IN VITRO CONSERVATION METHOD FOR PLANT GERMPLASM RESOURCES

Abstract

The present invention pertains to the technical field of plant reproduction using plant tissue culture techniques and specifically to methods for conserving plant germplasm resources as an alternative for maintaining germplasm banks in breeding programs. The invention discloses an in vitro conservation method for plant germplasm that proposes the use of gibberellin-inhibiting preservation agents in in vitro culture media, which advantageously allows for nearly 100% survival during the acclimatization stage after 180 days.

Description

IN VITRO CONSERVATION METHOD FOR PLANT GERMPLASM RESOURCES TECHNICAL FIELD The present invention is located in the technical field of agriculture, particularly within the area of ​​plant reproduction by plant tissue culture techniques and specifically is located in the field of methods for the conservation of plant germplasm resources. BACKGROUND Preserving plant genetic resources is important for breeding programs (Pañis et al., 2020). Medium-term conservation of species through plant tissue culture (PTC) is an alternative for establishing germplasm banks. Medium-term in vitro conservation has the advantages of requiring little storage space, immediate availability of the germplasm bank, control of storage conditions, and obtaining pest- and disease-free plant material (Pañis et al., 2020; Engels and Andreas, 2021). Furthermore, this conservation method facilitates easy transport and allows for the exchange of germplasm without international customs restrictions. Medium-term in vitro preservation involves slowing the growth of cells, tissues, organs, or whole plants. This technique commonly uses osmotic agents to reduce water availability (e.g., mannitol, sorbitol, and polyethylene glycol), refrigeration at low temperatures to reduce metabolism (2–10 °C), and growth-inhibiting compounds (e.g., abscisic acid, maleic hydrazide, ancymidol, N-dimethylaminosuccinamic acid, and paclobutrazol) (L. et al. 2017; Walters and Pence 2020; Priyanka et al. 2021; Subedi et al. 2021). Gibberellin (GA) synthesis inhibitors have been used to inhibit cell elongation and can affect chlorophyll content, stomatal density, growth, and differentiation (Subedi et al. 2021). The prior art includes various efforts aimed at providing new proposals for in vitro germplasm conservation of different plant species, such as the Chinese patent CN 111587688 (B), which describes an in vitro conservation culture method for Actinidia chinensis resources. The in vitro conservation and improvement method comprises the steps of preparing cuttings and reproductive stock; performing a bacteria-free graft; rooting the grafted plants; and transplanting the grafted seedlings. For its part, application CN 110089427 (A) provides a method for in vitro conservation of germplasm resources of Roxburgh anoectochilus shoots, comprising the following steps: selection and disinfection of the explant, induction of multiple shoots and proliferation, slow growth, restoration of the regeneration culture, rooting and transplanting after saving the germplasm.Publication CN 109221094 (A) discloses a method of cryopreservation of the in vitro stem apex of the banana, using banana plantlets 3.0 ~ 6.0 cm tall, pre-cultured for 2 days in sucrose culture medium containing 0.4 mol / L; at room temperature, the stem apex of 1 ~ 2 phyllopodium is removed from the strip; after the liquid-laden stem apex is loaded, it is manipulated with a vitrification solution, the drop is formed on a strip of aluminum foil; it is stored for at least 1 h in liquid nitrogen, thawed and washed with 1.2 mol / L sucrose culture solution, washing for 10 minutes each time;The stem apex, processed as described above, is placed in a sucrose MS culture medium containing 0.3 mol / L, cultured for 2 days in the dark, then transferred to a 6-BA MS culture medium containing 0.5 mg / L, cultured for 30-35 days, and once normal growth is restored, it is inoculated into a new culture medium to obtain multiple shoots, which are then grown in root media. It is stored for long-term storage in liquid nitrogen. Chinese patent application CN 109169277 (A) provides a method for the in vitro conservation of Dendrobium candidum germplasm resources that includes: pretreatment of Dendrobium candidum plants; acquisition of sterilizable material; inoculation of explants; slow preservation of germplasm; rejuvenation of germplasm; planting in water to strengthen seedlings for rooting; hardening; and transplanting. Growth and reproduction are effectively slowed to conserve material, the subculture interval is extended, and the number of subcultures is reduced, thus minimizing potential genetic variation. Patent CN 107079804 (B) provides a method for the in vitro conservation of Sargassum horneri germplasm resources, in which a germplasm conservation culture solution is prepared by adding 0.1 mol / L NaNO₃ to sterilized seawater, 30.1 mol / L NaH₂PCU, and 1 mg / L uniconazole. The method provided by the invention is used to carry out the in vitro conservation of Sargassum horneri, and can greatly extend the preservation time of the main branches of Sargassum horneri strains. The stored material can be quickly restored to growth and propagated in large quantities. Another document is Chinese patent CN 104756865 (B), which describes an in vitro preservation method for Hemiboea cavaleriei Levl. The method comprises the following steps: tissue culture and rapid propagation of Hemiboea cavaleriei Levl; production of a test tube seedling with 4 to 6 leaves from strong, differentiated cluster-bud seedlings obtained by tissue culture and rapid propagation; and inoculation of the test tube seedling with 4 to 6 leaves into a preservation medium for in vitro conservation. The preservation medium is an MS medium containing 1.0 mg / L of CCC, 0.3% AC, 3.5% cane sugar, and 0.4% agar; the pH value of the medium is 5.8, and the temperature of the medium is 18 ± 1°C. and the lighting intensity is 1400 to 1600 Ix and the lighting time is 8 to 10 h / d. Also included is Chinese patent CN 103651127 (B), which provides an in vitro preservation method for Chirita longgangensis. The in vitro preservation method comprises the following steps: selection of aseptic materials from Chirita longgangensis seedlings; inoculation of a portion of the test materials into different culture media; inoculation of a portion of the test materials into culture media containing different inhibitors; and exposure of a portion of the test materials to light at four different intensities. Chinese patent application CN 104920221 (A) discloses a method for in vitro preservation of potato germplasm. The method includes the steps of inoculating virus-free potato germplasm plantlets into a preservative culture medium in a culture vessel at normal temperature under sunlight for 30 days; adjusting the temperature to 15 to 19°C, adjusting the lighting time to 8 hours, and performing the culture for 60 days to obtain potatoes in test tubes; continuing the culture for 300 to 400 days; adjusting the temperature to normal, performing the culture for 100 to 200 days, allowing the virus-free plantlets to wilt and allowing the potatoes in test tubes to develop into seedlings; and either chipping the developed seedlings or directly inserting the potatoes in test tubes into the preservative culture medium and continuing the conservation process. However, the methods known to date are highly species-specific, which presents the disadvantage of requiring the standardization of various conservation protocols. Furthermore, some methods demand complex conservation conditions such as low temperatures. Another drawback is that some methods require constant subculture to maintain the viability of the specimens. OBJECT OF THE INVENTION Accordingly, the objective of the present invention is to provide a new in vitro method for preparing and preserving germplasm material that can overcome the disadvantages of the prior art. Therefore, a primary object of protection relates to an in vitro conservation method of plant germplasm material comprising the following steps: a) Obtain in vitro shoots in the multiplication stage; b) Grow in a preservation medium having a preservation agent selected from either paclobutrazol or trinexapac-ethyl, at a concentration of between 1 and 3 mg / L, preferably 1, 1.5, 2, 2.5 or 3. ML / a / ZUZZ / UU ll Yet another object of protection refers to an in vitro conservation method of plant germplasm material that allows the inhibition of shoot development in in vitro cultures without affecting their survival. Another object refers to an in vitro conservation method of plant germplasm material that allows maintaining in vitro cultures without subcultures until after 180 days. Additionally, the invention relates to an in vitro method of preserving plant germplasm material that allows for increased stomatal density. Also considered as an object of protection is a method of in vitro conservation of plant germplasm material that allows its regeneration in the acclimatization stage after the conservation period estimated at at least 180 days. Furthermore, the invention also contemplates as an object of protection an in vitro preservation medium for plant germplasm material characterized in that it has a preservation agent selected from paclobutrazol or trinexapac-ethyl, in a concentration of between 1 and 3 mg / L, preferably 1, 1.5, 2, 2.5 or 3 mg / L. The objectives of the present invention referred to above, and even others not mentioned, will be evident from the description of the invention and the figures that accompany it for illustrative and non-limiting purposes, which are presented below. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Effect of growth inhibitor concentration on in vitro preservation of different species, (ayd) Anthurium (Anthurium andreanum Lind.), (bye) sugarcane (Saccharum spp. Hybrid) and (cyf) agave (Agave potatorum Zuce.) after 180 days of culture. Doses from left to right in each figure: a) paclobutrazol (PBZ): 0, 1, 2 and 3 mg L-1 and df) trinexapac-ethyl (TNE) 0, 1, 2 and 3 mg L-1, white bar = 2 cm. Figure 2. Effect of paclobutrazol (PBZ) and trinexapac-ethyl (TNE) on total chlorophyll content, a) Anthurium (Anthurium andreanum Lind.), b) sugar cane (Saccharum spp. Hybrid) and c) agave (Agave potatorum Zuce.). Values ​​represent mean ± standard error. Different letters represent statistically significant differences (Tukey, p<0.05). Figure 3. Effect of paclobutrazol (PBZ) and trinexapac-ethyl (TNE) on stomatal density, a) Anthurium (Anthurium andreanum Lind.), b) sugar cane (Saccharum spp. Hybrid) and c) agave (Agave potatorum Zuce.). MA / a / ¿U¿¿ / UU Values ​​represent mean ± standard error. Different letters represent statistically significant differences (Tukey, p<0.05). Figure 4. In vitro regeneration and acclimatization of anthurium, sugarcane, and agave. Regenerated shoots: a) Anthurium (Anthurium andreanum Lind.) at 60 d, b) sugarcane (Saccharum spp. Hybrid) at 45 d, and c) agave (Agave potatorum Zuce.) at 60 d, black bar = 2 cm. Acclimatized plantlets: d) Anthurium (Anthurium andreanum Lind.), b) sugarcane (Saccharum spp. Hybrid), and c) agave (Agave potatorum Zuce.) at 90 d of ex vitro culture under greenhouse conditions, white bar = 5 cm. DESCRIPTION OF THE INVENTION The present invention relates to a method for the in vitro conservation of germplasm resources. In vitro conservation systems for ornamental crops such as anthurium (Anthurium andreanum L.), with C3 metabolism, and agro-food crops such as agave (Agave potatorum Zuce.) and sugarcane (Saccharum spp. Hybrid), with CAM and C4 metabolism, respectively, are important for safeguarding accession collections in the genetic improvement of these species. The advantage of this invention lies in demonstrating the efficiency of the growth inhibitors paclobutrazol (PBZ) and trinexapac-ethyl (TNE) as an alternative for the in vitro conservation of various plant species. PBZ and TNE are compounds that inhibit GA synthesis and have shown effects on the in vitro development of different species. However, it is important to establish in vitro conservation protocols for each species because the type of photosynthetic metabolism—3-carbon (C3) compounds phosphoglycerate, 4-carbon (C4) compounds oxaloacetic acid, and crassulacean acid metabolism (CAM)—defines the physiological response to the conservation methods used. To clarify the description of the present invention and for a better understanding thereof, it should be understood that the terminology used in this document is not intended to be limiting. A glossary of technical terms used in this specification is listed below. In this document, the singular forms a, an, and the include both singular and plural references unless the context clearly indicates otherwise. For example, a nanoparticle means one nanoparticle or more than one nanoparticle. The terms comprising, comprising as used herein are synonymous with including, includes or containing, contains, and are included or of open composition and does not exclude additional, unrecited members, elements, or stages of the procedure. It will be appreciated that the terms comprising, comprising and comprising as used herein encompass the terms consisting of, consists of, and consists of. The recitation of numerical intervals by endpoints includes all whole numbers and, where applicable, fractions subsumed within that range (e.g., 1 to 5 may include 1, 2, 3, 4 when referring to, for example, a series of items, and may also include 1.5, 2, 2.75, and MA / a / 2U22 / UU 1021 3.80, when referring to, for example, measurements). The recitation of endpoints also includes the endpoint values ​​themselves (e.g., 1.0 to 5.0 includes both 1.0 and 5.0). Any numerical range cited herein is intended to include all subintervals subsumed within it. Throughout this application, the term approximately is used to indicate that a value includes the standard deviation of error for the device or method used to determine the value. In this specification, the term "an embodiment" means a particular feature, structure, or characteristic described in connection with the embodiment that is included in at least one embodiment of the present invention. Therefore, the occurrences of the phrase "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be evident to a person skilled in the art from this description, in one or more embodiments.Furthermore, although some embodiments described herein include some, but not others, features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the invention and to form different embodiments, as understood by those in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination. In this regard, the present invention relates to an in vitro method for preparing and preserving germplasm material comprising the following steps: a) Obtain in vitro shoots in the multiplication stage; b) Grow in a preservation medium having a preservation agent selected from either paclobutrazol or trinexapac-ethyl, at a concentration of between 1 and 3 mg / L, preferably 1, 1.5, 2, 2.5 or 3. In one embodiment of the invention, a preservation medium for preparing and preserving germplasm material in in vitro cultures is also contemplated, having a preservation agent selected from either paclobutrazol or trinexapac-ethyl, at a concentration of between 1 and 3 mg / L, preferably 1, 1.5, 2, 2.5 or 3. Examples The examples presented are illustrative and not limiting, since a person skilled in the art will understand that there are variations that fall within the scope of protection of the present invention. Example 1. Preparation of plant material The process for preparing the plant material is described below, the steps of which are considered conventional in the field of development of the invention for obtaining explants. In vitro shoots (1.0 cm) of Anthurium andreanum L. were used as an example of C3 metabolism, obtained after four subcultures (every 60 d) in the multiplication stage in basal salts of MS medium (Murashige and Skoog, 1962) with 0.01% (w / v) thiamine HCI, 0.05% (w / v) nicotinic acid, 0.05% (w / v) pyridoxine HCI, 10.0% (w / v) myo-inositol, 0.2% (w / v) glycine, 30 g L-1 of sucrose and supplemented with 2 mg L-1 of benzylaminopurine and Phytagel™ (Sigma®) at 0.22% (w / v) were used as a gelling agent. As an example of C4 metabolism, in vitro shoots (1.5 cm) of Saccharum spp. Hybrid were used, obtained after four subcultures (every 45 d) in MS medium composed of 3 mg L1 of BAP, 3 mg L1 of indoleacetic acid (IAA, Sigma®) and 0.22% (w / v) Phytagel™. For the CAM metabolism study, 2.0 cm in vitro shoots of Agave potatorum Zuce. were used, obtained after four subcultures (every 45 days) in MS medium with 2 mg L1BAP, 1 mg L1IAA, 1 mg L1Kinetin (KIN, Sigma®) and 0.22% (w / v) Phytagel™. The pH of the culture media was adjusted to 5.8 and sterilized for 15 min in an autoclave (Felisa® FE299, JAL, MX) at 1.5 kg cm-2 pressure and 120°C. Example 2. Effect of growth inhibitors on in vitro growth rate To evaluate the effect of GA inhibitors on minimal growth, different concentrations (0, 1, 2, and 3 mg L⁻¹) of the compounds paclobutrazol (PBZ) and trinexapac-ethyl (TNE) were tested on nutrient-rich shoots of anthurium, agave, and sugarcane. The shoots were cultured in 22 x 220 mm test tubes with polypropylene caps (MOLLER®), containing 15 mL of semi-solid MS medium supplemented with 30 g L⁻¹ of sucrose, without growth regulators, and 0.22% (w / v) of Phytagel™ (Sigma®) in the case of anthurium and sugarcane, while for agave, 0.60% (w / v) of micropropagation grade agar (PhytoTech Labs®) was used as a gelling agent. The pH of the culture media was adjusted to 5.8 with 0.5 N NaOH and sterilized for 15 min in an autoclave (Felisa®) at 1.5 kg cm-2 pressure and 120°C. The culture vessels, with one sprout each, were incubated at 24 ± 2°C and maintained at LED light irradiance of 40-50 pmol m-2s-1 with a photoperiod of 16 h light.After 180 days of incubation, the percentage of survival, number of shoots, plant length, number of leaves, number of roots, root length, total chlorophyll content, and stomatal density were evaluated. The growth inhibitors PBZ and TNE had an effect on the different developmental variables evaluated in the species anthurium, sugarcane, and agave. This effect intensified as the concentrations of these compounds increased. The survival rate was only affected in anthurium, decreasing as the concentrations of PBZ and TNE increased. The sugarcane species MA / a / ¿U¿¿ / UU ll sugar and agave showed 100% survival in the different concentrations evaluated of PBZ and TNE (Table 1). yozí Table 1. Effect of growth inhibitors on the in vitro conservation of anthurium (Anthurium andreanum Lind), sugar cane (Saccharum spp. Hybrid) and agave (Agave potatorum Zuce.), after 180 days of cultivation. Species Inhibitor Concentration (mg L') Survival (%) Number of shoots Shoot length (cm) Number of leaves Number of roots Root length (cm) Anthurium PBZ 0 100 ± 0.00 a 1 25±0.13a 5 00 10.21 3 6 1610.20' 4.8310.20 ' 3.5410.16' 1 100 ± 0 00 3 1 5010 15a 3.40 ± 0 07 ° 441 ±0 19° 2 751 0 21 ° 2.251 0.25 = 2 61.10 i 2.77 : 1 2510.13' 1 3610.11 = 3 41 10 14== 1.3310 14 = 2.2510.25 = 3 47.22 12 78 = 11610 11a 1 27 10 78 = 3 33 1 0.14 = 0 58 1 0 19 = 0 19±0.06 = TNE 1 82.66 ±4.335 1 2510.13a 1.49 ± 0.12 = 4.16 10 27“ 1.00 i 0 21 = 0 58 ±0.07 = 2 47.22 12 78 = 1 3310 14a 1.35 i 0.09 = 3.6610 14°=° 1 00 ±0 24 = 0.18±0.04 = 3 19.99 ±2.54 3 1 00 10.00a 1.15 ± 0.46 = 3 16 i 0.11 3 0.50 1 0 15 = 0 7510.02 = Sugarcane PBZ 0 100 1 0.00a 10 70 10.68 = 9 40 1042 a 4 80 10 20 a 90 50 ±3 23' 7.601 0.38 a 1 100 1 0.00' 25 90 ± 1.08c 4 6510.25°= 3.70 10 26“ 24.00 1 1.19° 4.4610.24 = 2 100 ± 0.00a 20 70 1 1 19 = 320 10 16 = 3.4010 16“° 6.50 1 0 30 = 1.40 10.10 31 3 100 ± 0.00a 91010.52a 2 00 i0 16e 2 50 1 0.16 = 6.30 10 36 = 1.1310.11 = TNE 1 100 ± 0.00a 39 40 11.06 a 5 50 1023° 4.1010.23 a° 93.30 13.10 a 4.851 0.24 = 2 100 ± 0.00a 39 30 ±0 81 3 4.70 1.24 °= 3.40 10 16 °=° 89.80 ±965’ 3.9010.25 = 3 100 ± 0.00a 40 50 10.61 a 3.55 1 0 31 =° 2 70 1021 == 25.30 ±1.08° 2.2510.20 = Agave PBZ 0 100 1 0.00’ 7 20 10.57a 7 30 i 0 29 3 5 20 1 0.24 ’ 22.80 10 91 3 7.6010.33’ 1 100 ± 0.00a 5 30 10.22° 5 90 10.24 ° 5 00 1 0.25 3° 17 70 ± 1 03 3 6.3010.35 = 2 100 ± 0 003 3.70 ±0.30 = 5.25 1 0 21 K 4 20 ± 0 24 “ 18.40 ±1 28° 4.8010.38 = 3 100 ± 0.00a 1 4010.16° 2 58 ± 0.14° 3.30 10 21 == 6 50 ± 0 52 = 2.6910.17 = TNE 1 100 ± 0 003 4.40 ±0.33°= 6.0510 30° 4.40 10.26’= 17.80 ±0.89 = 7.281 0.32 ’= 2 100 ± 0.00a 2 30 10 15° 4 50 10.18 = 4.5010.14’° 18.10 10 76° 46010.19 = 3 100 ± 0.00a 1 4010.16° 2 5610 11 = 3 00 1 0.21 = 6.20 10 46 = 2.9010.16 =. Values ​​represent mean ± SE. Means with different letters in a column for each species are significantly different (Tukey, p<0.05). PBZ: Paclobutrazol and TNE: Trinexpac-ethyl. In anthurium, PBZ and TNE had no effect on the number of shoots. The greatest shoot length was observed in the control treatment, with an average height of 5 cm, followed by the PBZ 1 mg L⁻¹ treatment, with a height of 3.4 cm. The remaining treatments showed the shortest shoot length, with shoots less than 1.5 cm tall. For the number of leaves, the control treatment showed the highest number of leaves, with 6.16 leaves per shoot, followed by the PBZ 1 mg L⁻¹ treatment, with 4.41 leaves per shoot; while the remaining treatments had between 3.16 and 4.16 leaves per shoot. The greatest number and length of roots were observed in the control treatment, with 4.83 roots per shoot and an average length of 3.54 cm, followed by the PBZ 1 mg L⁻¹ treatment, with 2.7 roots per shoot and an average length of 2.25 cm. The remaining treatments showed between 0.5-1.33 roots per shoot, with a length between 0.18-0.75 cm (Fig. 1a, d). In sugarcane, the highest number of shoots per explant was observed in the TNE treatments, with 39 shoots per explant; while the lowest number of shoots was observed with 3 mg L⁻¹ of TNE, with 9.10 shoots per explant. For the shoot length variable, the largest shoots were found in the control treatment, with a length of 9.40 cm; while the shortest shoots were observed in the other treatments, with lengths between 3.20 and 5.5 cm. For the number of leaves, the treatments with the highest number of leaves per shoot were 0 and 1 mg L⁻¹ of TNE, with 4.8 and 4.10 leaves per shoot, respectively; while the remaining treatments showed the lowest number of leaves. Regarding the number of roots per explant, the treatments with the highest number of roots were the control treatment, 1 and 2 mg L⁻¹ of TNE, with 90.5, 93.3, and 89.8 roots per explant, respectively.The greatest root length was observed in the control treatment, with a length of 7.60 cm; while the shortest root length was observed in the rest of the plots with values ​​between 1.13-4.46 cm in length (Fig. 1b, e). In agave, the highest number of shoots per explant was observed in the control treatment, with 7.2 shoots per explant, followed by 1 mg L⁻¹ of TNE, with 5.3 shoots per explant. The longest shoots were observed in the control treatment, with a height of 7.30 cm, followed by 1 mg L⁻¹ of PBZ and 1 mg L⁻¹ of TNE, with heights of 5.90 and 6.05 cm, respectively; while the shortest shoots were observed in the remaining treatments, with heights between 2.56 and 5.25 cm. The highest number of leaves per shoot was observed in the control treatment, with 5.20 leaves per shoot. The lowest number of leaves was observed in the 3 mg L⁻¹ PBZ and 3 mg L⁻¹ TNE treatments, with 3.30 and 3 leaves per shoot, respectively. For the root count, the highest number of roots was observed in the control treatment, with 22.8 roots per shoot; while the lowest number of roots was observed in the 3 mg L⁻¹ PBZ and 3 mg L⁻¹ TNE treatments, with 6.50 and 6.203 roots per explant, respectively.The greatest root length was observed in the control treatment, with a length of 7.60 cm; while the shortest length was observed in the 3 mg L'1 of PBZ and 3 mg L~1 of TNE treatments, with 2.69 and 2.90 cm in length (Fig. 1 c, f). These results show that the present preservation method, unlike other studies, reduces the cost of the culture medium due to the low price of the preservatives PBZ and TNE. Additionally, other in vitro preservation methods use mannitol, sorbitol, PEG, and ABA, which are more expensive, or employ refrigeration chambers that consume a lot of energy. In sugarcane, the addition of PBZ and TNE to the culture medium inhibited in vitro plant development of this species without affecting shoot survival. These results indicate that PBZ and TNE can be used for the in vitro conservation of Saccharum spp. Furthermore, the inhibition of GA synthesis by PBZ and TNE promoted shoot proliferation in vitro. This was likely due to reduced competition among shoots for space, water, light, and nutrients, resulting in the formation of new shoots. In agave, the addition of PBZ and TNE showed no difference in survival rates. On the other hand, unlike sugarcane, a significant decrease in the number of A. potatorum shoots was observed in the PBZ and TNE treatments. This could be explained by the fact that there is no interaction between GA inhibitors and cytokinins or other developmental regulators that promote the formation of new shoots. wiA / aizvzzivu i qz i Example 3. Total chlorophyll Chlorophyll content was determined according to the methodology proposed by Harborne (1973). 250 mg of leaf tissue was collected and placed in a 10 mL amber bottle. 2.5 mL of 80% acetone was added, and the samples were incubated for 24 h at -4°C. After incubation, the samples were macerated, and the extract was placed in a funnel lined with filter paper (Whatman, grade 4). The filtrate was diluted to 6.25 mL with 80% acetone, and its absorbance was measured using a spectrophotometer at 645 and 663 nm. Chlorophyll content was calculated using the following formula: Total Chlorophyll (C)= ([(8.20 χ Α663) - (20.2 χ A645)] (V)) / ((1000 χ W)) Where: A663 and A64S: Absorbance, C = Concentration, V: Graduated volume in mL, W: Sample weight in g, 1000: Conversion factor Total chlorophyll content showed significant differences for anthurium and sugarcane at the different concentrations of PBZ and TNE evaluated. However, in agave, no significant differences in chlorophyll content were found at the different concentrations of PBZ and TNE (Fig. 2). In anthurium, the highest chlorophyll content was observed in the control treatment and the 1 mg L⁻¹ PBZ treatment, with 1.37 and 1.28 mg g⁻¹ of PF, respectively; while the lowest chlorophyll content was observed in the 2 and 3 mg L⁻¹ TNE treatments, with 0.29 and 0.27 mg g⁻¹ of PF, respectively. In sugarcane, the highest chlorophyll content was observed in the 2 and 3 mg L⁻¹ PBZ and 3 mg L⁻¹ TNE treatments, with 1.18, 1.21, and 1.25 mg g⁻¹ of PF, respectively. While the lowest chlorophyll content was observed in the control and 1 mg L⁻¹ of TNE, with 0.72 and 0.69 mg g⁻¹ of PF, respectively. Regarding agave, the chlorophyll content remained between 0.47 and 0.54 mg g⁻¹ of PF. In anthurium, the reduction in total chlorophyll was likely due to the degradation of photosynthetic pigments caused by a toxic effect, which is also reflected in the low survival rate of the explants. The phytotoxicity caused by TNE could be due to an increase in reactive oxygen species (ROS), leading to damage to the cell membrane, proteins, and DNA structure, as well as the inhibition of photosynthesis. This likely results in a decrease in the number and length of shoots, since a high concentration of ROS triggers cell apoptosis. In sugarcane, the increase in chlorophyll caused by PBZ and TNE could be explained by a greater accumulation of photosynthetic pigments caused by denser packing of chloroplasts in tissue cells. In agave, treatments with PBZ and TNE had no effect on chlorophyll content. In this species, CAM plants under in vitro conditions exhibit a slow growth rate without requiring photosynthetic pigments for their development. Furthermore, it is important to note that the carbon source in in vitro cultures is obtained by the explants directly from the sugars added to the MS culture medium. Example 4. Stomatal density To calculate stomatal density (SD), the third leaf from the apex to the base of the stem was taken from ten shoots of the different treatments for each species. SD was determined according to the methodology described by Xu and Zhou (2008) with slight modifications. A thin layer of nail polish was applied uniformly to the abaxial surface. After 3 min, the polish was gently peeled off and the leaf mounted on a slide. Leaf replicas were examined under a light microscope. The number of epidermal cells per mm² was counted from three random fields of one leaf at 10X and 40X magnification. SD was calculated using the formula suggested by Wilkinson (1980): SD= (NS / (EC + NS)) x 100s Where: SD: Stomatal density, NS: Number of stomata, EC: Number of epidermal cells Stomatal density (SD) showed differences between the species and concentrations of PBZ and TNE evaluated (Fig. 3). In anthurium, the highest SD was observed with 3 mg L'1 of PBZ, and 2 and 3 mg L'1 of TNE, with 19.69, 20.0 and 21.08%, respectively; while the lowest SD was found in the PBZ control treatment, with 13.61%. In sugarcane, the highest SD was found in the 3 mg L⁻¹ PBZ treatment, at 20.99%; while the lowest SD was observed in the control treatment, at 16.87%. For agave, the highest SD was found in the 2, 3 mg L⁻¹ PBZ and 2, 3 mg L⁻¹ TNE treatments, with values ​​between 21.86-23.01%; while the lowest SD was observed in the control treatment, 1 mg L⁻¹ PBZ and 1 mg L⁻¹ TNE, with 19.98, 20.28 and 20.20%, respectively (see figure 3). Stomatal density (SD) is a physiological parameter that measures the number of stomata per unit of leaf area and is affected by water availability, relative humidity, nutritional status, and light (irradiation, photoperiod, and intensity). This study shows an increase in SD in the evaluated species. Because PBZ and TNE reduce cell elongation, this leads to anatomical changes in roots, stems, and leaves. In leaves, the leaf blade is reduced, which affects the size of stomata and epidermal cells. The increase in SD in the evaluated species was due to the reduction in the leaf blade, resulting in a relative increase in the number of stomata and epidermal cells in the leaves. Example 5. In vitro regeneration and acclimatization After 180 days of storage, individual 2-cm-long shoots obtained from the best minimal growth treatment were cultured in multiplication medium for 60 days for anthurium and agave and 45 days for sugarcane. The culture conditions and concentrations of plant growth regulators were the same as in the multiplication stage. For the acclimatization stage, surviving seedlings of the three species from the different GA inhibitor treatments were used. For anthurium, seedlings were transferred to a sterile substrate composed of peat moss and volcanic rock (particle size 5-10 mm) in a 1:1 ratio. For agave, seedlings were transferred to a sterile substrate composed of common soil, perlite, and tezontle in a 1:1:1 ratio. Finally, for sugarcane, a sterile substrate with a mixture of peat moss and perlite in a 4:1 ratio was used. In all cases, the substrate was sterilized in an autoclave (Felisa®) for 15 min at 1.5 kg / cm² pressure and 120 °C, and plastic trays with 72 cavities were used. Finally, the plants were placed under greenhouse conditions (natural light irradiance of 130 pmol m⁻² s⁻¹, 30 ± 2 °C, and 60 ± 5% RH). Foliar fertilizer (Gro-green®) was applied (1 mg L⁻¹) once a week, and the plants were watered once a day. After 60 days of growth, the survival percentage was determined. Anthurium, sugarcane, and agave plantlets obtained during in vitro conservation were successfully regenerated by direct organogenesis. Survival rates of 97%, 96%, and 98% were observed in anthurium, sugarcane, and agave plantlets, respectively, during the acclimatization stage (Fig. 4). It was possible to regenerate shoots from plantlets obtained during in vitro conservation. The survival rates obtained during acclimatization demonstrate the efficiency of the protocol for the in vitro conservation of the evaluated species. Gibberellin synthesis inhibitors demonstrate an effect on growth reduction that counteracts abiotic stress. The PBZ and TNE treatments in in vitro-grown plants likely induced tolerance to factors involved in the acclimatization process, such as water availability, relative humidity, temperature, and changes in intensity, irradiance, and photoperiod. Furthermore, the biochemical effects of PBZ related to these factors include the detoxification of reactive oxygen species (ROS), increased antioxidant and chlorophyll content, increased abscisic acid, and decreased ethylene. Of the species evaluated, the highest survival rate obtained in agave (98%) was likely due to the fact that CAM plants maintain resistance mechanisms to the abiotic factors they encounter during acclimatization. The medium-term conservation of plant genetic resources is an important strategy for genetic improvement. Therefore, it is necessary to use biotechnological alternatives that allow for the establishment of in vitro plant conservation systems. Development-inhibiting compounds such as PBZ and TNE are a low-cost alternative for safeguarding accession collections in the species evaluated in this study. This protocol only requires two subcultures per year for the three species evaluated in this study. Between each subculture (180 days), the plants remain viable and can be used at any time for regeneration and subsequent acclimatization. Furthermore, no visible variations were observed in regenerated shoots or acclimatized seedlings. This invention determined that the addition of GA inhibitors, PBZ and TNE, to MS culture medium did not affect development, survival, total chlorophyll content, or stomatal density in anthurium, sugarcane, and agave seedlings. In anthurium, using 1 mg L⁻¹ of PBZ maintained 100% survival compared to the same dose in sugarcane and 3 mg L⁻¹ of PBZ or TNE in agave. Medium-term in vitro preservation aims to reduce explant growth while maintaining survival in confined spaces. GA inhibitors at appropriate concentrations could be used as an alternative for in vitro preservation without affecting the survival rate. The advantages of this method include reduced labor, low cost of reagents used, and minimal space requirements for in vitro conservation of plant germplasm. Although the foregoing description was prepared taking into account the preferred embodiments of the invention, those skilled in the art should be aware that any modification of form and detail will be considered within the spirit and scope of the present invention. The terms in which this specification has been drafted should always be taken in a broad and non-restrictive sense. The materials, form, and description of the elements may be varied provided that this does not alter the essential characteristics of the model. LITERATURE Engels JMM, Andreas WE (2021) A Critical Review of the Current Global Ex Situ Conservation System for Plant Agrobiodiversity. I. History of the Development of the Global System in the Context of the Political / Legal Framework and Its Major Conservation Components. Plants 10(8):1557. doi:10.3390 / plants10081557 Harbome JB (1973) Nitrogen compounds. Phytochemical Methods. Springer, The Netherlands, pp. 166211 L¡ J, Wu Y, Xie Q, Gong Z (2017) In: L¡, J., Li, C., Smith, S.M.B.T.-H.M. (Eds.), 5 - Abscisic acid. Academic Press, pp. 161-202. doi: 10.1016 / B978-0-12-811562-6.00005-0 Murashige T, Skoog F (1962) A revised médium for rapid growth and bioassays with tobáceo tissue culture. Physiol Plant 15:473-497 Pañis B, Nagel M, Van den houwe I (2020) Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and / or in Liquid Nitrogen. Plants 9(12):1634. doi: 10.3390 / plants9121634 Priyanka V, Kumar R, Dhaliwal I, Kaushik P (2021) Germplasm Conservation: Instrumental in Agricultural Biodiversity—A Review. Sustainability 13:6743. doi: 10.3390 / su13126743 Subedi M, Karimi R, Wang Z, Graf R, Mohr R, O'donovan J, Brandt S, Beres B (2021) Winter cereal responses to dose and application timing of Trinexapac-ethyl. Crop Science Walters C, Pence VC (2020) The unique role of seed banking and cryobiotechnologies in plant conservation. Plants People Planet 3:83-91. doi: 10.1002 / ppp3.10121 Wilkinson HP (1980) In: Metcalfe, C.R., Chalk, L. (Eds.), The Plant Surface (Mainly Leaf). Anatomy of the Dicotyledons. I. Clarendon Press, Oxford, pp. 97-165. Xu Z, Zhou G (2008) Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. J Exp Bot 59:3317-3325

Claims

1. A method for in vitro conservation of plant germplasm material, characterized in that it comprises the following steps: c) Obtaining in vitro shoots in the multiplication stage; d) Cultivating in a conservation medium having a preservation agent selected from paclobutrazol or trinexapac-ethyl, at a concentration of between 1 and 3 mg / L.

2. The in vitro conservation method of plant germplasm material according to claim 1, further characterized in that the plant germplasm is selected from plants with C3, C4 or CAM metabolisms.

3. The in vitro conservation method of plant germplasm material according to claim 1, further characterized in that the conservation medium is preferably MS.

4. The in vitro preservation method of plant germplasm material according to claim 1, further characterized in that the concentration of the preservation agent is preferably between 1, 1.5, 2, 2.5 or 3 mg / L.

5. An in vitro preservation medium for plant germplasm material characterized in that it has a preservation agent selected from either paclobutrazol or trinexapac-ethyl, in a concentration of between 1 and 3 mg / L, preferably 1, 1.5, 2, 2.5 or 3 mg / L.