 1.0 Thiamin HCl 10 d. , Carbon source Glucose 30 g / 1 TABLE 8 Germination medium of the embryo a. Main salts Component Concentration (mg / L) NH4N03 825 KN03 950 CaCl2.2H20 220 MgSO4.7H20 185 KH2PO4 85 b. Secondary salts Component Concentration (mg / L) Kl 0.425 H3BO3 3.6 2-367 MnS0 .4H20 11.15 ZnS04.7H20 4.3 CuS04.5H20 0.0125 CoCl2.6H20 0.0125 Na2.EDTA 18.65 FeS? 4.7H20 13.9 c. Organic Myo-inositol 25 d. Vitamins Component Concentration (mg / L) Nicotinic Acid 0.5 Pyridoxine HCl 0.5 Thiamine HCl 5 e. Carbon source Sucrose 20 g / 1 According to one embodiment of the invention, the basal embryogenic mass can be subjected to additional rounds of inositol deprivation and synchronized embryogenesis and regeneration of subsequent plants.

DETAILED DESCRIPTION In the method of the present invention, the seeds are surface sterilized before use in the in vitro culture to free them from bacterial / fungal contaminants. Surface sterilization involves treating the seeds with a solution containing any sterilizing agent such as sodium hypochlorite, calcium hypochlorite, mercuric chloride, alcohol, cetrimide, etc. Surface sterilization of the seeds can be done by treating the seeds with a 0.05-0.5% w / v solution of mercuric chloride in water for 3-11 minutes with continuous agitation, then washing thoroughly with sterile distilled water (4-8 times) , followed by the immersion of the seeds is rectified alcohol (50-100% volume / volume) for 10-20 seconds and then roasting them in the flame of an alcohol burner for 5-10 seconds. The superficially sterilized seeds can be placed for germination in filter paper containers moistened with germination medium of seeds containing Murashige and Skoog salts at half their concentration, Gamborg B5 medium at half their concentration, 100 mg / l of inositol and any source of Carbon such as glucose or sucrose of 1 to 3% weight / volume, adjusting the pH of the medium to 5.2-6.0 and sterilizing as a result of the autoclave at 121 degrees C, at 1.1249 kgf / cm2 (16 psi) for 16 minutes. For germination, the seeds can - . 5 -367 incubate at a temperature of 23-33 ° C in the light (at 30-60 μmol / m2 / second intensity) or in the dark until the seeds germinate and form mature seedlings. The explants (pieces of cotyledons, segments of hypocotyls or segments of mesocotyls) can be obtained preferably from seedlings of 6-12 days after germination by cutting with a sharp sterile scalpel and knife, and in an aseptic medium, i.e. laminar flow, known in the art. The cut explants can be placed in • the medium containing Murashige and Skoog salts at the concentrations given in Table 2, Gamborg B5 vitamins at the concentrations given in Table 3, 100 mg / L inositol, a carbon source, preferably glucose at 1.5-4.5% w / v, a gelling agent, preferably 0.6 to 0.8% w / v or phytagel of 0.15-0.29% w / v, and plant growth regulators 2, 4D 0.44 to 4.44 μM and BA 0.22 to 2.22 μM. The pH of the medium is adjusted to 5.4-6.2 before autoclaving at 121 ° C, 1.1249 kgf / cm2 (16 psi) for 16 minutes. The composition of the medium provided for the induction of the callus is presented in Table 5. The cultures were incubated at a temperature in the range of 23-33 ° C in white fluorescent light of at least 90 μmol / m2 / second of low intensity a photoperiod of 16 hours for a period of 3-5 weeks. At this time, sufficient callus is formed on the cutting edges of the explants. The calluses can be yellowish to brown in appearance and have a friable texture. The calluses developed on the cutting edges of the explant can be transferred to a basal liquid medium, the composition of which is given in table 6, at a packing density of 600 to 1000 mg of callus per 50 ml of medium in an Ehrlenmeyer flask. of 250 ml. The medium does not contain any regulator or gelling agent and its pH is adjusted to 5.2-6.0 before autoclaving at 121 ° C, 1.1249 kgf / cm2 (16 psi) for 16 minutes. The callus cells can be shaken in this medium at 110-130 strokes per minute on a rotary shaker set at 23-33 ° C temperature, 20-40 μmol / m2 / second intensity under a photoperiod of 16 hours. The cells can be cultured in this medium and incubation conditions for a period of 12-32 days, until the embryogenic clusters are formed in the cell suspension culture. The cell suspension developed by stirring the basal liquid medium can be screened to select the embryogenic clusters / cell groups developed in the culture. It can be passed, through a combination of metal sieves of 10, 40 and 100 mesh sizes. The 10 mesh size collects the most tissue clusters 52-367 large and 100 mesh size collects fine suspension of cells. The fraction of cells that contain the smallest clusters, which can become embryogenic, can be collected on a screen with a size of 40 mesh. The selected fraction of cells can be transferred to the fresh basal liquid medium and subcultured regularly at an interval of 8-12 days. or it can be transferred to the inositol deprivation medium, medium which is the basal liquid medium minus inositol, the composition is given in Table 7, for a period of 8-12 days, followed by the replacement of the inositol in the basal liquid medium and then subculturing regularly at an interval of 8-12 days. In both cases, the somatic embryo develops, but with a different destiny of development. While in the first, the embryos of all stages of development can be obtained in each subculture cycle of 8-12 days with similar frequency, in the last, the development of the embryos is synchronized and almost all the embryos remain in the same development stage. The strategy in the present method is to select the second process to cultivate the embryogenic mass. In addition, after mature bipolar embryos in the torpedo stage are removed from the suspension for germination, the basal embryogenic mass can 52-367 undergo additional cycles of inositol deprivation, synchronization of development and collection of mature embryos for germination. Embryogenic clusters and synchronized embryos can be grown in agitated medium on a rotary shaker at 110-130 strokes per minute and at 23-33 ° C temperature, 20-40 μmol / m2 / second light under a photoperiod of 16 hours. The mature bipolar somatic embryo can be removed from the liquid medium and transferred to a solid support, preferably vermiculite saturated with germination medium of the embryo, composition of which is given in Table 8. The pH of medium is adjusted to 5.2 6.0 before autoclave at 121 ° C, 1.1249 kgf / cm2 (16 psi) for 16 minutes. The cultures can be incubated at 23-33 ° C temperature at a light intensity of at least 60 μmol / m2 / second, and a photoperiod of 16 hours. The fresh liquid medium can be added to the embryos that germinate on a weekly basis. The embryos were cultured in germination medium of the embryo for a sufficient period of time to form seedlings in the 4-5 leaf stage with well-developed roots. At this stage, the seedlings can be removed and can be transferred to a potting mix that is a sterile mix of garden soil: sand: vermiculite: moss in a ratio of 2: 1: 1: 1. 52-367 Good moisture conditions can be provided to newly developed seedlings, covering the plants with transparent polythene bags, the inner surface of which is sprayed with water. After cultivating the seedlings under these conditions and at a temperature of 23-33 degrees C and fluorescent light of at least 90 μmol / m2 / second under a photoperiod of 16 hours for a period of time sufficient for acclimatization, the plants, if you want, can be transferred to the field. The present invention will now be described in greater detail with reference to the following non-limiting examples.

EXAMPLE I Dependent media response to the induction of somatic embryogenesis Seeds of cotton plants were treated (G. hirsutum L. Coker 312) with 0.1% w / v mercuric chloride for 7 minutes, washed 6 times with sterile distilled water, followed by immersion of the seeds in rectified alcohol for 10 seconds and "subjected to The sterile seeds were placed for germination in filter paper containers moistened with the germination medium of the seeds containing salts - . 5 -367 of Murashige and Skoog to half their concentration, Gamborg B5 vitamins at half their concentration, 100 mg / l inositol and 2% w / v sucrose (the pH of the medium was adjusted to 5.6 before autoclaving). For germination, the seeds were incubated at 28 ± 2 ° C in white fluorescent light (30 μmol / m2 / sec) under a 16-hour photoperiod. The cultivation continued until the seeds germinated to provide radicles and plumules with well expanded cotyledons. The 9-day seedlings were used to provide the hypocotyl segment and the cotyledon piece as the explants. The explants were cut with the help of a sterile sharp scalpel. The explants were placed in the callus induction medium, CIM1 containing Murashige and Skoog salts, Gamborg B5 vitamins, 100 mg / l inositol, 3% w / v glucose, 750 mg / l MgCl 2 and 0.22% weight / volume of fitagel, supplemented with 2.4-D 2.2 μM and BA 0.88 μM (the pH of the medium was adjusted to 5.8 before autoclaving). The explants were incubated in this medium at 28 ± 2 ° C temperature at 90 μmol / m2 / second of light intensity under a photoperiod of 16 hours for 3-4 weeks. The calluses developed on the cutting edges of the explants were cut and inoculated in a basal liquid medium containing salts of Murashige and Skoog, vitamins - . 5 -367 Gamborg B5, 100 mg / l inositol, 3% w / v glucose, pH 5.6 at a packing density of 800 mg of callus per 50 ml of medium in 250 ml Ehrlenmeyer flasks. The medium was sterilized by autoclaving at 121 ° C, 1.1249 kgf / cm2 (16 psi) for 16 minutes. The cultures were shaken on a rotary shaker at 120 rpm and at 28 ± 2 ° C temperature, 30 μmol / m2 / second of light intensity under a photoperiod of 16 hours. The culture of the explants for the induction of the calluses was also done in medium CIM2, CIM3 and CIM4 containing Murashige and Skoog salts, Gamborg B5 vitamins, 100 mg / l inositol, 3% w / v glucose, 750 mg / l of MgCl2 and 0.22% weight / volume of fitagel supplied with different combinations of the growth regulator, such as 2,4-D 0.45 μM plus Kin. 2.32 μM (in CIM2), NAA 10.7 μM plus Kin. 4.64 μM (in CIM3); NAA 2.68 μM plus 2iP 2.4 μM (in CIM4). The cultures were incubated under the same conditions of temperature and light. The calluses were transferred to the basal liquid medium and cultured in a similar manner. After 20-22 days of growth of the culture in the liquid medium, the suspension generated thereof was sieved through metal sieves of different pore sizes (10, 40 and 100 mesh, from SIGMA chemical company, St. Louis). The largest cell clusters were collected on the size 10 mesh and the fine suspension that was 52-367 collected on the mesh size 100 was discarded. The smallest cell clusters collected in the size 40 mesh were subcultured in fresh basal liquid medium and scored for the presence of a somatic embryo. The frequency of induction of somatic embryogenesis (SE) was calculated based on the presence of embryogenic clusters and the number of somatic embryos obtained per explant was scored accordingly in each combination of the medium. The results obtained are summarized in Table 9.

TABLE 9 Response of embryogenesis of different types of explants in selected media compositions 52-367 *% of explants showing induction of embryogenic callus. The critical difference for the induction of somatic embryogenesis (SE) and the formation of mature embryos was 17.69 and 3.43 for the cotyledon explants (n = 5, repeated twice) and 13.14 and 2.16 for the hypocotyl (n = 6, repeated three times), respectively. It is evident from the results that variations in the combinations and concentrations of growth regulators provide a different response with respect to the frequency of the induction of somatic embryogenesis and the number of mature somatic embryos obtained by explant. Thus, this finding can be used to develop the most optimal conditions for the regeneration of the cotton plant EXAMPLE 2 The procedure of Example 1 was repeated, except that the seeds germinated and grew for 9 days until the development of radicles and plumule with well expanded cotyledons, in the dark. The same results were obtained essentially.

EXAMPLE 3 The procedure of Example 1 was repeated, 52-367 except that the germination medium of the seeds contained 2% w / v glucose as the carbon source. The same results were obtained.

EXAMPLE 4 The procedure of Example 2 was repeated, except that the germination medium of the seeds contained 2% glucose instead of sucrose. Similar results were obtained.

EXAMPLE 5 The procedures of Examples 1 and 2 were repeated with the Coker 310 cotton variety. Similar results were obtained.

EXAMPLE 6 The procedure of Example 1 was repeated to the extent of obtaining embryogenic clusters in suspension, derived from the calli generated in a combination of 2,4-D and BA. The embryogenic clusters were subcultured in the basal liquid medium (BM) devoid of inositol. The embryogenic clusters subcultured in the basal liquid medium containing inositol were used as control. The clusters were inoculated at a packing density of approximately 800 mg of cells per 50 ml of medium and 52-367 incubated on a rotary shaker under the same conditions of light, temperature and photoperiod as in Example 1. After 1 to 2 subcultures of 10 days in inositol-free medium, the cultures were returned to the basal liquid medium containing inositol . The frequency and number of embryos at different stages of development per unit mass were graded in both conditions. The data obtained are presented in Table 10: TABLE 10 Synchronization of somatic embryogenesis by inositol deprivation 2-367 It is clear from the results that without inositol deprivation, synchronized embryogenesis is not obtained. Therefore, continuous subcultures of the embryogenic mass in the basal medium containing inositol provided embryos at all stages of development at almost the same frequency. However, when the embryogenic mass was cultured in inositol-free basal medium during a single 10-day cycle and then returned to the basal medium containing inositol, it produced a synchronized development of the somatic embryo (100% of embryos in the globular stage after of a subculture in basal medium, 91.66% embryos in the bud stage after 2 subcultures in basal medium and 81.99% of embryos in the torpedo stage after 3 subcultures). The total number of mature embryos produced per explant was increased to 4-5 times the highest value. The embryogenic clusters, when subcultured in an inositol-free medium for 2 hours 52-367 cycles, showed a reduction in homogeneity in the stages of development of the embryos. Therefore, a single cycle of inositol deprivation is required to induce a high level of synchrony in embryogenesis.

EXAMPLE 7 Seeds of cotton plants (G. hirsutum L. Coker 312) were sterilized and germinated as in Example 1. The explants were immersed in Agrobacterium suspension for 10-15 minutes, dried with blotting paper and inoculated into a coculture medium comprising Murashige and Skoog salts, Gamborg B5 vitamins, 100 mg / l inositol, 3% w / v glucose, 750 mg / l MgCl 2 and 0.22% w / v phytagel, supplemented with 2,4 -D 2.2 μM and BA 0.88 μM (the pH of the medium was adjusted to 5.8 before autoclaving). The cultures were incubated at 28 ± 2 ° C temperature at 90 μmol / m2 / second of light intensity under a photoperiod of 16 hours for 3 days. After 3 days, the explants were washed with sterile water, dried with blotting paper again and inoculated into the medium having a composition similar to the co-culture medium, except that it is further supplemented with 250 mg / l of augmentin and 50 mg / l of kanamycin. The explants were cultured according to Example 1 and Example 6. Similar results were obtained with respect to 52-367 to somatic embryogenesis and regeneration of transformed plants. The Agrobacterium strain used in this experiment was a common laboratory strain LBA 4404 harboring a derivative of a binary pIG vector that has nptll as a selection marker and gusA with an intron as a reporter gene. It is clear from the regeneration obtained with the transformed explants that cultivating the putative transformed cells in liquid medium in the presence of a selection agent (kanamycin in this case) is less likely to produce false-positive selections, since all the embryos recovered were GUS tve and showed a homogeneous blue color after the histochemical test. In addition, the frequency of the induction of somatic embryogenesis and the number of somatic embryos per explant were obtained with the transformed explants and the results were similar, both with treatment and without treatment with Agrobacterium tumefaciens.

EXAMPLE 8 The procedure of Example 7 was repeated, except that the explants were cocultivated in the dark. Essentially similar results were obtained. 52-367

Claims (2)

CLAIMS! 1. A method for the regeneration of cotton via somatic embryogenesis with a substantially synchronized development of the embryos after a short-term deprivation of inositol, the process comprises the steps of: (i) cutting the explant from the germinated cotton seedlings , selected from the group consisting of cotyledon, hypocotyl, mesocotyl and mixtures thereof; (ii) culture the explant for the purpose of induction of callus in a first solid medium, in a culture medium containing glucose as the carbon source, supplemented with Gamborg B5, 2,4-D and BA vitamins and inositol , at a temperature between 23 to 33 ° C at a light intensity of at least 90 μmol / m2 / second under a photoperiod of 16 hours over a period of 3-5 weeks, to allow undifferentiated calluses to form explant (iii) transferring the callus from the first solid callus induction medium to a liquid medium comprising a basal medium containing glucose as the carbon source and supplemented with Gamborg B5 vitamins and culturing the suspension generated therefrom at a temperature of 23 to 33 ° C at a reduced light intensity of 20-40 μmol / m2 / second, under a photoperiod of 16 hours for a sufficient time to form embryogenic clusters; (iv) sieving the cell suspension through metal sieves of different pore sizes to select the embryogenic cells and / or clusters and subculturing the embryogenic callus containing the somatic embryo in basal medium; (v) subjecting the embryogenic mass / clusters to inositol deprivation, after subculturing it in the basal medium without inositol for a sufficient period of time and then returning the culture of inositol containing the medium to allow the somatic embryo to synchronize its development; (vi) transfer the bipolar somatic embryo to a germination medium of the embryo on a support and culture the embryos in germination medium of the embryo until the seedling stage developed sufficiently to transfer it to the soil; (vii) also transfer the seedlings to a mix for pots for acclimation and then to the field. 2. The method according to claim 1, wherein the explants are derived from cotton or from any other seedling of a plant. 3. The method according to claim 1, wherein the explant is derived from cotton cv Coker 312 and the 52-367 seedlings are developed by (i) sterilizing the cottonseed in a sterilization solution of 0.1% HgCl2 for 5-10 minutes, preferably 7 minutes, (ii) rinsing the seed in sterile water 4-6 times , (iii) toast the seed in the flame of an alcohol burner for 5-10 seconds, (iv) inoculate the seed in the germination medium of the seed, (v) cultivate the seed in the germination medium of the seed in light or dark at a temperature of 23 degrees to 33 degrees C for a period of 6-12 days, preferably 9-10 days, (vi) cutting off the seedling explant. The method according to claim 3, wherein the germination medium of the seed is a liquid medium comprising Murashige and Skoog salts and Gamborg B5 vitamins at half their concentration. The method according to claim 3, wherein the carbon source in the germination medium of the seed is selected from the group consisting of sucrose and glucose at a range of 1 to 3% w / v. The method according to claim 1, wherein the first solid means for induction of callus comprises the following components of the Murashige and Skoog medium:
1. Component Concentration (mg / L) a. Salts from Murashige and Skoog medium (1962) NH4NO3 1650 KNO3 1900 CaCl2.2H20 440 MgSO4.7H20 370 KH2PO4 170 Kl 0.83 H3BO3 6.2 MnS04.4H20 22.3 ZnS04.7H20 8.6 Na2Mo04.2H20 0.25 CuSO4H20 0.025 CoCl2.6H20 0.025 Na2. EDTA 37.3 FeS04.7H20 27.8 b. Organic Myo-inositol 100 7. The method according to claim 1, wherein the Gamborg B5 vitamins, when included, comprise: 52-367 Component Concentration (mg / L) Nicotinic Acid 1.0 Pyridoxine HCl 1.0 Thiamin HCl 10 8. The method according to claim 1, wherein 2,4-D as the auxin supplied exogenously in the first solid inducement medium of the callus is selected from a range of 0.44 to 4.4 μM, preferably 1.76 to 2.64 μM. The method according to claim 1, wherein BA, such as the cytokinin provided exogenously in the first solid callus induction medium is selected from a range of 0.22 μM to 2.2 μM, preferably 0.66 μM to 1.00 μM. The method according to claim 1, wherein the gelling agent in the first solid callus induction medium is selected from a group consisting of agar in the range of 0.6-0.8% w / v, preferably 0.7% and fitagel in the range of 0.15-0.29% weight / volume, preferably 0.22% weight / volume. The method according to claim 1, wherein the first solid medium for induction of callus contains glucose as the primary carbon source. 12. The method according to claim 1, in 52-367 where the explants are cultivated in the callus induction medium at a temperature between 23 to 33 ° C, preferably between 27 to 29 ° C at a light intensity of at least 90 μmol / m2 / second, under a 16-hour photoperiod for a period of no more than 3-5 weeks, to allow undifferentiated callus to form from any explant. The method according to claim 1, which essentially includes the step of transferring the callus from the first solid medium for induction of callus to a liquid medium in Ehrlenmeyer flasks, at a packing density of 600 to 1000 mg of callus / 50 ml of medium, preferably 800 mg / 50 ml and shake the culture in this and all subsequent steps until the somatic embryos are removed for germination, on a rotary shaker at 110-130 rpm. The method according to claims 1 and 13, wherein the means of induction of embryogenesis is a liquid basal medium comprising M and S salts, Gamborg B5 vitamins, inositol and glucose as the carbon source. The method according to claims 1 and 13, wherein the embryogenic mass and the somatic embryos of the cell suspension of the plant, generated therefrom in the liquid medium, are incubated at a temperature of 23 to 33 ° C, preferred way of 27-29 ° C at an intensity of 52-367 light of 20-40 μmol / m2 / second, typically 27-33 μmol / m2 / second, under a photoperiod of 16 hours. The method according to claim 1, wherein the embryogenic mass / clusters are subjected to inositol deprivation for a period of 8 to 12 days, preferably 10 days in inositol deprived medium, comprising basal salts MS, vitamins Gamborg B5, glucose as the carbon source but not inositol, leading to synchronization of somatic embryo development. The method according to claim 1, wherein the first solid medium for induction of callus has a pH in the range of 5.4-6.2 and all the liquid medium in the process has a pH in the range of 5.2-5.8, being sterile as a result of autoclaving at 121 ° C, 1.1249 kgf / cm2 (16 psi) for 16 minutes. The method according to claim 1, wherein the potting mix comprises soil for garden: sand: moss: ermiculite, typically in a ratio of 2: 1: 1: 1. The method according to claim 1, wherein the timing of the development of somatic embryogenesis is used for the multiplication of selected cultivated cotton plants, or for the development of cultivated transgenic cotton plants. 20. The method according to claim 1, in 52-367 where inositol deprivation is applied to plant species other than cotton, to improve embryogenesis in tissue culture. The method according to claim 1, wherein the culture medium and the basal medium comprise Murashige and Skoog medium. 22. The method according to claim 1, wherein the period of time sufficient to form the embryonic clusters comprises 12-32 days. 23. The method according to claim 1, wherein the subculture of the embryogenic callus containing the somatic embryo in the basal medium is carried out at intervals of 8-12 days. The method according to any preceding claim, wherein the embryogenic mass / clusters are subjected to inositol deprivation for a period of 8 to 12 days, preferably 10 days. 25. The method according to claim 1, wherein the support for the germination medium of the embryo comprises vermiculite.
2. 2-367