PROCESS FOR THE CONSERVATION OF GERMPLASM OF PLANT SPECIES BY SPRAY DRYING OF SOMATIC CELLS UNTIL THEIR REACTIVATION IS OBTAINED FOR THE OBTAINING OF SEEDLINGS.

Abstract

This process yields totipotent plant cells capable of regenerating plantlets, similar to propagation, in vitro micropropagation, and synthetic seed production. It leverages spray drying as a technology for preserving plant germplasm and as a novel method not currently available. The process begins with somatic cells in suspension and involves inducing friable embryogenic calluses from seeds, leaves, stems, roots, or any plant explant capable of generating cells in suspension, followed by obtaining the cell pack. This cell pack is mixed with encapsulating agents and then spray-dried. Finally, the spray-dried cells are rehydrated and reactivated to continue their morphogenic development, resulting in plantlets capable of acclimatizing to greenhouse and field environments.

Description

PROCESS FOR THE CONSERVATION OF GERMPLASM OF PLANT SPECIES BY SPRAY DRYING SOMATIC CELLS UNTIL THEIR REACTIVATION IS OBTAINED FOR OBTAINING SEEDLINGS TECHNICAL FIELD The present invention is developed within the technical area of ​​biotechnology, plant tissue culture and plant material conservation. It comprises a process for microencapsulation by spray drying of embryogenic somatic cells obtained from callogenic cultures and cells in suspension until plant plantlets are obtained. This process involves the formulation of encapsulating agents and the configuration of spray drying parameters. Through the described process, the operations for rehydration, reactivation and in vitro regeneration of plantlets from spray-dried somatic cells are integrated. This invention enriches the conservation processes of plant material or germplasm existing until now, since the processed and stored cells retain the capacity to regenerate an entire plant. OBJECT OF THE INVENTION Germplasm conservation represents an alternative for rescuing endangered or threatened species, storing species of agroindustrial interest, and exchanging plant material. The main methods for germplasm conservation are cryopreservation and freeze-drying. These methods require specific conditions, such as freezing the plant material and using protective substances in both processes. However, cryopreservation requires storage conditions at sub-zero temperatures using liquid nitrogen, which requires the mandatory use of a cold or refrigeration room. These traditional methods entail costs in reagents, maintenance, conservation, and infrastructure, making them unaffordable in different regions of the world.Therefore, spray drying represents an alternative for the conservation of plant germplasm, since different equipment can be used at the laboratory, pilot plant, or industrial level. In addition, it is possible to adapt the operating conditions such as the inlet and outlet temperatures of the dryer, the concentrations of encapsulating agents, and the use of growth regulators and other additives according to the plant species to be conserved. Similarly, spray-dried products can be preserved under vacuum at room temperature, but when they are kept refrigerated, viability increases, that is, shelf life is prolonged, reducing the costs of cryopreservation and lyophilization and increasing the number of parameters used in the process and in the conservation of plant germplasm. BACKGROUND Spray drying, also known as spray drying, is a unit operation by which a liquid product containing solids is atomized in a stream of hot gas to obtain a powder instantly. The obtained powder can be called particles, capsules or microcapsules. In this process, the particles are formed at the same time as they are dried (Barbosa Cánovas, et al. (2005). Food Powders: Physical properties, Processing and Functionality. New York: Kluwer Academic / Plenum Publishers). Dehydrated enzymes, detergents, plant extracts and soluble proteins are examples of products produced by spray drying (Peighambardoust et al., 2011) Application of spray drying for preservation of lactic acid starter cultures: A review. Trends Food Sci Tech 22(5):215-224). Likewise, spray drying has also been frequently used to preserve lactic acid bacteria (Peralta et al., 2017) Spray-dried adjunct cultures of autochthonous non-starter lactic acid bacteria. Int J Food Microbiol 255:17–24). Water loss during spray-drying from the cell decreases their water activity, cell metabolism is delayed, and they eventually enter a state of dormancy. Peralta et al. (2017) showed that spray-drying does not affect the metabolic activity of the microorganisms tested. Yeasts have also been spray-dried. Aponte et al. (2016, Impact of different spray-drying conditions on the viability of wine Saccharomyces cerevisiae strains.World J Microb Biot 32:13) evaluated the viability of several yeast species after spray drying and during storage, finding that yeast was viable after drying and that the fermentation yield of encapsulated yeast was statistically equal compared to fresh cultures, thus concluding that drying did not affect yeast metabolism. Studies on the use of spray drying for the preservation of microorganisms have shown that many factors influence microbial survival. Santivarangkna et al. (2008, Santivarangkna, Kulozik U, Foerst P (2008) Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J Appl Microbiol 105(1 ).1-13) showed that the outlet air temperature influences microbial survival. In eukaryotic cells, such as Beauveria bassiana (Liu and Liu (2009) Formulation and characterization of the microencapsulated entomopathogenic fungus Metarhizium anisopliae MA12. J Microencapsul 26(5):377-384), Saccharomyces cerevisiae (Aponte et al. 2016) and Saccharomyces boulardii (Duongthingoc et al. (2013) Effect of whey protein agglomeration on spray dried microcapsules containing Saccharomyces boulardii.Food Chem 141(3):1782-1788), lower inlet and outlet air temperatures used during spray drying promote better results. Other authors reported that the composition of the material used for encapsulation is very important to increase microbial survival during storage (Jafari et al. (2008) Encapsulation efficiency of food flavors and oils during spray drying. Dry Technol 26(7):816-835). These materials can be used alone or as a mixture, but complex mixtures of several materials have been reported, such as maltodextrins, gums, skim milk, soy protein and milk, whey, among many others. The interaction between the encapsulating agents and the cell wall (Eckert et al. (2017) Microencapsulation of Lactobacillus plantarum ATCC 8014 through spray drying and using dairy whey as wall materials. LWT - Food Sci Technol 82:176183), is mainly by hydrogen bond interactions. Regarding the preservation of plant cells, cryopreservation is used in the conservation of plant germplasm; however, it is a process with high energy consumption and can therefore be very expensive, in addition to the fact that the products must be kept at very low storage temperatures (-80°C). On the other hand, chemical encapsulation in alginate beads for the production of synthetic seeds of plant species represents a method of plant propagation and germplasm conservation and is one of the most commonly used methods; however, given the characteristics of the obtained product, cell viability is lost in one or two months of storage. Therefore, this invention solves the problem of maintaining plant cell viability in storage from microencapsulated somatic cells stored under vacuum. This invention presents a process that does not exist in the state of the art, which allows obtaining encapsulated plant cells that retain their viability, as well as the process for hydration, reactivation and redifferentiation of the in vitro culture until the development of seedlings, which is not limited to a genus or species, but can be applied to somatic cells, calluses or embryos of any plant species. DESCRIPTION OF THE FIGURES Figure 1. The general process for obtaining microcapsules containing cells for germplasm preservation is shown in the figure. a) Disinfect seeds, b) Obtain seedlings, c) Induce embryogenic callus, d) Induce cells in suspension, e) Obtain the cell package, f) Determine viability, g) Prepare encapsulating agents, h) Resuspend cells, i) Spray dry, j) Preserve germplasm in microcapsules. Figure 2. Flow diagram of the process for obtaining seedlings from rehydrated microencapsulated cells, k) Hydrate microcapsules. i) Obtain the cell package, m) Reactivate the cell package, n) Obtain seedlings with roots, o) Condition the seedlings, p) Acclimatize the seedlings, q) Plant in the field. DETAILED DESCRIPTION The present invention consists of a process for microencapsulating and preserving somatic cells of plant species that maintain the ability to regenerate a complete seedling after the process and during storage. Figure 1 shows the general process for obtaining microcapsules containing cells for germplasm conservation. The main raw material is embryogenic cells in suspension obtained from friable embryogenic callus. Callus comes from leaf, stem, or root explants of seedlings obtained in vitro after seed disinfection and germination. This invention consists of the formulation of the encapsulating agent and the configuration of the operating parameters of spray drying to obtain viable and totipotent microencapsulated cells. The embryogenic cell suspensions are sieved to obtain a cell package homogeneous in size, this cell package is mixed with the solutions of encapsulating agents such as maltodextrin and gum arabic, the mixture is processed in a spray dryer regulating the temperature of the inlet and outlet air. At the end of the process, the product (microcapsules) is obtained, which consists of a powder where the microencapsulated plant cells are contained. The product's humidity, water activity and cell viability are determined, subsequently the powder is stored in refrigeration.The stored powder (microcapsules) is rehydrated to remove the encapsulating agent 10 that protects the cell, and the cell package is obtained. This is reactivated in a solid culture medium so that the cells redifferentiate and regenerate complete, rooted plantlets. The seedlings are acclimatized in a greenhouse and sown in the field for normal growth and development. Figure 2 shows the flow diagram of the process for obtaining plantlets from rehydrated microencapsulated cells. Therefore, this invention proposes a new method for preserving and storing plant material, from which plants can be obtained that can be cultivated in the field, but with the innovation of using spray-drying of cells protected by encapsulating agents under certain operating conditions. In accordance with the present invention, the steps comprising the complete process are presented below: a) Disinfect the seeds to obtain aseptic plants: the seeds are disinfected in an ethanol-water solution between 60% and 96% (v / v) for a period of 5-20 minutes, then 3 washes of 3 minutes each are carried out with sterile distilled water, then the seeds are immersed in sodium hypochlorite at concentrations between 1% and 100% (w / v) for 5-20 minutes. Finally, three washes of 3 minutes each are carried out in sterile distilled water. The percentage of disinfection obtained is between 90% and 100%. b) Obtain in vitro seedlings: the disinfected seeds are placed in glass jars with 20 mL of sterile MS (Murashige & Skoog) culture medium at 50% (w / v) of salts supplemented with 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent. The final pH of the sterile culture medium must be adjusted to 5.710.2. The jars with the seeds and the sterile culture medium are incubated for 2-3 weeks in a bioclimatic chamber at a controlled temperature of 2211 ° C, with a photoperiod of 16 hours of light and 8 hours of darkness until seed germination, in this way a germination percentage of healthy seedlings in vitro between 80% and 95% is obtained in order to generate healthy and aseptic explants. c) Inducing embryogenic callus from plant explants: The seedlings germinated in the previous step are used to obtain 1 cm long leaf, stem and / or root explants. Five explants are planted in glass flasks containing 20 mL of sterile Gamborg culture medium supplemented with 2 mg / L of 2,4-dichlorophenoxyacetic acid (other auxins such as naphthaleneacetic acid or other cytokinins such as thidiazuron or benzylaminopurine can also be used), 3% (w / v) sucrose and 0.25% (w / v) anionic polysaccharide as a gelling agent. The final pH of the sterile culture medium should be adjusted to 5.710.2. The flasks containing the explant and the culture medium are incubated in a bioclimatic chamber at a controlled temperature of 22 ±1°C, with a photoperiod of 16 hours of light and 8 hours of darkness for 3-4 weeks.The above allows percentages of friable embryogenic callus formation greater than or equal to 35% for the leaves, greater than or equal to 65% for the root and greater than or equal to 90% for the stem. d) Inducing suspension cells from friable embryogenic calli: the friable embryogenic calli obtained from the previous phase are used to induce suspension cell cultures in Gamborg liquid medium supplemented with 1 mg / L of 2,4-dichlorophenoxyacetic acid (it is also possible to use other auxins such as naphthaleneacetic acid or other cytokinins such as thidiazuron or benzylaminopurine), and 3% (w / v) of sucrose, the final pH of the medium must be adjusted to 5.710.2. To obtain cell concentrations higher than 1x106cells / mL, subcultures are carried out for periods of 7-15 days each and the cell density is determined every 7-10 days in a Neubauer chamber. The cultures and subcultures are incubated in a bioclimatic chamber at a controlled temperature of 2211 °C with a photoperiod of 16 hours of light and 8 hours of darkness, shaking between 50-150 rpm. e) Obtain the cell pack for spray drying: The suspension cell cultures containing the embryogenic somatic cells are sieved under sterile conditions, using 200 mesh sieves with a particle diameter of 0.74 pm. 40 mL of the broth obtained after sieving is centrifuged between 4°C and 10°C and between 3000-5000 rpm for 5-30 minutes so that the somatic cells are separated from the culture broth and the cell pack is obtained, which will be spray dried. f) Determine cell pack viability for spray drying: The viability of the cells in the cell pack is necessary to ensure that the cells to be processed are alive. This viability is determined by reacting 0.4% trypan blue, mixed 1:1 (v / v) with the cell sample. The reaction is allowed to develop for 1 minute and the cells are observed under an optical microscope at 100x magnification using a Neubauer chamber. Cell viability under the conditions in which the cultures were grown is 100%. g) Prepare the encapsulating agents for spray drying: for spray drying of the cell pack, solutions of encapsulating agents are used, including 30% (w / v) maltodextrin, 14% (w / v) gum arabic and 0.4% (w / v) xanthan gum, these are prepared separately, hydrated for 24 hours in distilled water, the pH is adjusted to 5.7 ± 0.2, subsequently they are mixed in a 1: 1 (v: v) ratio and homogenized with the help of an orbital shaker between 80-100 rpm for 1-3 hours and sterilized at 15 lb / in2 for 15 minutes. The mixtures of encapsulating agents can be maltodextrin-gum arabic or maltodextrin-xanthan gum. Other encapsulating agents may be used such as polysaccharides, alginates, whey, skimmed milk and / or vegetable proteins in concentrations of 1 to 50% (w / v). h) Resuspend the cells in the encapsulating agents: the cell package obtained in item e) is resuspended in the mixture of sterile encapsulating agents obtained in item g), the mixture is subjected to orbital shaking at 80-100 rpm for 30-60 minutes to homogenize the cell package and not damage the somatic cells obtained. i) Spray drying plant cells to obtain powdered microcapsules: Somatic cell drying is carried out by feeding the mixture obtained in section h) into a spray dryer using an inlet air temperature between 80-200°C and an outlet air temperature between 50-90°C. This wide temperature range is justified by the different configurations of the equipment for spray drying and by the possible combinations of inlet and outlet air temperatures that allow obtaining a product in powder form with a yield greater than 50%. This powder is the microcapsules that contain plant somatic cells with a viability between 60-100%. The size of the microcapsules is between 1-40 pm. j) Preserve spray-dried germplasm. Microcapsules should be maintained at a humidity level between 0 and 15%, with a water activity level between 0.1 and 0.5, and stored in airtight, vacuum-sealed bags and refrigerated at a temperature of 4°C to 10°C. Dehydrated somatic cells have a viability of at least 90% for a period of 6 months. k) Hydrate the microcapsules containing the spray-dried cells: 5 grams of the powder obtained as a product with the microencapsulated cells are rehydrated in sterile water or sterile liquid MS culture medium (adjusted to pH between 5.7 and 7) supplemented with cefotaxime and ethylene bisdithiocarbamate manganese with zinc salts at concentrations between 100-800 ppm. maintained under orbital agitation between 50-150 rpm for 1-24 hours. This process allows the encapsulating agent that keeps the cell protected to disintegrate. I) Obtaining the cell pack after spray drying: The suspension obtained after rehydration of the microcapsules is centrifuged at 3000-5000 rpm, between 4°C-10°C for 5-30 minutes to separate the dispersed encapsulating agents from the cells without detriment to cell viability, to obtain the cell pack that was processed by spray drying. The cell viability of this cell pack is determined according to step f). m) Reactivate the cell package to obtain embryogenic structures: the obtained cell package is reactivated in 20 mL of sterile culture medium, solid MS with 3% (w / v) sucrose and 0.25% (w / v) anionic polysaccharide as a gelling agent, supplemented with 200 ppm cefotaxime and 400 ppm ethylenebisdithiocarbamate manganese with zinc salts to prevent the growth of bacteria, fungi and yeasts. Reactivation can be increased if growth regulators such as thidiazuron are used. benzylaminopurine, 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid. The culture is incubated in a bioclimatic chamber at 25 ° C with a 16-hour photoperiod for 4 weeks. After this period, redifferentiation and formation of embryogenic structures are observed. n) Obtain rooted seedlings from the reactivated cell pack: the obtained embryos are transferred to solid MS medium with 3% (w / v) sucrose and 0.25% (w / v) anionic polysaccharide as a gelling agent and incubated at 22±2°C with a 16-hour photoperiod for 2 weeks. After this period, the seedlings are separated under aseptic conditions and transferred to flasks containing 20 mL of sterile solid MS medium at 50% salts with 3% (w / v) sucrose and 0.25% (w / v) anionic polysaccharide as a gelling agent and incubated for 14 days at 25°C with a 16-hour photoperiod. After this period, 100% of the regenerated seedlings have rooted, developing roots between 2-5 cm in length and 3-5 leaves with a height of between 3-10 cm. o) Condition the seedlings. Seedlings obtained and fully rooted at 20-30 days of age are removed from the growing medium and the roots are washed with sterile water. The seedling is then transferred to half-kilo pots containing substrate, into which 300-500 grams of substrate mix, 40% peat and 60% perlite, are added. p) Acclimatize the seedlings. Seedlings are acclimatized in a greenhouse by covering the pot in step o) with a solid polypropylene container of 500 to 1000 g capacity, with three to five holes of 5 to 10 mm diameter drilled in the base. This provides a microclimate around the seedling with a relative humidity above 75%. Seedlings are watered once a week. 80-95% acclimatization is achieved with seedlings that have a root length of 5 cm and three leaves of 5 cm in height. q) Planting in the field: the seedlings acclimatized in the greenhouse are transferred and sown in the field directly in the soil, but a mixture of 30% peat and 70% perlite can be used. Likewise, the soil can be supplemented with worm humus so that the plants have a development and growth that allows the elongation and growth of the main root of the plant. The seedlings have between 5-8 leaves with a height of between 10-15 cm. Spray-drying is a process that has not been used to date for the stabilization and preservation of plant cells. Somatic cells dehydrated using this process have a viability of at least 90% for 6 months under refrigeration storage at 4°C to 10°C, and the ingredients, at the concentrations employed, protect the cells during the drying process, allowing said cells to maintain the capacity to regenerate a complete plant. The survival and metabolic capacity of bacteria, fungi, and yeasts after spray-drying are widely reported in the state of the art.However, plant cells are metabolically different from bacteria, fungi, and yeast, so our innovation lies in developing a process that keeps cells viable after spray-drying and allows these dried cells to maintain their redifferentiation capacity using rehydration and reactivation processes. Example. Description of the process for obtaining carrot seedlings from spray-dried somatic cells. a) Disinfect the seeds to obtain aseptic plants: 10 grams of carrot seeds are disinfected in 70% (w / v) ethanol for 5 minutes, then three washes of 3 minutes each are carried out with sterile distilled water and they are immersed in 5% (w / v) sodium hypochlorite for 15 minutes and rinsed 3 times for 3 minutes with sterile distilled water, obtaining 100% disinfection of the seeds. b) Obtain in vitro seedlings: To obtain aseptic seedlings, 5 disinfected seeds are placed in each glass jar containing 20 mL of sterile MS (Murashige & Skoog) culture medium at 50% (w / v) of salts supplemented with 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent. The final pH of the medium must be adjusted to 5 7 ± 0.2. The jars with the seeds and the sterile culture medium are incubated for 21 days in a bioclimatic chamber at a controlled temperature of 22 ± 1 ° C, with a photoperiod of 16 hours of light and 8 hours of darkness. Obtaining 83% germination in the seeds. c) Inducing embryogenic callus from plant explants: Embryogenic callus induction is carried out from aseptic seedling explants, cuttings of approximately 1 cm in length are made from roots, stems and leaves, the explants are sown in Gamborg solid culture medium supplemented with 3% (w / v) sucrose, 0.25% (w / v) anionic polysaccharide as a gelling agent and 2 mg / L 2,4-dichlorophenoxyacetic acid. 5 explants are placed in each culture flask. The cultures are incubated in a bioclimatic chamber for 4 weeks at 22 ±1°C, with a photoperiod of 16 hours of light and 8 hours of darkness. After this period, 35% of calluses are obtained from leaf explants, 65% of calluses from root explants, and 90% of calluses from stem explants. d) Inducing suspension cells from friable embryogenic calli: 3 grams of friable embryogenic calli obtained from stem explants are transferred to 100 mL of Gamborg liquid culture medium supplemented with 3% (w / v) sucrose, 0.25% (w / v) anionic polysaccharide as a gelling agent, and 1 mg / L of 2,4-dichlorophenoxyacetic acid. Subcultures are carried out every 15 days, and cell density is assessed every 7 days by counting in a Neubauer chamber. The cultures and subcultures are incubated in a bioclimatic chamber for 4 weeks at 100 rpm. During this period, a cell concentration of 8.25x10° cells / mL is reached. II e) Obtain the cell pack for spray drying: Liquid cultures with suspended cells are sieved under sterile conditions using a 200 mesh screen with a particle diameter of 0.74 pm to homogenize the size of the cells to be processed. This step is intended to ensure that the cells can freely pass through the spray dryer. The broth obtained after sieving is centrifuged at 4°C and 3,500 rpm for 20 minutes. The liquid phase is removed, and the sedimented cell pack is obtained. f) Determine cell package viability for spray drying: Cell viability is determined in the broth obtained after sieving. Take 1 mL of sieved broth and mix it with 0.4% (w / v) trypan blue in a 1:1 ratio. The mixture is allowed to react for 1 minute using the exclusion technique. Place 20 pL of the mixture in a Neubauer chamber and observe under a microscope. Unstained cells are viable. The cell package has 100% viability. g) Prepare the encapsulating agents for spray drying: the encapsulating agents used are 30% (w / v) maltodextrin and 14% (w / v) gum arabic. These agents are hydrated for 24 hours separately in distilled water and the pH is adjusted to 5.7, subsequently they are mixed in a 1:1 (v:v) ratio, homogenized with the help of an orbital shaker at 80 rpm for 1 hour and sterilized at 15 lb / in2 for 15 minutes. A mixture of encapsulating agents, maltodextrin-gum arabic with 44% total solids is obtained. h) Resuspend the cells in the encapsulating agents: The cell package with 100% viability is added to 100 mL of the encapsulating agent mixture prepared in step g) of this example. The resulting suspension is stirred at 100 rpm for 30 minutes to homogenize the cell package. i) Spray-drying plant cells to obtain powdered microcapsules: The suspension obtained in step h) of this example, containing the plant cells, is fed to a spray dryer with an inlet air temperature of 120°C and an outlet air temperature of 50°C. The product obtained is a powder containing the microcapsules that contain and protect the plant cells. The powder has a yield of 70%. j) Preserving spray-dried germplasm: The germplasm contained in the microcapsules obtained after spray-drying has a moisture content of 3.27% and a water activity of 0.186. The microencapsulated cells maintain 100% viability immediately after the process. The germplasm is stored in vacuum-sealed airtight bags and refrigerated at 8°C, maintaining a viability of 92% after 6 months of storage. k) Hydrate the microcapsules containing the spray-dried cells: The microencapsulated cells contained in the powder are rehydrated under sterile conditions. To do this, 3 grams of powder are added to 20 mL of sterile liquid MS culture medium supplemented with 200 ppm of cefotaxime and 400 ppm of ethylene bisdithiocarbamate manganese with zinc salts to prevent contamination. The suspension is stirred at 100 rpm for 24 hours to disintegrate the encapsulating agents and release the cell inside the microcapsule. I) Obtaining the cell pack after spray drying: To obtain the cell pack after spray drying, 40 mL of the rehydrated suspension is taken according to the previous step. The suspension is centrifuged at 4°C and 3,500 rpm for 20 minutes, the supernatant is removed, and the sedimented cell pack is obtained. The viability of this cell pack is determined using the trypan blue exclusion technique, and the cells maintain 100% viability after spray drying. m) Reactivate the cell package to obtain embryogenic structures: the cell package recovered after spray drying is reactivated by placing it in 20 mL of sterile solid MS culture medium supplemented with 3% (w / v) sucrose, 0.25% (w / v) anionic polysaccharide as a gelling agent, 200 ppm cefotaxime and 400 ppm manganous ethylenebisdithiocarbamate with zinc salts, it is incubated in a bioclimatic chamber at 25°C with a 16-hour photoperiod for 4 weeks. After this period, embryos are observed in the globular, heart, torpedo and cotyledonary stages. n) Obtain rooted seedlings from the reactivated cell pack: the cotyledonary stage embryos are sown on solid MS medium supplemented with 3% (w / v) sucrose, 0.25% (w / v) anionic polysaccharide as a gelling agent and incubated for 2 weeks in a bioclimatic chamber at 25°C with a 16-hour photoperiod. After this period, 100% of fully rooted seedlings are obtained. The seedlings are separated under aseptic conditions and transferred to flasks containing sterile solid MS medium at 50% salts with 3% (w / v) sucrose, 0.25% (w / v) anionic polysaccharide as a gelling agent and incubated for 14 days at 25°C with a photoperiod preferably of 16 hours of light. After this period, seedlings with roots 3 cm long and 3 leaves 5 cm high are obtained. o) Condition seedlings: Seedlings that are fully rooted after 20 days are separated from the culture medium and washed with sterile water to remove any remains of the culture medium. A seedling is planted in a pot with 300 grams of substrate, of which 40% is peat and 60% perlite. p) Acclimatization of the seedlings: The conditioned seedlings are transferred to a greenhouse, covering the seedling with a solid 600 g polyethylene container, which has four 7 mm diameter holes in its base. This allows a microclimate to be maintained around the seedling and a relative humidity of 85%. The seedlings are watered with running water every 7 days. 80% acclimatization is achieved. The seedlings have a root length of 4 cm and have 4 leaves of 5 cm in height. q) Planting in the field: Seedlings that have been acclimatized after 14 days in the greenhouse and have a root length of approximately 5 cm and a height of approximately 10 cm are sown directly into the ground. At this stage, after 14 days in the field, seedlings with six leaves, 10 cm high, are obtained.

Claims

1. A process for the conservation of germplasm of plant species by spray-drying somatic cells until their reactivation to obtain seedlings, characterized in that it comprises the following steps.a) Disinfect seeds to obtain aseptic plants; b) Obtain in vitro plantlets; c) Induce embryogenic calli from plant explants; d) Induce suspension cells from friable embryogenic calli; e) Obtain the cell pack for spray drying; f) Determine viability of the cell pack for spray drying; g) Prepare encapsulating agents for spray drying; h) Resuspend cells in the encapsulating agents; i) Spray dry plant cells to obtain powdered microcapsules; j) Preserve spray dried germplasm; k) Hydrate microcapsules containing spray dried cells; l) Obtain the cell pack after spray drying; m) Reactivate the cell pack to obtain embryogenic structures; n) Obtain rooted seedlings from the reactivated cell pack; o) Condition the seedlings; p) Acclimatize the seedlings and q) Plant in the field.

2. The process according to claim 1, characterized in that in step a) the seeds are disinfected in an ethanol-water solution between 60% and 96% (v / v) for a period of 5-20 minutes, then 3 washes of 3 minutes each are carried out with sterile distilled water.

3. The process according to claim 2. characterized in that the seeds are then immersed in sodium hypochlorite in concentrations between 1% and 100% (w / v) for 5-20 minutes, then three washes of 3 minutes each are carried out in sterile distilled water.

4. The process according to claim 1, characterized in that in step a) the percentage of disinfection obtained is between 90% and 100%.

5. The process according to claim 1, characterized in that in step b) the disinfected seeds are placed in glass jars with 20 mL of sterile MS culture medium, 50% (w / v) of salts supplemented with 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent.

6. The process according to claim 5, characterized in that the flasks with the seeds and the sterile culture medium are incubated for 2-3 weeks in a bioclimatic chamber at a controlled temperature of 22±1°C, with a photoperiod of 16 hours of light and 8 hours of darkness until the seeds germinate.

7. The process according to claim 5, characterized in that the final pH of the sterile culture medium must be adjusted to 5.7±0.

2.

8. The process according to claim 1, characterized in that in step b), the germination percentage of healthy seedlings in vitro is between 80% and 95%.

9. The process according to claim 1, characterized in that in step c) the germinated seedlings are used to obtain leaf, stem and / or root explants of 1 cm in length.

10. The process according to claim 1, characterized in that in step c), five explants are sown in glass flasks containing 20 mL of sterile Gamborg culture medium supplemented with 2 mg / L of 2,4-dichlorophenoxyacetic acid, 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent.

11. The process according to claim 10, characterized in that the final pH of the sterile culture medium must be adjusted to 5.7±0.

2. I6 12. The process according to claim 10, characterized in that the flasks containing the explant and the culture medium are incubated in a bioclimatic chamber at a controlled temperature of 22 ±1°C with a photoperiod of 16 hours of light and 8 hours of darkness for 3-4 weeks.

13. The process according to claim 12, characterized in that a friable embryogenic callus formation greater than or equal to 35% for the leaves, greater than or equal to 65% for the root and greater than or equal to 90% for the stem is obtained.

14. The process according to claim 1, characterized in that in step d) the friable embryogenic calli obtained are used to induce suspension cell cultures in a Gamborg liquid medium supplemented with 1 mg / L of 2,4-dichlorophenoxyacetic acid and 3% (w / v) of sucrose.

15. The process according to claim 10 and 14, characterized in that another auxin such as naphthaleneacetic acid or other cytokinins such as thidiazuron or benzylaminopurine can be used.

16. The process according to claim 14, characterized in that the final pH of the medium must be adjusted to 5.7±0.

2.

17. The process according to claim 14, characterized in that to obtain cell concentrations greater than 1x106 cells / mL, subcultures are carried out in periods of 715 days each and the cell density is determined every 7-10 days in a Neubauer chamber.

18. The process according to claim 14 and 17, characterized in that the cultures and subcultures are incubated in a bioclimatic chamber at a controlled temperature of 22±1°C with a photoperiod of 16 hours of light and 8 hours of darkness, with agitation between 50-150 rpm.

19. The process according to claim 1, characterized in that in step e) the suspension cell cultures containing the embryogenic somatic cells are sieved under sterile conditions, using 200 mesh sieves with a particle diameter of 0.74 pm. I7 20. The process according to claim 19, characterized in that 40 mL of the broth obtained after sieving is centrifuged between 4°C and 10°C and between 3000-5000 rpm for 5-30 minutes to obtain the cell package.

21. The process according to claim 1, characterized in that in step f) the viability is determined by reacting 0.4% trypon blue, mixed 1:1 (v / v) with the cell sample, the reaction is allowed to develop for 1 minute and the cells are observed in an optical microscope at 100x magnification with the aid of a Neubauer chamber.

22. The process according to claim 21 characterized in that the cell viability under the conditions in which the cultures were developed is 100%.

23. The process according to claim 1, characterized in that in step g) solutions of encapsulating agents are used, including 30% (w / v) maltodextrin, 14% (w / v) gum arabic, and 0.4% (w / v) xanthan gum.

24. The process according to claim 23, characterized in that the encapsulating agents are hydrated for 24 hours in distilled water, the pH is adjusted to 5.7 + 0.2, subsequently mixed in a 1:1 (v:v) ratio and homogenized with the aid of an orbital shaker between 80-100 rpm for 1-3 hours and sterilized at 15 lb / in? for 15 minutes.

25. The process according to claim 24, characterized in that the mixtures of encapsulating agents can be maltodextrin-gum arabic or maltodextrin-xanthan gum.

26. The process according to claim 23 characterized in that other encapsulating agents such as polysaccharides, alginates, whey, skimmed milk and / or vegetable proteins can be used in concentrations of 1 to 50% (w / v).

27. The process according to claim 1, characterized in that in step h) the cell package obtained in item e) is resuspended in the mixture of sterile encapsulating agents obtained in item g), the mixture is subjected to orbital shaking at 80-100 rpm for 30-60 minutes to obtain somatic cells.

28. The process according to claim 1, characterized in that in step i) the drying of somatic cells is carried out in a spray dryer using an inlet air temperature between 80-200°C and an outlet air temperature between 50-90°C.

29. The process according to claim 28, characterized in that microcapsules with a yield greater than 50% are obtained by spray drying.

30. The process according to claim 29, characterized in that microcapsules with cells with a viability between 60-100% are obtained.

31. The process according to claim 29, characterized in that the size of the microcapsules is between 1-40 pm.

32. The process according to claim 1, characterized in that in step j) the microcapsules are maintained at a humidity between 0 and 15% and a water activity between 0.1 and 0.

5.

33. The process according to claim 1, characterized in that in step j) the microcapsules are protected in airtight bags, vacuum sealed and refrigerated at a temperature in a range of 4°C to 10°C.

34. The process according to claim 1, characterized in that in step j) the dehydrated somatic cells have a viability of at least 90% for a period of 6 months.

35. The process according to claim 1, characterized in that in step k) 5 grams of the powder obtained as a product with the microencapsulated cells are rehydrated in sterile water or sterile liquid MS culture medium supplemented with cefotaxime and manganese ethylene bisdithiocarbamate with zinc salts in concentrations between 100-800 ppm, maintained in orbital agitation between 50-150 rpm for 1-24 hours.

36. The process according to claim 35 characterized in that the pH is adjusted between 5.7 and 7.

37. The process according to claim 1, characterized in that in step I) the suspension obtained after rehydration of the microcapsules is centrifuged between 3000-5000 rpm, between 4°C-10°C for 5-30 minutes to obtain the cell package.

38. The process according to claim 37, characterized in that once the cell package is obtained, cell viability is determined according to step f).

39. The process according to claim 1, characterized in that in step m) the cell package obtained is reactivated in 20 mL of sterile culture medium, solid MS with 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent, supplemented with 200 ppm of cefotaxime and 400 ppm of ethylenebisdithiocarbamate manganese with zinc salts.

40. The process according to claim 39 characterized in that the culture is incubated in a bioclimatic chamber at 25°C with a 16-hour photoperiod for 4 weeks.

41. The process according to claim 1, characterized in that in step m) the reactivation can be increased if growth regulators such as thidiazuron, benzylaminopurine, 2.4-dichlorophenoxyacetic acid, naphthaleneacetic acid are used.

42. The process according to claim 1, characterized in that in step n) the embryos obtained are transferred to solid MS medium with 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent and are incubated at 22±2°C with a photoperiod of 16 hours of light for 2 weeks.

43. The process according to claim 42, characterized in that the seedlings are separated under aseptic conditions and transferred to flasks containing 20 mL of sterile MS solid medium at 50% salts with 3% (w / v) of sucrose and 0.25% (w / v) of anionic polysaccharide as a gelling agent and are incubated for 14 days at 25°C with a photoperiod of 16 hours of light.

44. The process according to claim 43, characterized in that 100% rooting of the regenerated seedlings is achieved, which develop roots between 2-5 cm in length and have 3-5 leaves with a height between 3-10 cm.

45. The process according to claim 1, characterized in that in step o) the seedlings with 20-30 days of age are removed from the culture medium and the roots are washed with sterile water.

46. ​​The process according to claim 45, characterized in that the seedling is transferred to pots with a half-kilo capacity of substrate to which 300-500 grams of substrate mixture are transferred, of which 40% is peat and 60% perlite.

47. The process according to claim 1, characterized in that in step p) the seedlings are acclimatized in a greenhouse.

48. The process according to claim 47 characterized in that the seedlings contained in the pot are covered with a solid polypropylene container of 500 to 1000 g capacity.

49. The process according to claim 48, characterized in that three to five holes of 5 to 10 mm in diameter are made in the base of the polypropylene container.

50. The process according to claim 45, characterized in that the microclimate around the seedling provides a relative humidity greater than 75%.

51. The process according to claim 1 characterized in that in step p) the irrigation of the seedlings is carried out once a week.

52. The process according to claim 1. characterized in that in step p) an acclimatization of 80-95% is achieved with seedlings that have a root length of 5 cm and have 3 leaves of 5 cm in height.

53. The process according to claim 1, characterized in that in step q) the greenhouse-acclimated seedlings are transferred and sown in the field directly in the soil.

54. The process according to claim 53, characterized in that a mixture of 30% peat and 70% perlite can be added to the soil where the seedling is planted, as well as supplemented with worm humus.