Method for regenerating plants by means of somatic embryogenesis, applicable to gymnosperms and angiosperms

Abstract

The invention relates to a new method for embryo maturation, applicable to gymnosperms and angiosperms, the embryo maturation being previously induced in embryonic tissue or competent cells of the selected genotypes or specimens to generate complete plants. The method comprises complete embryo maturation of more than 90% of the treated immature embryos or competent cells. This efficiency is provided by applying proteins obtained from somatic or zygotic embryos alone, or together with maturation-promoting proteins, in a culture medium supplemented with nitrate (100%) or in combination with an ammonia source, one or more carbon sources, and maturation promoters such as abscisic acid (ABA) or analogues thereof.

Description

[0001] PLANT REGENERATION PROCESS VIA SOMATIC EMBRYOGENESIS APPLICABLE TO GYMNOSPERMS AND ANGIOSPERMS

[0002] FIELD OF INVENTION

[0003] The invention relates to a novel method for carrying out the regeneration process with genetic diversity applicable to angiosperms and gymnosperms; which allows regeneration by somatic embryogenesis of plants of all types of higher species, via stimulation with maturation-inducing proteins obtained from genotypes with mass production of somatic embryos; through stimulation with maturation-promoting proteins and a medium supplemented with nitrate as the main source of nitrogen, assimilable carbon sources, and a promoter that signals maturation or its analogues.

[0004] BACKGROUND OF THE INVENTION

[0005] The propagation of commercially available gymnosperms and angiosperms is increasingly important to meet the demand for products for human society. Conventional seed propagation has its drawbacks when it comes to mass propagation of desirable genotypes. To this end, asexual reproduction processes are being innovated to propagate commercial plants that are otherwise unfeasible. To this end, using simple selection techniques, only those plants that demonstrate homogeneity in their growth and development characteristics are propagated.Protocols have been invented to regenerate a large number of species, however, only a few ensure the mass production of specimens with genetic diversity (Ramírez-Serrano, 2001; Ramírez-Serrano and Soltero 2007), or the simple maintenance of the embryogenic capacity of genotypes (Ramírez-Serrano, 2001; Ramírez-Serrano, González-Álvarez and Pelayo Ortiz 2003); despite the advances, there is no widely known method that can be applied to the regeneration of higher plants using somatic embryos. Hence the importance of knowing the promoters or inducers of zygotic embryogenesis at all its stages, which is homologous to somatic embryogenesis.That according to the research carried out are several types of proteins that promote the maturation of zygotic embryos, where energy sources are not limited, contrary to the availability in the media formulated for the maturation of somatic embryos, because they must store sufficient energy for germination, which under in vitro conditions, is limited by the low efficiency in the generation of required proteins, which is improved by the addition of amino acids (Nuutila et al., 2000), however they are only required in the first stages of maturation. Or specifically, the culture medium is supplemented with arabinogalactans (promoter proteins), to stimulate the development of somatic embryos in Picea abies (Egertsdotter and von Arnold, 1995). Regarding the induction of maturation, the genes that determine it are known (Yu and Paulis, 1993); likewise, it was proven that the change from totipotent to competent cells in Chichorium sp., is through 15 pro-embryogenic polypeptides; differences in the proliferation of embryogenic and non-competent cells have also been established given by the metabolism of polypeptides with a weight of 45 to 55 kDa, as in Oriza sativa L. (Chen and Luthe 1987), Zea mays L. (Franz et al. 1989) and Daucus carota L. (Sung and Okimoto 1981); in this last species it was found that also proteins called arabinocalactans (AGPs) are decisive for the maturation of embryos (Kreuger and Van Horst, 1993; Toonen et al., 1997), these results agree with other extracellular proteins determined chitinases (De Jong et al., 1992), with a similar effect phytosulfoquin-α (Kobayashi et al. 1999). However, malformations and inability to germinate occur.Therefore it is considered that the nitrogen source is crucial, nitrate is the solution to this problem, due to its absorption process that allows it to be stored in the vacuoles, and subsequently metabolized into ammonium, which does not cause toxicity when there is abundance of the latter source within the cell, and in this way produce the proteins required for germination, which is essential to obtain a complete plant with the ability to absorb water and mineral salts and perform photosynthesis, demonstrated with the somatic embryos of Pinus sylvestris L. obtained in a medium with a low ratio of ammonium to nitrate (Ramírez-Serrano, 2003) due to its storage in the vacuoles prior to its conversion into ammonium; previously said storage was established in leaves by Martinoia and Collaborators, (1981) and in roots by van der Leich et al.(1998), later, Crowford (1995) determined its importance as a nutrient and signal for plant growth, its storage in the vacuole, as well as its metabolism of nitrite to ammonium in the plastid, it is also the signal to increase the expression of nitrate reductase, nitrite reductase and Ntr genes; where it is decisive along with the addition of carbon sources in order to provide sufficient energy for the production or metabolism of the rest of the primary metabolites, to be stored in the cells of the embryo. Supplementation with abscisic acid (ABA) is also required as a regulator of protein production and storage in the terminal phase of embryo maturation.

[0006] The objective of this protocol was to innovate the known methodology, in order to obtain the maturation of embryos from a diverse range of genotypes or selected specimens of higher plants, applicable to gymnosperms and angiosperms.

[0007] BRIEF DESCRIPTION OF THE INVENTION

[0008] According to the present invention, there is a somatic embryo maturation process that can be applied to higher plants in the plant kingdom (gymnosperms and angiosperms). This is achieved through treatment with proteins that have been determined as inducers or promoters of maturation, even in recalcitrant lines, which induce the maturation of immature embryos or incompetent cells that do not respond to previous maturation stimuli. Also, through supplementation with nitrogen sources such that it can be stored in the vacuoles and under no circumstances is there toxicity in the cells due to excess ammonium, which is crucial to trigger the maturation of previously induced and programmed embryos according to the taxon of plant organism.This medium is also supplemented with a carbon source, which must provide the energy to metabolize the remaining primary metabolites required to be stored as reserves in the embryo for germination; also a maturation promoter, which is a phytohormone or its analogues that signals to promote nutrient storage in the embryo's cells; it should be noted that this process does not address the induction, proliferation, and maintenance of the embryogenic capacity of competent cells or embryos stimulated to mature. This process alone allows for the maturation of more than 90% of treated genotypes or specimens belonging to 95% of the selected parents.

[0009] The terminology protein treatment is to embed immature embryos or competent cells or in the process of changing from totipotent to competent in a solution obtained from embryos in the early stages of maturation, which contain the promoter proteins, in addition to the "supplementation with nitrogen sources" refers in this process of somatic embryogenesis to the use of one or two sources of nitrate, or in combination with an ammonia source, so that it can be stored in the vacuoles as nitrate to later metabolize it in the ammonia source for the production of amino acids, which is encouraged by the energy of the carbon source (maltose, fructose, sucrose, starch), which allows the late maturation promoter (ABA or its analogues) to be activated and the signal is maintained, production and storage of primary metabolites in the cell, and in case of embedding immature embryos or recalcitrant cells to the previous stimuli,via stimulation with metabolized or analogous proteins to induce embryo maturation or generate competent cells to develop a mature embryo with the capacity to germinate and become complete autotrophic plants.

[0010] DRAWINGS

[0011] Figure represents a pre-cotyledonary Gymnosperm somatic embryo (Ep).

[0012] Figure Ib represents a mature Gymnosperm somatic embryo (EM).

[0013] Figure 1c represents a mature somatic embryo mass (MEM).

[0014] Figure Id represents maturing embryos of Angiosperm originating from reprogrammed somatic cells (Ei).

[0015] Figurale represents germinated somatic embryos, originating from competent somatic cells of angiosperm (EG).

[0016] Figure 2a represents the germination of somatic embryos of Gymnosperm (EMG).

[0017] Figure 2b represents the development of Gymnosperm seedlings under in vitro conditions as pre-adaptation prior to transplantation to normal environmental conditions (stage not treated in this application) (Pl).

[0018] Figure 2c represents the development of mature Angiosperm somatic embryos to seedlings under in vitro conditions (Pl).

[0019] Figure 2d depicts the development of adapted plants from mature somatic embryos of Angiosperm (stage not addressed in this application) (Pía). DETAILED DESCRIPTION OF THE INVENTION

[0020] According to the present invention, there is a somatic embryo maturation process that can be applied to higher plants in the plant kingdom (gymnosperms and angiosperms). This maturation process is promoted by treatment with proteins obtained from the lysis of immature embryos (zygotic and somatic), plus supplementation with promoter proteins such as arabinogalactans, chitinases, and extracellular proteins from somatic embryos.The above is obtained via nitrogen supplementation in such a way that it can be stored in the vacuoles and under no circumstances promote a high availability of ammonium, which is not stored, because it is metabolized into amino acids, therefore, its excess causes toxicity in the cells; for which it is supplemented with one or two sources of nitrogen, in the second case, the ratio of ammonium to nitrate that does not produce toxicity, which can vary from 0 / 100 to 49 / 51, where the compounds can be calcium nitrate, potassium nitrate, ammonium nitrate; which is crucial to trigger the maturation of previously induced and programmed embryos according to the taxon of plant organism (Figures a to l).This medium is also supplemented with a carbon source, which must provide the energy to metabolize the rest of the primary metabolites required to be stored as reserves in the embryo for germination, such as maltose, sucrose, fructose, glucose, starch in an amount that can vary from 10 to 50 g / l, individually or in a mixture; also a maturation promoter, which is a phytohormone such as ABA and / or its analogues, which, at a concentration or dose of 20 pM to 100 pM, signal to promote nutrient storage in the embryo's cells; it should be noted that this promoter does not work with recalcitrant seeds because they lack this promoter evolutionarily; and to stimulate immature embryos or recalcitrant competent cells, which do not respond to previous stimuli for maturation, they are embedded with proteins considered to inducers or promoters of embryo maturation or cells recalcitrant to previous stimuli.It should be noted that this process does not address the induction, proliferation, or maintenance of the embryogenic capacity of competent cells or embryos stimulated to mature. This allows for the maturation of more than 90% of the treated genotypes or specimens belonging to the 100% of the selected progenitors.

[0021] The present invention requires understanding and controlling critical physiological factors that affect the proliferation and maturation of somatic embryos that are homologous to zygotic embryos; also, nitrogen assimilation under in vitro conditions, which is one of the keys to the effectiveness of this formula, which can be applied to higher plants, applicable to both gymnosperms and angiosperms.

[0022] Zygotic embryogenesis in gymnosperms occurs by simple fertilization within a completely female structure (female gametophyte). Subsequently, a pre-embryonic structure of 16 elongated cells develops in the archegonium, which can generate a clone, thus generating zygotic polyembryogenesis in gymnosperms. In contrast, in angiosperms, fertilization is double, carried out by two pollen grains, one fertilizing the ovule and the second uniting with a binucleate cell to generate the protection of the embryo, two cotyledons or the endosperm, which is an evolutionary strategy to ensure energy expenditure in a seed; double fertilization occurs; without this event, no structure is generated; however, in gymnosperms there may be seedless cones and / or with empty gametophytes, which can develop into a seed, paradigmatically without an embryo, energy spent for no purpose.And from a biotechnological perspective, complete seeds cannot be generated in vitro by their nature, never a female gametophyte (gymnosperms), nor the cotyledons or endosperm (angiosperms), which are structures united but independent of the embryo when they are generated, in any case. On the other hand, the assimilation of nitrogen is crucial, hence the importance of inhibiting its absorption as ammonium, which enters directly into the amino acid synthesis pathway, because it cannot be stored and in excess produces toxicity; quite the opposite of nitrate, which must be metabolized to ammonium, but can be stored in the vacuoles, which explains why nitrogen formulation is crucial for the maturation of embryos in gymnosperms and angiosperms, which have the same assimilation and absorption of nitrogen.

[0023] Therefore, the terminology "modified formula with nitrogen sources" is used, which refers in this process of somatic embryogenesis to the use of a single source, or two sources of nitrate; or nitrate in combination with an ammonia source, in such a way that it can be stored in the vacuoles as nitrate to later metabolize it in the ammonia source for the production of amino acids, which is encouraged by the energy of the carbon source (maltose, fructose, sucrose, glucose, starch), in such a way that it is activated and maintained by the maturation promoter signal that can be ABA and its analogues, for the production and storage of primary metabolites in the cell, and in the case of embedding immature embryos or cells that require stimulation with exuded proteins such as arabinogalactans or analogues, chitinases and their analogues, by mature embryos to stimulate the production of primary metabolites.

[0024] This invention has several important features. One or more nitrate sources are used: the use of proteins metabolized by immature and mature embryos, when embryos of genotypes with a low response to maturation are soaked in this protein solution to induce it; nitrogen sources, which can be supplemented in a single compound such as nitrate or potassium nitrate, or either of the two in combination with ammonium nitrate, to promote its storage, and subsequent transformation into ammonium for the metabolism of amino acids and proteins, according to the growth of the embryo, such that the carbon sources that can be maltose, fructose, glucose, sucrose, starch, are the energy source, both combined when required in embryos or competent cells, to the activity of the maturation promoter ABA or its analogs.It should be noted that for embryos of recalcitrant species this phytohormone is not a promoter, since it does not germinate "per se", but grows from fertilization to senescence.

[0025] The term “embryo maturation” is the application of this process to immature gymnosperm embryos or competent cells programmed to generate complete plants, which germinate under in vitro conditions (Figures 2a to 2c), and adapt to normal environmental conditions (Figure Id), not addressed in this application.

[0026] REFERENCES

[0027] Boyer, C., Hilbert, JL and Vasseur, J.1993. Embryogenesis-related protein synthesis and accumulation during early acquisition of somatic embryogenesis competence in Chichorium. Plant Sci 93:41-53.

[0028] Chen, L. and Luthe, D.1987. Analysis of proteins from embryogenic and non-embryogenic rice (Oryza sativa L.) calli. Plant Sci 48:181-188.

[0029] Crowford, N.M.1995. Nitrate: Nutrient and Signal for Plant Growth. The Plant Cell 7: 859-868.

[0030] De Jong, A.J., Cordewener, J., LoSchiavo, F., Terzi, M., Vandekerckhove, J. y DeVruesm S.C.1992. A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4: 425-433.

[0031] Egertsdotter, U. y von Arnold, S.1998. Development of somatic embryos in Norway spruce. J. Exp. Bot. 49(319): 155-162

[0032] Franz, P., De Ruijter, N. y Schel, J.1989. Isozyme as biochemical and cytological markers in embryogenic callus cultures of maize Zea mays L.). Plant Cell Rep 8: 67-70.

[0033] Kobayashi, T., Eun, C.H., Hanai, H., Matsubayashi, Y. y Kamada , H.1999. Phytosulfokine-a, apeptidyl plant growth factor, stimulates somatic embryogenesis in carrot. J. Exp. Bot. 50: 1123-1128.

[0034] Kreuger, M. y Van Holst, GJ1993. Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L. Planta 189: 234-248.

[0035] Martinoia, E., Heck, U. and Wiemken. Vacuoles as storage compartments for nitrate in barley leaves. Nature 289: 292-294.

[0036] Nuutila, A.M., HAMALAINEN, J. and Mannonen, L.2000. Optimization of media nitrogen and copper concentrations for regeneration of green plants from polyembryogenic cultures of barley (Hordeum vulgare L.) Plant Sci. 151: 85-92. Toonen, M., Schmidt, E., Van Kammen, A. and De Vries, SC1997. Promotive and inhibitory effects of different arabinogalactan proteins on Daucus carota L. somatic embryogenesis. Planta. 2003: 188-195

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Claims

CLAIMS 1. A process and mature embryos obtained from immature embryos or competent cells with the ability to develop into higher gymnosperm and angiosperm plants by this invention.

2. A maturation process applicable to immature embryos, competent cells and recalcitrant lines of gymnosperms and angiosperms according to claim 1, characterized by the treatment with a crude solution of proteins obtained from somatic and zygotic embryos of 10-1000 pl, and also supplemented with arabinogalactans and / or chitinases and / or extracellular proteins and / or analogous in a ratio of 99 / 1 to 1 / 99, on the immature embryos or competent cells or to induce them; the basic culture medium is supplemented with a single source of nitrate without ammonia 00 / 0, or two such as ammonium / nitrate in a ratio of 49 / 51 to 01 / 99, one or several carbon sources that can be maltose and / or fructose and / or glucose and / or sucrose and / or starch that varies between 1 and 5 %; a maturation promoter and signal such as abscisic acid or its analogues at doses of 20 and 120 pM, according to claim 1.

3. Mature somatic embryos obtained from immature embryos, competent cells and recalcitrant lines of gymnosperm and angiosperm according to claim 1 and, which were treated according to claim 2.

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