Supplementary composition and nutritional matrix composition, synthetic seed, method for culturing plant and use of supplemental composition

Abstract

The invention belongs to the technical field of biology. More precisely, a gel-like nutritional substrate containing at least meta-topoforest and melatonin and a method for producing the same are described. Also described is an artificial seed comprising a container filled with a nutritional substrate comprising at least melatonin and an intertopological forest in which a plant propagule is placed. The present invention also describes a method of in vitro culturing a plant in which the in vitro cultured meristem and / or embryonic tissue is placed on a nutritional substrate comprising at least meta-topoforest and melatonin, which later is exposed to stress conditions common in the field, enabling the propagule to grow into an adult plant. Preferably, the plant meristem and / or embryogenic tissue used is from a plant species that can be asexually propagated.

Description

TECHNICAL FIELD

[0001] The present invention relates to the biotechnology field. More precisely, a nutritional substrate is described, which consists of a basal medium for growing plants and at least one supplementary composition comprising at least one citocinin and melatonin. An artificial seed is also described, which comprises a container filled with the nutritional substrate, in which a plant propagule is placed for germination and generation of new adult plants (or adult plants) under in vitro environmental conditions present in the field. The synthetic seeds produced with the nutritional substrate of the present invention can be planted in greenhouses or in sunny fields. The present invention also describes a method for plant cultivation, in which plant propagules from in vitro culture are placed in the nutritional substrate to directly pass to in vitro environments, where they will be exposed to stress conditions common in the field. BACKGROUND

[0002] Plant tissue culture techniques have been widely used, not only for commercial plant propagation, but also for plant genetic manipulation by, for example, genetic transformation, mutagenesis and gene editing methods, in addition to being an important tool for conventional plant breeding and seedling hygiene, enabling healthier plantations.

[0003] These tissue culture systems are commonly known as micropropagation systems, in which plant tissues are grown in vitro in a suitable medium, from which they proliferate and regenerate into mature plants. For example, micropropagation systems can maintain the continuous production and quality of plant material for commercial plantations. Plant propagation and regeneration by tissue culture techniques are well known in the art and many publications present techniques and methods for micropropagation of a wide variety of plant species, in which in vitro micropropagation via organogenesis or somatic embryogenesis is successfully reported. In the field, micropropagated sugarcane plants have shown good results in terms of, for example, tillering and productivity.

[0004] Despite the successful reports, the use of these techniques on an industrial scale is an activity that still presents many challenges and there is no consensus even on the parameters that should be observed to define a process for a particular species.

[0005] The state of the art is contradictory when it comes to defining the best practices applicable to plant tissue culture, especially when it comes to crops such as sugarcane, which have such complex genomes. Usually, protocols are defined for specific genotypes and new protocols are optimized / developed with each new variety bred. Thus, in vitro culture today is still an empirical process, in which, for each species, or even for each variety (genotype) within the same species, the following conditions are minimally tested: (I) different in vitro culture techniques (organogenesis, direct and indirect somatic embryogenesis); (II) source of explants; (III) composition of the culture medium (vitamins, carbon source and fitorreguladores); (IV) hormonal balance; and (V) environmental conditions (Peres, 2002).

[0006] With regard to item (IV) mentioned above, there are numerous hormones and fitorreguladores known in the state of the art. Once the action of these compounds in the various stages of micropropagation is discovered, it opens up countless possibilities for controlling, optimizing and developing new processes. However, despite the many understandings that have been generated since then, the type of compound, its concentration, combination and time of use are still the subject of many current research and development.

[0007] For example, the exogenous application of cytokinins is widely used to stimulate the productive response of plants and to promote their growth under adverse environmental conditions (Marenco & Lopes, 2007). Another important function of the exogenous application of cytokinins is to delay plant senescence by accumulating and maintaining photosynthetic pigments in plants (Van Staden et al., 1988). Cytokinins have specific effects of increasing the rate of amino acid incorporation, delaying senescence and increasing nutrient uptake (Salisbury & Ross, 1992). They have been used in various crops to accelerate growth rate, induce root formation, prevent fruit abscission and adjust the ratio of male to female flowers in inflorescences (Kumar et al., 2011).

[0008] However, the use of cytokinins alone is not enough to guarantee the development and regeneration of plant explants and / or propagules into adult plants, as other stimuli must also be considered, especially when the seedlings from the explants will be subjected directly from in vitro culture to the abiotic stresses of the field. In addition, there are various cytokinins involved in the development process of plants, which need to be properly selected independently or in combination, qualitatively and quantitatively.

[0009] Therefore, the present application teaches a new plant culture composition comprising a combination of plant growth regulators and other compounds with the aim of providing essential nutritional and stimulatory conditions to explants and / or plant propagules to obtain robust metabolic and physiological properties when placed directly in the field under in vitro conditions.

[0010] The following briefly presents the prior art documents closest to the claimed invention.

[0011] Document US2018206427 (A) discloses a plant propagation system based on a detachable support that can be used for micropropagation of plants, wherein the plants are placed in a liquid medium containing a plant development stimulating compound such as meta-topolina.

[0012] Publication WO13016198 (Al) relates to compositions, methods and systems for plant propagation, in particular bamboo. In the medium for root development and plant maintenance, the cytokinin other than thidiazuron (TDZ) is selected from the group consisting of, for example, N6-benzylaminopurine (BAP), meta-topolina (mt), zeatin, zeatin riboside, dihydrozeatin, kinetin, isopentenyladenine, adenine hemisulfate, etc.

[0013] Nawaz et al. (2016) propose the possibility of using melatonin for other crops with seed industry potential, especially exogenously applying this compound to fruits, seeds and even roots. However, this study does not disclose or suggest the combination of melatonin and meta-topolina, much less the concentrations described in the present invention. SUMMARY

[0014] The present invention describes a supplemental composition comprising a combination of meta-topolina and melatonin. This composition can be added to a nutrient medium for growing explants and / or plant propagules, whether or not these explants and / or plant propagules have previously been grown in vitro. This nutrient medium is composed of a base composition comprising all the necessary nutritional sources for growing plants, such as base salts, vitamins, carbon sources, antioxidants, etc., as well as one or more plant growth regulators and plant hormones. The supplemental composition of the present invention can comprise other plant growth regulators and plant hormones in addition to the amino acid composition and the silicon source.

[0015] The supplemental composition of the present invention promotes a higher survival rate of explants and / or plant propagules under in vitro conditions when they are directly transferred to the field, even under abiotic stress.

[0016] The action of the supplementary composition is based on the redirection of the carbon skeleton to accelerate metabolism and growth, followed by enzyme / protein stabilization under stress, delay of senescence, increase in photosynthetic pigment content, induction of the expression of heat shock proteins and promotion of the cell wall structuring of roots and leaves. These properties are desirable because they increase the survival rate during the acclimatization phase of the plant growth under the action of the supplementary composition of the present invention, since during this phase there is greater water loss due to the low functionality of the stomata and the thin layer of cuticular waxes.

[0017] In a first aspect, the present invention provides a supplementary composition characterized by comprising the combination of meta-topolin and melatonin. In more detail, the concentration of melatonin in the supplementary composition is characterized by being between 5 and 25 µM, preferably between 10 µM and 15 µM, even more preferably the concentration of melatonin is 10 µM. Similarly, the concentration of meta-topolin is characterized by being between 3.5 and 7 µM, preferably between 4 and 6 µM, even more preferably the concentration of meta-topolin is 5 µM.

[0018] In addition, the supplementary composition comprises L-alanine, L-proline and glycine and potassium silicate.

[0019] In a second aspect, the present invention provides a nutritional substrate composition characterized by comprising the supplementary composition and a base composition for the growth of plants.

[0020] Preferably, the base composition of the plant culture is characterized by comprising base salts, vitamins, antioxidants, sugars, adsorbents and gelling agents.

[0021] More preferably, the base composition of the plant culture is characterized by the base salts being Hoagland salts or MS salts; the vitamins being B5G formula; the antioxidants being citric acid, ascorbic acid and polyvinylpyrrolidone (PVP); the sugars being sucrose; the adsorbents being activated carbon; and the gelling agents being agar and porcine skin gelatin.

[0022] The third aspect of the present invention also relates to a synthetic seed characterized by comprising (a) a container filled with the nutritional substrate composition as defined above; (b) a plant propagule or plant explant inserted into the nutritional substrate inside the container of step (a).

[0023] Preferably, the propagule or plant explant is a somatic embryo, zygotic embryo, meristem, sementes verdadeiras and / or totipotent plant explant.

[0024] Furthermore, preferably, the plant species can be selected from the group consisting of wheat, barley, maize, rice, oat, pasture grass, peat, miscanthus, sugar cane, soybean, soy, pea, tomato, rape, grape, potato, pineapple, strawberry, orange, cassava, tobacco and ornamental species. In particular, the plant species is a monocotyledon, more preferably sugar cane.

[0025] A fifth aspect of the present application relates to a method for growing a plant, characterized in that it comprises (a) in vitro culturing of a plant explant to produce plant propagules; (b) inserting the plant propagules of step (c) into a nutrient medium as defined above; (c) subjecting the nutrient medium of step (b) to in vitro field conditions; and (d) regenerating into adult plants.

[0026] Finally, the present application provides a sixth aspect, which relates to the use of a supplement composition as defined above, characterized in that it is for the manufacture of a nutrient medium for growing a plant. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 are the results of the percentage of germination (A); shoot length (cm) (B); and leaf area (cm 2 ) (C) after 21 days of in vitro culturing of embryogenic cell suspension (ES suspension).

[0028] Figure 2 are the visual aspects of a tray (A) from treatment 1 ; a tray plug (B) from treatment 1 ; a tray (C) from treatment 2; a tray plug (D) from treatment 2; a tray (E) from treatment 3; a tray plug (F) from treatment 3; a tray (G) from treatment 4; a tray plug (H) from treatment 4 after 21 days of in vitro culturing.

[0029] Figure 3 are the relationships between the leaf area and root area (cm 2 ) of embryogenic cell suspensions for each treatment after 21 days of in vitro culturing.

[0030] Figure 4 shows the visual appearance of leafy area evidence (C) and root area evidence (D) after 21 days of embryogenic cell aggregates in the nutrient medium of the present application supplemented with the supplement composition of treatment 13 after 7 days (A); after 14 days (B).

[0031] Figure 5 shows the shoot height (cm) (A); leaf area (cm 2(B); photosystem II quantum yield (Fv / Fm) (C) and pigment quantification (Chlorophyll a; Chlorophyll b and Carotenoids) (D). Means followed by the same letter are not different (p < 0.05) according to Scott-Knott test.

[0032] Figure 6 is the percentage of survival of plants from somatic embryo clusters in vitro cultured in the field at 21, 30, 45, 60 and 90 days after planting.

[0033] Figure 7 shows the survival rate (%) after 21, 30, 45 and 60 days (DAP) after planting ES clusters in the field. DETAILED DESCRIPTION

[0034] The growth of plants, organs, tissues and plant cells depends on the development of optimized media for the perfect interaction of components such as carbon sources, nutrients, minerals, nitrogen sources and growth regulators, which enable these tissues to maintain and develop into new adult plants.

[0035] The type of components and their concentrations in the growth medium can vary according to the purpose of the crop and there is no consensus in the literature about the best composition for each plant species and purpose.

[0036] For example, in plant culture of plant species in which micropropagation of asexual reproduction is desired, plant explants will be preferentially cultured in media containing de-differentiation stimuli to produce totipotent structures such as callus and / or somatic embryos, which will then be cultured into new plants under differentiation stimuli.

[0037] Generally, in indirect somatic embryogenesis processes, where explants are first transformed into callus for differentiation into somatic embryos for regeneration, the induction of calogênese can occur by the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D; auxin; WO9001058; Guiderdoni & Demarly, 1988, Plant Cell Tiss. Organ Cult. 90: 71 ; Ali et al., 2007, Pak. J. Bot. 39: 1961 ). 2,4-D is also the plant growth regulator of choice for direct somatic embryogenesis processes, used alone and often in combination with other hormonal agents (cytokinins, such as kinetin and BAP, IAI and GA3), where explants do not pass through a callus stage (Snyman et al., 2001, Acta Hortic 56: 105; Franklin et al., 2006, Plant Growth Regul. 50: 111 ; Behera & Sahoo, 2009, Nat. Sci. 7: 1 ). Different concentrations of 2,4-D have been reported and it is the hormonal agent used at different stages of the process, from the induction of callus to the beginning of the somatic embryo maturation stage (Naz et al., 2008; Jahangir et al., 2010). There is still no consensus on how to use this hormonal agent and many variations have been observed, including genotype dependence (Ho & Vasil, 1983; Brisibe et al., 1994, Jiménez, 2001 ).

[0038] In addition to auxin 2,4-D, other hormones are often used in in vitro micropropagation processes. Among them, naphthalene acetic acid (NAA) (Irvine et al., 1991, Plant Cell Tiss. Organ Cult. 26: 115; Lakshmanan et al., 2006, Plant Cell Rep. 25: 1007) and benzylaminopurine (BAP) (Ali et al., 2008, Pak. J. Bot. 40: 139; Ather et al., 2009, Pak. J. Bot. 41: 815) are efficient bud inducers. Indole butyric acid (IBA) (Khatri et al., 2002, Asin J. Plant Sci. 1: 41), gibberellic acid (GA3) (Ather et al., 2009, Pak. J. Bot. 41: 815; Dash et al., 2011, Asian J. Biotechnol. 3: 378) and NAA (Biradar et al., 2009, Karnataka J. Agric. Sci. 22: 21; Ali et al., 2010, Pak. J. Bot. 42: 3783) have been reported for rooting. Abscisic acid (ABA) is characterized as a desacelerador de crescimento, which has profound effects on embryogenesis, such as stimulating the differentiation of embryogenic callus (Kaur & Kapoor, 2016). The cytokinin zeatin ((E)-2-methyl-4-(7H-purin-6-ylamino)but-2- ene-1-ol) is an inducer of cell division, breaking of apical dominance, organogenesis in in vitro cultures, flowering and chloroplast development. It is widely used in plant explant regeneration media.

[0039] However, some of these compounds, such as benzyladenine (BA) and 2,4-D, address the problems of genetic alteration and abnormal growth in plants grown in vitro, while others, such as zeatin, are much more expensive than benzyladenine (BA) and make micropropagation difficult to scale up. As such, a cytokinin that is a substitute for those mentioned above, such as topolin, can be used.

[0040] Therefore, the present application describes a nutritional medium comprising at least a basal composition and a supplementary composition, which enables the culture, germination, regeneration, micropropagation, protection, storage, cryopreservation, rustification and acclimatization of plant explants and / or plant propagules, previously already or not in vitro cultured, conferring metabolic and physiological properties to them (such as robustness and root volume, increased leaf area, optimized photosynthetic system, among others), making them sufficient to resist, survive and develop in an in vitro environment under various environmental stresses.

[0041] The basal composition in the nutritional medium of the present application comprises, but is not limited to, basal salts, vitamins, antioxidants, carbon sources, adsorbents and gelling agents. The supplementary composition of the present application comprises at least, but not limited to, a combination of one or more cytokinins with melatonin, and optionally an amino acid composition and a silicon source. The cytokinin is preferentially topolin, more specifically m- topolin. In addition, the nutritional medium can comprise other compounds commonly used in culture media and synthetic seeds, such as, but not limited to, other plant growth regulators, antibiotics, antibacterials, antifungals, nematicides, fertilizers, herbicides, bioinoculantes, among others.

[0042] The basal salts in the basal composition in the nutritional medium of the present application can be any salt that provides the organic or inorganic micronutrients and macronutrients necessary for the maintenance, culture and development of plant explants. Examples of inorganic compounds are nitrogen (N), potassium (K), phosphorus (P), calcium (Ca), sulfur (S) and magnesium (Mg), which are usually required in millimolar amounts. The optimal concentration of each nutrient varies greatly depending on the plant species, type of crop, among others. Plant cells can grow with nitrate (NO -3 ) as the sole nitrogen source, however, the addition of ammonium salts (NH 4+ ) to the culture medium usually presents a beneficial effect, making it an essential nutrient for some species. Other reduced nitrogen sources can also be added to the culture medium. Nitrate is usually used at a concentration between 25 and 40 mM. The amount of ammonium salt can vary between 2 and 20 mM. Plant cells can grow in a culture medium in which the sole nitrogen source is ammonium salt, producing citrate, succinate, malate or other acids in the TCA cycle. Other nitrogen sources such as urea, glutamine or hydrolyzed casein, among others, can also be used to grow plant cells.

[0043] Preferably, the basal salt component in the nutritional medium of the present application is one of the MS salts and Hoagland's basal salts (or Hoagland's medium) (Hoagland & Arnon, 1950), aiming at the directional growth and stabilization of the osmotic potential of the culture medium.

[0044] Vitamins are essential for plant growth and development and are synthesized endogenously by plants. However, when plant cells or tissues are placed in in vitro media, some vitamins become limiting and need to be supplemented. For example, there is a minimum concentration of the vitamin thiamine and increasing it can be used to optimize growth and development of in vitro grown plant explants. Other vitamins that plants use for growth and that can constitute the base composition in the nutritional medium of the invention are niacin, pyridoxine, calcium pantothenate, biotin, p-amino-benzoic acid, riboflavin, ascorbic acid, citric acid, vitamin B6, folic acid, etc.

[0045] Preferably, the vitamins added to the base composition of the nutritional medium are the B5G formulation (Gamborg et al., 1968). The main difference between the widely used MS and B5 vitamin formulations is related to the different concentrations of the individual components. For example, the concentration of the vitamin thiamine is 10 times higher in the B5 solution. This fact can be related to the better performance of the nutritional medium supplemented with this vitamin complex, since thiamine is an essential cofactor for aerobic respiration enzymes in plants, playing a greater stimulatory role in growth and germination (Goyer, 2017).

[0046] The antioxidant present in the base composition of the nutritional medium of the invention can be any organic or inorganic compound that reacts with the metals present in the medium, preventing the metals from becoming available for oxidation (Matkowski, 2008). The antioxidant can be PVP (polyvinylpyrrolidone), which adsorbs the exudates released by the explants, which can cause oxidation of the plant material in the early stages of development (Matkowski, 2008), as well as citric and ascorbic acids, cysteine, niacin, glutathione, lipoic acid, salicylic acid, tocopherols, polyamines, etc.

[0047] The carbohydrate source in the medium is the direct energy supplier for cell growth before the plant acquires autotrophy. In addition, the carbohydrate source influences the development of the highly embryogenic cultures and can act as a morphogenetic agent and plant growth regulator (Pien et al., 2001).

[0048] The carbon source in the basic composition present in the nutritional medium of the present application can be any carbohydrate that can be used and metabolized by plant cells. In this regard, sucrose is the most commonly used carbohydrate in sugarcane micropropagation protocols (Lipavská, H. & Konrádová, H., 2004), although some studies report a positive influence on embryogenesis of cultures using maltose or corn syrup instead of sucrose (Gill et al., 2004; D2, Kaur & Kapoor, 2016). In addition to sucrose and maltose, mannitol stands out for its osmoregulatory properties, which are used in many studies to cause osmotic stress and induce the formation of somatic embryos (George et al., 2008).

[0049] Thus, the carbon source in the basic composition in the nutritional medium of the present application is preferably sucrose or a conventional sugar, but can also be glucose, fructose, maltose or a combination thereof. Other carbohydrates such as lactose, galactose, starch and sorbitol have also been tested and can also constitute the basic composition.

[0050] Adsorbents are responsible for adsorbing and reducing the availability of exogenous auxins in the culture medium and inducing the process of in vitro rooting (Pan & Van Staden, 1998). Adsorbents also act to control the influence of phenolic substances released by explants into the culture medium, which are usually associated with an increase in the embryogenic potential of these cultures (Kaur & Kapoor, 2016). These compounds can be, for example, activated carbon and graphite. Preferably, the basic composition in the nutritional medium of the present application comprises activated carbon.

[0051] Finally, the gelling agent can be all those gelling agents that are in a solid / gelled state at room temperature. The correct gelling agent must be chosen taking into account several properties, including: not containing large amounts of impurities that would unbalance the chemical and physical properties of the culture medium; maintaining a complexation that does not interfere with the availability of minerals in the culture medium and maintaining the "state" of water in the culture medium (substrate potential). In addition, by applying the concept of IPN (Interpenetrating Polymer Network) to the construction of the gelled medium, by mixing two gelling agents, there is greater durability at higher temperatures, while not limiting aeration. The most commonly used gelling agents in culture media are agar, gelatin, porcine gelatin, bovine gelatin, gelrite, agarose, phytagel, tapioca or corn starch, among others. Preferably, for the nutritional medium of the present application, the gelling agent consists of a combination of bacteriological agar and porcine gelatin.

[0052] Thus, the basic composition in the nutritional medium of the present application comprises, but is not limited to, the following components in the following concentration ranges:

[0053]

[0054] Preferably, the concentrations of each component of the base composition in the nutritional matrix of the present application can vary according to the following ranges:

[0055]

[0056] Furthermore, the base composition in the nutritional matrix of the present application can comprise casein hydrolysate, which not only acts as a nitrogen source, but also as a general adjuvant for growth, induction, maintenance of embryogenic potential and plant regeneration (Gandonou et al., 2005).

[0057] Furthermore, the base composition can comprise bioinoculants, biostimulants, fertilizers, plant growth regulators or / and phytohormones, growth-regulating compounds such as auxins, especially but not limited to auxin 2,4-D, naphthalene acetic acid (NAA), benzylaminopurine (BAP), indolebutyric acid (IBA), gibberellic acid (GA3), abscisic acid (ABA) and indoleacetic acid (IAA), antibiotics, antifungals, antibacterials, nematicides, herbicides, etc.

[0058] In the implementation of the present application, the base composition can also comprise rooting inducers such as: IAA (indoleacetic acid), phenolic compounds, TIBA (2,3,5-triiodobenzoic acid), synthetic anti-gibberellins and gibberellin biosynthesis inhibitors, naphthalene acetic acid (ANA) and indolebutyric acid (AIB).

[0059] The supplementary composition of the present application comprises at least one combination of a cytokinin and a melatonin, wherein the cytokinin is preferably a topolin.

[0060] According to Aremu et al. 2012, it is known that topolins modulate the amount of some secondary metabolites responsible for the adaptation and survival of plants to environmental changes, such as phenolic compounds. However, it has not yet been shown the type of topolin and its concentration, much less in combination with melatonin and at early stages of development, as is the case of the crops of the present application.

[0061] In this sense, the present application describes a supplementary composition comprising at least one topolin in combination with melatonin. The topolin is any one of m-topolin (mT), m-topolin riboside (mTR), m-methoxytopolin (MemT), m-methoxytopolin riboside (MemTR), m-methoxytopolin 9-tetrahydropyran-2-yl (MemTTHP) or an agriculturally stable salt thereof.

[0062] The concentration of topolin in the nutrient medium is in the range of 1 to 15 µM, more preferably in the range of 3.5 to 10 µM, even more preferably the concentration of topolin in the present invention is in the range of 4 to 7 µM. More specifically, the concentration of topolin is in the range of 5 to 6 µM, even more specifically the concentration of topolin is 5 µM.

[0063] Moreover, it is known that melatonin has some important biological effects on plants, such as maximizing / inducing root and shoot growth, activating seed germination and delaying induced leaf senescence. Exogenously, melatonin acts to reinforce plants that are undergoing heat stress (by inducing the expression of heat shock proteins HSP70 and HSP90) (Arnao & Hernandez-Ruiz, 2014; Nawaz et al., 2016). Nawaz et al. 2016 revealed that it is known that Poaceae and Brassicaceae have high concentrations of melatonin in the roots and that its concentration is influenced by genotypes and environmental factors. Moreover, the authors revealed that there are differences in the concentration of melatonin between different cultivars of the same species and that it is related to the germination of the seeds of the fruits. There are indications that low concentrations of exogenous melatonin improve the resistance of plants to water stress by enhancing the activity of antioxidant enzymes. For this reason, Nawaz et al. 2011 suggested that a good strategy for the future would be to coat seeds and fruits with this compound. Arnao & Hernandez-Ruiz et al. 2018 described that the biological functions of melatonin are indeed related to the formation of roots and the stimulation of the development of the area of the plants, but that such compounds in concentrations greater than 10 µM in the roots act in the opposite way, inhibiting plant growth.

[0064] However, there is no prior art document that indicates the optimal concentration of melatonin to achieve both effects: stimulation of growth / regeneration and greater adaptability and resistance to stress. Moreover, there is no report that growth media already containing cytokinins and other plant growth regulators can be supplemented with melatonin to increase / enhance the physiological characteristics responsible for the growth of plant explants and resistance.

[0065] Therefore, the supplementary composition of the present invention comprises at least one combination of a cytokinin, preferably topolin, more preferably m-topolin, with melatonin. Preferably, melatonin is present in a concentration in the range of 5 to 25 µM. More preferably, the concentration of melatonin is in the range of 8.5 µM to 22.5 µM, even more preferably the concentration of melatonin is in the range of 10 to 20 µM, even more particularly the concentration of melatonin is in the range of 10 µM to 15 µM, and even more specifically the concentration of melatonin is 10 µM.

[0066] Moreover, the supplementary composition of the present invention can comprise other compounds, such as, but not limited to, specific amino acids and silicon sources.

[0067] The amino acid mixture contributes to the detoxification of reactive oxygen species (ROS), the protection of membrane integrity and the stabilization of enzymes / proteins under stress. The mixture can include any of the following: valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartate, glutamate, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, serine. In addition, the amino acids can come from a complex amino acid source such as, but not limited to, casein hydrolysate, yeast extract, BSA, amines and organic acids.

[0068] For the present invention, the combination of the proteogenic amino acid L-proline, necessary for accelerating metabolism and growth (Franzoni et al., 2019), with L-alanine and glycine enhances the technical effect of the composition for growth supplementation and greater tolerance to stress in the plant explants in culture.

[0069] Finally, the supplementation composition can comprise a silicon source, silicon being a chemical element involved in functions related to the structuring of the cell walls of the roots and leaves, thus favoring photosynthesis, increasing the total chlorophyll content and providing more erect leaves, maximizing their architecture due to the accumulation of cellulose, hemicellulose and lignin in the cells (Epstein, 1999).

[0070] These properties are desirable because they increase the survival rate during the acclimatization phase of micropropagated plants, since during this phase there is greater water loss due to the low functionality of the stomata and the thin layer of cuticular wax (Silva, 2007).

[0071] In addition, the silicon source acts to reduce transpiration and increase the resistance of the plant to the attack of pathogens, since the silicon deposited in the cell wall in the form of amorphous silicon dioxide can act as a mechanical barrier to fungal invasion.

[0072] Thus, the silicon source in the supplementation composition of the present invention can be any silicate (magnesium, calcium, aluminum, iron, manganese, etc.), preferably potassium silicate.

[0073] Thus, in addition to topolin, more specifically meta-topolin, and melatonin, the supplementation composition can also comprise, but not limited to: potassium silicate, proline, alanine and glycine.

[0074] Potassium silicate is present in a concentration ranging from 1 to 10 µM, preferably ranging from 2 to 8 µM, more preferably ranging from 4 to 6 µM. Proline is present in a concentration ranging from 0.1 to 10 mM, preferably ranging from 0.1 to 2 mM, more preferably ranging from 0.2 to 2 mM. Alanine is present in a concentration ranging from 0.05 to 5 mM, preferably ranging from 0.1 to 2 mM, more preferably ranging from 0.1 to 0.8 mM. Glycine is present in a concentration ranging from 0.1 to 4 mM, preferably ranging from 0.3 to 2.5 mM, more preferably ranging from 0.4 to 2 mM.

[0075] In one embodiment of the present application, the nutrient medium can be placed in any container, tube, package, capsule, etc. for the formation of artificial seeds, wherein explants or plant propagules are placed for culture, regeneration, storage, hardening-off, acclimatization and other purposes. Preferably, any container is filled with the nutrient medium of the present application, wherein plant propagules are inserted into the nutrient medium inside the container, forming artificial seeds.

[0076] Another embodiment of the present application comprises a method for culturing, regenerating, storing, hardening-off and acclimatizing plant explants or propagules for direct transfer to an external in vitro environment, wherein abiotic stresses can be encountered.

[0077] More preferably, the method of the present application is aimed at culturing plant propagules and comprises the following steps:

[0078] (a) in vitro culturing of plant explants to produce plant propagules;

[0079] (b) inserting plant propagules from step (a) into the nutrient medium of the present application;

[0080] (c) placing the nutrient medium from step (b) under in vitro field conditions; and

[0081] (d) regeneration of adult plants.

[0082] Thus, the present application describes a nutrient medium characterized by comprising a base composition for growing plants and a supplementary composition, wherein the supplementary composition comprises at least meta-topolin and melatonin. A synthetic seed is also described, comprising a container filled with the nutrient medium of the present application and a plant propagule inserted into the nutrient medium inside the container. Furthermore, the present application describes a plant culturing method comprising in vitro culturing of plant explants to produce plant propagules and inserting / placing them into the nutrient medium of the present application, followed by placing this nutrient medium containing plant propagules under in vitro field conditions for regeneration of adult plants. Finally, the present application uses the supplementary composition of the present application to manufacture a nutrient medium for growing plants.

[0083] A "plant propagule" is any plant tissue that has the capacity or potential to regenerate into a mature plant. Plant propagules can be leaf tissue, meristematic tissue, embryonic tissue, tissue from a true seed, terminal bud, root bud, cotyledon, hypocotyl, etc. Preferably, the plant propagules of the present application are calli that have been previously cultured in vitro under callus induction conditions, or are embryogenic tissues that have or have not been previously cultured in vitro to become embryogenic.

[0084] In the present application, the plant propagules can be obtained from a gymnosperm or an angiosperm of the monocotyledonous or dicotyledonous class, preferably from a monocotyledonous plant. Even more preferred are monocotyledonous plants from the family Poaceae. Preferably, the plant of interest is a monocotyledonous plant. Even more interestingly, the plant of choice is sugarcane (Saccharum spp.), which can be a natural sugarcane plant, an improved variety, a genetically modified sugarcane plant (by genome editing or conventional transgenesis), etc.

[0085] A "plant explant" is any plant tissue that has been directly excised from any part of a plant, and differs from a "plant propagule" in that it does not necessarily have inherent regenerative capacity. For example, a leaf explant is a plant explant according to the present application, but it is not a plant propagule because it does not yet have the capacity to directly regenerate into a mature plant. Every plant propagule of the present application is a plant explant, but not every plant explant is a plant propagule according to the present application.

[0086] Regeneration refers to the process of development of a plant propagule or explant into a mature plant in vitro or ex vivo.

[0087] "Ex vivo conditions" refer to environmental water, light and other conditions existing in an environment with little or no control, such as a greenhouse, a plant house, a field, etc.

[0088] Example

[0089] Example 1. Testing different ranges of melatonin concentrations to accelerate plant germination or development, optimize root formation, and increase... Resistance to abiotic stresses.

[0090] In these experiments, aggregates of somatic embryos from sugarcane variety 1 of the embryogenic callus induction pathway were used. Newly matured somatic embryo clusters were selected for testing different concentrations of melatonin added to basal nutrient solution 1 (Table 1)

[0091] Table 1 - Composition of basal medium 1

[0092]

[0093] Treatments 1 to 4 consisted of different concentrations of melatonin: 0, 5, 10, and 30 µM, which were directly microfiltered (0.22 µmol pore size) into the nutrient solution after autoclaving.

[0094] Subsequently, each treatment was poured into a tray with 126 wells (0.9 mL per well), where each replicate consisted of a column with 8 wells, each well containing a somatic embryonic cluster, for a total of 14 replicates per tray and 3 trays per treatment. The trays were placed in a controlled growth chamber at 28°C and maintained for 21 days.

[0095] The variables analyzed were the germination percentage, green area percentage, and root volume, calculated using an image analysis system after 21 days of in vitro culture.

[0096] Following this period, the gelled nutrient substrates for each treatment were placed in plastic containers and stored in a greenhouse to assess the survival rate, shoot length (cm), and leaf area (cm²) of plants derived from cultured somatic embryos after 30 days of in vitro culture. 2 ), number of tillers, chlorophyll a, chlorophyll b and carotenoids.

[0097] Significant differences were found in germination variables analyzed after 21 days of in vitro culture, with the highest germination percentage (90%) observed at a concentration of 10 µM melatonin (treatment 3). Figure 1 C), in which leaf and root growth is more uniform ( Figure 2 For the variables analyzed after 30 days of in vitro culture in a greenhouse, the bud length (0.9 cm) in treatment 3 (10 µM melatonin) was [missing value]. Figure 1 B) and leaf area (45 cm²) 2 () Figure 1 C) are also the highest.

[0098] These results indicate that the use of 10 µM melatonin in the supplemental composition optimized in vitro development, and that germinating plants exhibited enhanced adaptive systemic responses to impending or future abiotic stresses in the field.

[0099] Example 2. Optimizing the composition by supplementing with specific amino acids.

[0100] Regarding the formulation of the basal nutrient solution, Hoagland's salts (Hoagland & Arnon, 1950); the vitamin formulation B5G (Gamborg et al., 1968), and the antioxidant PVP (polyvinylpyrrolidone); citric acid and ascorbic acid were pre-selected for their excellent performance in all the previously tested treatments (they produced the medium 1). However, the previously tested concentration ranges for the supplements: the aromatic cytokinin inter-topolin; the melatonin (N-acetyl-5-methoxytryptamine) and the amino acids (proline, glycine and alanine) should be extended to further enhance the plant resistance to the simulated water stress in the greenhouse.

[0101] The experiment used somatic embryo clusters from sugarcane variety 1. Newly mature somatic embryo clusters were selected for testing.

[0102] The formulations of the tested supplement compositions are those described in Table 2 for the supplement of the medium 1 (Example 1).

[0103] Table 2 - Concentration and composition of the compositions added to the basal medium of the nutrient medium 1.

[0104]

[0105] The in vitro experiment consisted of 6 replicates per treatment, where each replicate consisted of a Petri dish (100 x 20) with 5 somatic embryo clusters per dish. The variables evaluated in vitro were: the percentage of germination calculated using an image analysis system after 21 days of culture; the leaf area and the root area. After this period, the plant individuals from each treatment were individualized and selected as 6 replicates per treatment, with 5 plants per replicate, which were transplanted into 25-hole plastic pots filled with wet substrate (peat moss peat pH 5.8 EC 0.3) and stored in a greenhouse for the evaluation of survival, shoot length (cm), leaf area (cm 2 ), number of tillers and quantification of chlorophyll a, chlorophyll b and carotenoids after 30 days of in vitro culture.

[0106] After the period of this bioassay evaluation in the greenhouse, the continuous water deficit of 96 hours, followed by hydration (process repeated 4 times) was initiated, according to Pereira et al. (2005), without irrigation, adjusted according to the proportion of available water.

[0107] Water stress was monitored using a portable modulated fluorimeter (Opti-Sciences, model OSI-FL, Hudson, USA) to measure initial fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv) and maximum quantum yield of PSII (Fv / Fm) according to the portable modulated fluorimeter after the leaves had been adapted to the dark (H" 30 min). The measurements were taken before the beginning of the water deficit period and after each rehydration phase, always between 9 and 11 a.m., thus reflecting the instantaneous state of the photosynthetic apparatus. Two readings were taken for each plant, always in the middle of the +2 leaves.

[0108] During the experimental period, the environmental conditions inside the greenhouse were measured daily with a hygrometer, represented by the average temperature and the average relative humidity, between 29.2 + / - 3°C and 63.4 + / - 5%. The experimental design was completely randomized. The analysis of variance was performed using the SISVAR statistical software (Ferreira, 2014) and the means were compared at 5% probability using the Tukey test.

[0109] Treatment 13 (1 mM proline; 0.4 mM alanine; 2 mM glycine; 20 µM melatonin and 5 µM m-tubulin) stood out in terms of uniformidade between leaf area and root area ( Figure 3 ). This result is important because the use of this treatment can optimize the production of synthetic seeds, since the somatic embryo aggregates should show root growth, but at the same time show efficiency and standardization of the leaf system before being inserted into the pre-established prototype. In Figure 4 the visual aspect of this treatment is shown.

[0110] Example 4. Optimizing the composition of potassium silicate supplements

[0111] Similarly to Examples 2 and 3, the presence of silicon in the supplementation solution was also tested in vitro. The test used ES clusters from sugarcane variety 1. New mature somatic embryo clusters were selected and previously dried. The base medium tested was Medium 1 containing different concentrations of potassium silicate (K2Si03) (0, 2, 4 and 6 µM), which, after autoclaving, was microfiltered (pore size 0.22 µmol) directly into the nutrient solution.

[0112] Subsequently, each treatment was poured into Petri dishes (100 x 20), in which each repetition consisted of a dish with 10 somatic embryo clusters per dish, totaling 50 repetitions per concentration. After inoculation, the plates were kept in a conventional growth chamber, with a 16-hour photoperiod and a light intensity of 82.5 W m -2 s -1 provided by white fluorescent lamps, at a temperature of 27 ± 2°C.

[0113] After this period, the germination percentage of the clusters in each treatment was evaluated. Subsequently, plug seedlings were selected and inserted into plastic containers (i.e., artificial seeds), with 20 replicates in each treatment, planted in 5 L pots in a greenhouse.

[0114] The evaluations were performed on days 21, 30, 45 and 60 and the following parameters were measured: percentage of survival, length of the sprout (cm), leaf area (cm 2 ), Fv / Fm and quantification of chlorophyll a, chlorophyll b and carotenoids. Root analysis data were obtained using the WinRHIZO root analysis system. Anatomical analysis was performed on the +1 leaf collected and embedded at 30 and 90 DAP and the following anatomical characteristics were analyzed under an optical microscope: thickness of palisade parenchyma and spongy parenchyma (leaf margin characteristics).

[0115] The treatments tested are described below:

[0116]

[0117] Significant differences were observed in the germination variables analyzed after 21 days of in vitro culture. K2SiO3 at concentrations of 2 µM and 4 µM did not differ from the control treatment, only K2SiO3 at a concentration of 6 µM showed phytotoxicity in the ES clusters, resulting in greater oxidation in the material.

[0118] For the survival variable analyzed on days 21, 30, 45, 60 and 90 after planting the artificial seeds in the greenhouse, the material showed high survival percentages in treatments 1, 2 and 3 ( Figure 5 ). It can also be seen that, throughout the evaluations, treatment 2 showed greater stability in terms of plant survival.

[0119] However, for the plant height variable, treatments 2 and 3 had the highest average values (Table 3), which can be explained by the fact that the concentration of K2SiO3 tested has a direct impact on the cell structure of the leaves, which allows for greater leaf angle, directly reflecting the length of the sprout.

[0120] Table 3: Height of the sprout (cm) on days 21, 30, 45, 60 and 90 after planting the artificial seeds. According to the Scott-Knott test, there was no difference between the average values followed by the same letter (p-value < 0.05).

[0121]

[0122] Other parameters were only analyzed on day 30 ( Figure 7), it can be seen that the height, leaf area and Fv / Fm of treatments 2 and 3 were significantly different from the control.

[0123] The results show that treatments 2 and 3, which received K2SiO3 at each concentration, had a higher level of chlorophyll b, which would result in greater field adaptation, since the amount and quality of light is constantly changing, favoring the performance of the plant.

[0124] Finally, the results of the root analysis can be seen in Table 4 below and in Figure 6 .

[0125] Table 4 - Data of the variables length (cm), average diameter (mm) and volume (cm3) of the roots of 90 DAP extracted from the WinRhizo system. The images were colored in the software so that it was easier to see the main root (blue), secondary roots and root hairs (red and yellow). According to the Scott-Knott test, the means followed by the same letter do not differ (p-value < 0.05). 3

[0126]

[0127] It can be seen that treatments 2 and 3 had the greatest root length, average diameter and volume. The images show that the robustness of the roots can be attributed to the incorporation of silicon into the cells.

[0128] Regarding the anatomical characteristics, it was observed that the palisade parenchyma cells presented, on average, one layer in the control treatment, two layers in treatment 2 and three to four layers in treatment 3 (data not shown).

[0129] The spongy parenchyma cells present the intercellular space characteristics of leaves grown in high humidity environments. The greater thickness of these leaf margin tissues, such as parenchyma, gives the plant a greater chance of survival during the transfer from the in vivo environment to the in vitro environment, resulting in higher photosynthetic activity, which is an important factor for successful transplantation (Braga et al., 2009).

[0130] Thus, it can be concluded that the supplementation of 4 µM potassium silicate in the supplementing composition of the present application enables a high percentage of in vitro germination and improves the leaf and root structure of the plant, contributing to the maximum enhancement of the resistance of the propagule after exposure to in vitro conditions in the field.

[0131] Example 4. In vitro acclimation of sugarcane somatic embryonic clusters (ES) with and without supplemental composition. Comparison of the nutrient substrates of the present invention

[0132] ​For these experiments, medium 2 (a medium having the supplementary composition of the present invention) and medium 3 (similar to medium 2 but without the supplementary composition) were used. Medium 3 represents a formulation commonly reported in the prior art for plant cell regeneration and / or in vitro culture processes, while medium 2 of the present invention has a supplementary composition added to its composition consisting of L-proline (1 mM), L-alanine (0.4 mM), glycine (2 mM), cytokinin-toporpine (5 µM), melatonin (10 µM), and potassium silicate (3 µM).

[0133]

[0134] Plant gels are only used for gelation media 3.

[0135] The experiment consisted of three treatments: the first treatment used medium 2, the second treatment used gelled medium 3, and the last treatment used the same medium 3 but in liquid form. (All treatments used fresh, mature, and dried ES sugarcane from the RB7515WT variety.)

[0136] Treatments 1 and 2 were poured into Petri dishes (100 × 20), with each replicate consisting of a dish containing 10 ES clusters, for a total of 30 replicates per treatment. Treatment 3, on the other hand, was performed by moistening sterile filter paper with liquid medium 3 on flip-top plates (placas modelo clamshells) for approximately 21 days. After inoculation of the ES clusters, the plates and flip-tops were stored in a standard growth chamber under a 16-hour photocycle and 82.5 W m² light provided by a white fluorescent lamp. -2 s -1 The light intensity at a temperature of 27±2℃.

[0137] Following this period, seedling trays and plants were selected and inserted into predefined containers, 100 replicates per treatment, and planted in the field at Plot 21. Survival rates were assessed at 21, 30, 45, and 60 days.

[0138] It can be seen that the difference between the treatments used was not in the germination rate of somatic embryonic clusters (data not shown), but in the survival rate of the material, which was assessed at 21, 30, 45 and 60 days after planting in the field. Figure 7 ).

[0139] Medium 2 from treatment 1 provided substantially better survival of somatic embryonic clusters under stressful field conditions, and plants regenerated from this material showed higher vigor.

[0140] These data show that supplementation with medium 2 allows for stronger in vitro hardening of somatic embryonic clusters during the transition from controlled in vitro culture to the external environment, where more demanding conditions are imposed on plant development and sufficient morphophysiological adaptations are required to resist stress.

[0141] Therefore, it is evident that the supplementary composition contained in nutrient matrix 2 exhibits superior technical performance compared to matrix 3. This supplement provides enhanced in vitro hardening and adaptability when sugarcane somatic embryoclusters are transferred directly from in vitro culture to the ex vivo environment (field). This is because this supplement particularly allows for optimization of the formation of more robust root systems, which can enhance systemic resistance to immediate stresses from in vitro culture and stresses faced in vitro (especially water).

[0142] The examples described represent the preferred scope, and it should be understood that the scope of the invention covers other possible variations and is limited only by the content of the claims, including possible equivalents.

Claims

1. A supplement composition characterized in that, The supplementary composition comprises a combination of meta-topolin and melatonin.

2. The composition of claim 1, wherein, The concentration of melatonin is 5 to 25 µM, preferably 10 µM to 15 µM, even more preferably the concentration of melatonin is 10 µM and the concentration of meta-topolin is 3.5 to 7 µM, preferably 4 to 6 µM, even more preferably the concentration of meta-topolin is 5 µM.

3. The composition according to any one of claims 1 to 2, characterized in that, The composition comprises L-alanine, L-proline and glycine and potassium silicate.

4. Nutrient medium composition, characterized in that, The nutrient medium composition comprises: a) a supplementary composition as defined in any one of claims 1 to 3; and b) a basal composition for plant culture.

5. The nutrient medium composition according to claim 4, characterized in that, The basal composition for growing plants comprises basal salts, vitamins, antioxidants, sugars, adsorbents and gelling agents.

6. Nutrient medium composition according to any one of claims 4 to 5, characterized in that, The basal salts are Hoagland salts or MS salts; the vitamins are B5G formula; the antioxidants are citric acid, ascorbic acid and polyvinylpyrrolidone (PVP); the sugars are sucrose; the adsorbents are activated charcoal; and the gelling agents are agar and porcine skin gelatin.

7. Synthetic seed, characterized in that, The synthetic seed comprises: a) a container filled with a nutrient medium composition as defined in any one of claims 4 to 6; and b) a plant propagule or a plant explant inserted into the nutrient medium inside the container from step (a).

8. The synthetic seed of claim 7, wherein, The plant propagule or explant is a somatic embryo, a zygotic embryo, a meristem, a true seed and / or a totipotent plant explant.

9. The synthetic seed of any one of claims 7 to 8, wherein, The plant species selected is wheat, barley, maize, rice, oat, grass, peat, miscanthus, sugar cane, soybean, Glycine max, pea, tomato, rape, grape, potato, pineapple, strawberry, orange, cassava, tobacco and ornamental species.

10. The synthetic seed of claim 9, wherein, The plant species is a monocotyledon, more preferably sugar cane.

11. A method for plant growth, characterized by, The method for growing plants comprises: a) in vitro culturing a plant explant to produce a plant propagule; b) inserting the plant propagule from step (a) into a nutrient medium as defined in any one of claims 4 to 6; c) placing the nutrient medium from step (b) under in vitro field conditions; d) regeneration of adult plants.

12. Use of a complementary composition as defined in any one of claims 1 to 3, characterized in that, The supplementary composition is used for the manufacture of a nutrient medium for growing plants.

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