Method for obtaining viable herbicide-resistant transgenic agave sisalana plants

Abstract

The present invention pertains to the fields of agricultural biotechnology or plant biotechnology. Specifically, it relates to the genetic modification of plants, in which genes of interest are inserted to impart desirable characteristics thereto. The present invention relates, in particular, to a method for obtaining genetically modified Agave sisalana (A. sisalana) plants. More specifically, the method according to the invention relates to the development of an unprecedented protocol for obtaining transgenic A. sisalana plants via infection by Agrobacterium tumefaciens (A. tumefaciens), with increased efficiency in the creation of transgenic plants.

Description

METHOD FOR OBTAINING VIABLE TRANSGENIC AGAVE SISALANA PLANTS RESISTANT TO HERBICIDE FIELD OF THE INVENTION

[0001] The present invention falls within the field of agricultural biotechnology or plant biotechnology. Specifically, it relates to the genetic modification of plants, where genes of interest are inserted to confer desirable characteristics.

[0002] The present invention relates, in particular, to a method for obtaining genetically modified Agave sisalana (A. sisalana) plants. More specifically, the method disclosed by the present invention relates to the development of a novel protocol for obtaining transgenic A. sisalana plants via infection by Agrobacterium tumefaciens (A. tumefaciens), with increased efficiency in obtaining viable transgenic plants. FUNDAMENTALS OF THE INVENTION

[0003] The following paragraphs are intended to introduce the reader to a more detailed description, but without intending to limit the subject matter claimed in this disclosure.

[0004] The genus Agave is endemic to the Americas, belonging to the subfamily Agavoideae, and comprises approximately 210 species distributed from the southern United States to Colombia and Venezuela, with Mexico being the main distribution center of the genus. Agave plants are widely used for the preparation of alcoholic beverages, fibers, food, medicines, ornamental plants, and are prominent in the tequila, mezcal, and natural fiber industries. The species of the genus Agave have two notable groupings: species that accumulate high concentration of digestible sugar in their stems, such as Agave tequilana (Sugar Agave), and species that have a high fiber content in their leaves, such as A. sisalana (Fiber Agave).

[0005] Brazil is the world's largest producer of natural sisal fibers, with an area of ​​200,000 hectares in the semi-arid region (FAO, 2020), where no other crop would be economically viable. It is estimated that today 500,000 people depend on this activity for their livelihood (Conab, 2016), generating an annual income of around US$78 million (Conab, 2018). Brazil exported 60,000 tons of products derived from the fiber, mainly from the state of Bahia, which is the largest producing region. However, only 4% of the leaves from a commercial *A. sisalana* plantation are used for the production of commercial fibers (Suinaga et al., 2007), with the remainder considered bagasse and left in the field to rot, although this large quantity of biomass could be used for bioenergy production.

[0006] Initial studies have shown the potential use of agave species in bioethanol production due to their significant biomass production, efficient physiological characteristics for energy conversion (CAM metabolism), and ability to survive in arid climates, leading to lower environmental impacts during production compared to sugarcane molasses or other bioethanol sources.

[0007] In this context, commercial plantations of *A. sisalana* play a fundamental role, since they are already established in the semi-arid regions of Brazil for fiber production and already produce a large amount of biomass (bagasse), which is currently wasted. On the other hand, few Efforts in managing and improving this crop have been made in recent years. In particular, weed competition is a significant problem in the production of these plants.

[0008] Among the plants, the emergence of weeds is noticeable, causing a problem of competition for nutrients between the weeds and the *Agave sisalana* plant. In addition to the anatomical characteristics of *Agave* plants, which prevent the use of agricultural machinery for weed control, the application of systemic herbicides, such as glyphosate, which acts to control weeds via direct application to the soil, causes root deformation in *Agave* plants, potentially leading to necrosis. Thus, the development of genetically modified plants resistant to glyphosate is of great interest for industrial-scale use, as it represents a strategy that overcomes all the obstacles related to the use of currently known herbicides.

[0009] In this sense, a necessary and promising alternative is the development of genetic transformation protocols for A. sisalana plants. Exploring this technical field enables the introduction of genes of interest into A. sisalana, which are capable of improving certain characteristics of these plants, such as herbicide resistance, in addition to allowing the emergence of other new characteristics not intrinsic to the species, such as resistance to pests and diseases.

[0010] Thus, the present invention solves the technical problem of obtaining viable shoots of A. sisalana after transformation via A. tumefaciens. The transformation method The genetics of A. sisalana from the present invention enables greater efficiency in obtaining shoots during the regeneration of genetically transformed plants, resulting in the production of transgenic A. sisalana plants transformed with genes of general agronomic interest.

[0011] Thus, the objective of the present invention is to describe an innovative and surprising strategy for the genetic transformation of plants of the A. sisalana group, which is justified by the absence of related protocols for obtaining viable plants with improved agronomic characteristics of interest, such as resistance or tolerance to drought, diseases, pests, and herbicide resistance. STATE OF THE ART

[0012] The potential use of transgenic Agave species has been gaining greater relevance recently, due to overcoming several obstacles in the crop, mainly its monocarpic nature and long life cycle. In this context, some studies in the literature propose the production of transgenic Agave plants. Thus, although few genetic transformation studies have been published for the Agave genus, none of these studies indicate the use specifically of A. sisalana. In this respect, it is important to highlight that the Agave genus is genotype-dependent and, therefore, the development of genetic transformation studies and protocols for each species of the genus is essential.

[0013] The Mexican patent document MXJL02000044, entitled "Transforms! on genetics in the agave genre and production of transgenic plants resistant to The article "herbicidas" describes the use of biolistics and infection via Agrobacterium, containing PPT / Bar marker genes and uidA (p-glucuronidase) as a reporter gene, from embryogenic calli, but does not describe the species used in this technology, its cultivation conditions, nor does it show whether the use of this strategy is actually capable of generating transformed plants (Cabrera-Ponce et al., 2002).

[0014] From calluses dedifferentiated from explants, Flores-Benitez et al. 2007, in turn, tested two methods of transforming Agave plants: biolistics and via A. tumefaciens. In experiments using A. tumefaciens, transgenic Agave salmiana plants were obtained, with a transformation efficiency of 2.7%, using the uidA gene (p-glucuronidase) as a reporter gene. In this study, an exposure time of 30 min was used for Agrobacterium, using the following co-culture culture medium: MS + 5 pM BAP + 2.7 pM NAA + 100 pM acetosyringone, callus induction medium: MS + 5 pM BAP + 2.7 pM NAA + 10 mL / L cocktail 20 + 50 mg / L kanamycin + 250 mg / L cefotaxime, and rooting medium: MS 50% + 1.14 pM IAA + 12.5 pM BAP + 50 mg / L kanamycin.

[0015] In turn, in the article by Gao et al., 2014, entitled "Expression of a hevein-like gene in transgenic Agave hybrid No. 11648 enhances tolerance against zebra stripe disease", 7 transgenic lines expressing the hevein-like gene were obtained, aiming to increase resistance to Phytophthora nicotiana in plants of the Agave hybrid genotype H11648. In this study, embryogenic calluses were subcultured in SH culture medium and not the commonly used MS culture medium. In this study, exposure to bacteria was 10 min, callus induction medium: SH + 13.2 pM BAP + 2.68 pM NAA + 0.45 pM 2,4-D + 6.5 g / L carrageenan, shoot induction medium: SH + 6.66 pM BAP + 2.68 pM NAA + 6.5 g L⁻¹ carrageenan + 2 mg L⁻¹ PPT, and rooting induction: SH + IAA.

[0016] The study on genetic transformation of Agave, mediated by A. tumefaciens, entitled "Development of an Agrobacterium tumefaciens mediated transformation protocol for two Agave species by organogenesis," published in 2016, was carried out via organogenesis from bulbil meristems of Agave tequilana and Agave desmettiana, using marker genes (PPT / Bar). Although the data on genetic transformation efficiency are unclear, it was observed that the regeneration of transgenic plants was more efficient in A. desmettiana, strengthening the genotype-dependent trait in Agave plants. In this study, MS culture medium supplemented with BAP (9 mg / ml), IBA (0.6 mg / ml), and acetosyringone was used.

[0017] As previously mentioned, only 4 (four) studies related to the development of genetic transformation protocols for Agave are currently known, and none of them include the species A. sisalana, nor the methods disclosed in the present invention, which therefore represent disruptive and superior methods in relation to the knowledge already available in the art.

[0018] Therefore, the aforementioned precedents, even when combined with each other, in any combination thereof, would not motivate a person skilled in the art to deduce or obtain the... The teachings of this patent application. In particular, the aforementioned prior art is silent regarding the disclosure expressed in this application, namely, a viable method for the genetic transformation of plants, especially A. sisalana, with evident improvement of agronomic characteristics of interest, such as resistance and / or tolerance to diseases, pests, and herbicide resistance. SUMMARY OF THE INVENTION

[0019] The present invention proposes a surprising and innovative method for obtaining genetically modified plants of the species A. sisalana, via A. tumefaciens. In particular, the present invention proposes a method for the genetic transformation of plants of the species A. sisalana, via A. tumefaciens, which express the cp4-epsps gene that, in turn, has the function of generating herbicide tolerance from the synthesis of the enzyme 5-enolpyruvateshikimate-3-phosphate synthase (EPSPS), in order to obtain shoots resistant to the herbicide glyphosate.

[0020] Based on the genetic transformation method of the present invention, it is possible to maximize the production of transgenic A. sisalana plants by combining the use of different explant sections for genetic transformation experiments, as well as different concentrations of plant growth regulators during the post-infection stages of transformed and non-transformed (control) explants that underwent contact and / or infection with A. tumefaciens, aiming to obtain a greater number of shoots and, consequently, possible transgenic cells. This strategy increased the chances of regenerating a complete A. sisalana plant.

[0021] According to Gao et al., 2013, the main factors influencing the efficiency of genetic transformation of the Agave genus include Agrobacterium concentration, infection time, acetosyringone concentration, pre-culture time, co-culture time, antibiotic treatment time, and plant growth regulator concentration. However, optimizing these factors for specific A. sisalana cultivars is not a trivial task for a specialist in the field and, for this reason, requires specific and in-depth studies in this regard.

[0022] In this respect, the present invention proposes maximizing the production of transgenic A. sisalana plants through the combination and action of several factors. To this end, extensive laboratory experimentation was carried out, resulting in an efficient protocol for the genetic transformation of A. sisalana. During this experimental process, different explant sections were tested for genetic transformation experiments, different infection times, different concentrations of plant growth regulators, and different co-cultivation times during the post-infection stages of transformed and non-transformed (control) explants, which underwent contact and / or infection with A. tumefaciens.

[0023] Specifically, the method for obtaining genetically modified A. sisalana plants of the present invention comprises a differential resting step for the explants, which occurs immediately after the exposure of the A. sisalana explants to a culture medium containing A. tumefaciens. The resting step described in the method of the present invention is proposed to "dry" the explants and "pause" them. infection by Agrobacterium, so that they could be exposed to a selective agent (antibiotic) at a later stage, that is, in the selection culture medium.

[0024] In the resting phase of the method for obtaining genetically modified A. sisalana plants of the present invention, a specific incubation time of 5 days was employed for the explants with A. tumefaciens. Specifically, the resting phase occurred in the dark, so that the possibly transformed cells could organize themselves before the selective necrosis of the non-transformed explants. The incubation time of the explants with A. tumefaciens was directly influenced by the type of bactericidal agent used.

[0025] Based on the genetic transformation protocol for A. sisalana plants disclosed by the present invention, it was possible to obtain a greater number of shoots and, consequently, transgenic cells of this group, increasing the chances of regenerating a complete A. sisalana plant. This fact further clarifies the importance of the present invention in the context of plant transformation, such as that of the genus Agave, since it refers to a genotype-dependent plant and, for this reason, requires specific genetic transformation protocols for each of its species.

[0026] In this respect, in short, the method for obtaining genetically modified A. sisalana plants of the present invention comprises the following steps: a) Cutting triangular sections of explants for greater exposure to Agrobacterium through a greater number of tissue wounds; b) Exposure time of the explants to infection by a) Agrobacterium through increased wound growth, followed by cultivation in co-culture medium with the addition of growth regulators at specific times and component concentrations; b) Establishment of the culture in a "resting" medium for explants with growth regulators and bactericidal agents at specific times and component concentrations; c) Cultivation in a selection medium for explants including the gene construct selection agent at specific times and component concentrations; d) Cultivation in a regeneration medium for explants with the addition of growth regulators at specific times and component concentrations; e) Elongation of the shoots obtained during the previous steps.

[0027] In step (a), meristematic regions of bulbils are cut using a scalpel. The cuts are triangular sections of this region with a size ranging from 1 to 3 cm, preferably around 1 cm.

[0028] Next, in step (b), the explants are exposed to an infection medium with A. tumefaciens for a period of approximately 10 to 25 minutes, preferably approximately 15 minutes. Then, the explants are cultured in a co-culture medium comprising approximately 4 to 8 g / L of MS salts (Murshigue & Skoog, 1962), approximately 20 to 50 g / L of sucrose, preferably approximately 3 mg / mL of dichlorophenoxyacetic acid (2,4-D), and approximately 8 to 10 g of agar / liter as a solidifying agent, kept in the dark for approximately 3 days. Specifically, in step (b) the explants are cultivated in a co-culture medium comprising approximately 4.88 g / L of MS salts (Murshigue & Skoog, 1962), approximately 30 g / L of sucrose, preferably approximately 3 mg / mL of dichlorophenoxyacetic acid (2,4-D), and approximately 8 to 10 g of agar / liter as a solidifying agent, kept in the dark for approximately 3 days.

[0029] In step (c) the explants were grown in a culture medium called resting medium, which comprises approximately 4 to 8 g / L of MS salts (Murashigue & Skoog, 1962), approximately 20 to 50 g / L of sucrose, preferably, approximately 3 mg / ml of 2,4-D, approximately 100 mg / L of proline and approximately 0.5 mg / L of casein and kept in the dark immediately for a period of approximately 5 days. Particularly, in step (c) the explants were cultured in resting culture medium comprising approximately 4.88 g / L of MS salts (Murashigue & Skoog, 1962), approximately 35 g of sucrose, preferably, approximately 3 mg / ml of 2,4-D, approximately 100 mg / L of proline and approximately 0.5 mg / L of casein kept in the dark immediately for a period of approximately 5 days.

[0030] After the resting period of step c), the explants were exposed to the selection agent at a concentration of approximately 1 mg / ml, exemplified by geneticin, referred to as step d), aiming to obtain transformed explants containing the insertion of the gene of interest, since only these survive to the detriment of the others. However, other possible candidates for selection agents for use in the method of the invention include kanamycin, or even other related selective agents.

[0031] In step (e) the explants are transferred to regeneration culture medium, which comprises approximately 4 to 8 g / L of MS salts (Murshigue & Skoog, 1962), approximately 20 to 50 g / L of sucrose, preferably about 1 mg / mL of 6-Benzylaminopurine (BAP), and approximately 8 to 10 g of agar / liter as a solidifying agent. The explants are maintained under these conditions for 5 subcultures, each lasting 15 days. Specifically, in step (e) the explants are transferred to a regeneration culture medium comprising about 4.88 g / L of MS salts (Murshigue & Skoog, 1962), about 30 g / L of sucrose, preferably about 1 mg / mL of 6-Benzylaminopurine (BAP) and about 8 to 10 g of agar / liter as a solidifying agent and maintained under these conditions for 5 subcultures, each of 15 days. In the present invention, said subculture means that every 15 days the explants are transferred to a fresh culture medium, comprising the same components as the main step in which it is carried out, in this case, the same components as step (e).Specifically, in step (e) the explants are kept under the conditions described above for 5 periods of 15 days, i.e. 75 days.

[0032] Step (f) consists of obtaining the transformed shoots. In this step, the surviving shoots obtained are transferred to a new culture medium called elongation for development. This culture medium comprises approximately 4 to 8 g / L of MS salts (Murshigue & Skoog, 1962), approximately 20 to 50 g / L of sucrose, preferably approximately 1 mg / mL of indolebutyric acid (IBA), and approximately 8 to 10 g of agar / liter as a solidifying agent. The shoots were maintained for approximately 3 subcultures, each of approximately 15 days. Specifically, in step (f), the shoots obtained are transferred to the elongation culture medium. comprising approximately 4.88 g / L of MS salts (Murshigue & Skoog, 1962), approximately 30 g / L of sucrose, preferably approximately 1 mg / mL of indolebutyric acid (IBA), and approximately 8 to 10 g of agar / liter as a solidifying agent, and maintained for approximately 3 subcultures, each of approximately 15 days. In the present invention, said subculture means that every 15 days the explants are transferred to a fresh culture medium comprising the same components as the main step in which it is carried out, in this case, the same components as step (f). Particularly, in step (f) the explants are maintained under the conditions described above for 3 periods of 15 days, i.e., 45 days. BRIEF DESCRIPTION OF THE FIGURES

[0033] The figures described are presented for a full and complete understanding of the purpose of this invention.

[0034] Figure 1 shows a photograph related to the different sections during the infection process by A. tumefaciens.

[0035] Figure 2 presents a graph related to the number of responsive explants (shoots obtained) after 60 days of co-culture, using the different sections, after the genetic transformation method (using A. tumefaciens and MS culture medium (control)). D 440 = number of discs introduced; T 440 = number of triangular sections introduced.

[0036] Figure 3 refers to a photograph related to regenerated shoots obtained from the use of 1 mg / L BAP supplementation in a medium called regeneration.

[0037] Figure 4 refers to a photograph of the elongation of transgenic shoots in a culture medium called lengthening, using only IBA.

[0038] Figure 5 presents a graph relating the type of explant cut (circular or triangular) to transformation efficiency.

[0039] Figure 6 presents a graph relating the incubation time of A. tumefaciens to transformation efficiency.

[0040] Figure 7 presents a graph related to the effect of the resting phase on obtaining necrotic explants.

[0041] Figure 8 refers to (A) a photograph of the transgenic A. sásalana plant, expressing the cp4-epsps gene, which confers resistance to the herbicide glyphosate and (B) a transgenic plant acclimatized in a greenhouse. DETAILED DESCRIPTION OF THE INVENTION

[0042] Unless otherwise specified, the terms used throughout this descriptive report have their common meanings in the art, within the context of the disclosure, and in the specific context in which each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in this descriptive report, to provide additional guidance to the technician regarding the description of the disclosure. Publications cited herein are specifically incorporated by reference in their entirety.

[0043] It will be appreciated that the same thing can be said in different ways. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed here. No special significance should be placed on whether a term is elaborated or discussed. Synonyms for certain terms are provided here, but the use of some synonyms does not preclude the potential use of others that may not be listed here.

[0044] In general, the present invention consists of an innovative and surprising method for the genetic transformation of plants of the species A. sisalana due to the following specificities: i) type of explant cut used in the genetic transformation method, triangular cuts from the meristematic region of A. sisalana bulbils; ii) infection time by A. tumefaciens for about 15 minutes in the dark; iii) co-culture of the explants with A. tumefaciens in a culture medium comprising about 4 to 8 g / L of MS + about 3 mg / ml of 2,4-D and an incubation time of about 3 days; iv) resting stage in a culture medium comprising about 4 to 8 g / L of MS + about 3 mg / ml of 2,4-D + about 100 mg / L proline + about 0.5 mg / L of casein, with the addition of a bactericidal agent, with an incubation time of about 5 days.

[0045] However, in order to obtain an efficient method for the genetic transformation of A. sisalana, as described in the present invention, the following preliminary steps were necessary. Evaluation of the different sections of the explant

[0046] Aiming to evaluate the effects of using different sections of the A. sisalana explant, different types of cuts were analyzed, since the infection caused by A. tumefaciens is caused by the production of Acetosyringone activates vir genes, genes responsible for virulence, found in the bacterial machinery. Therefore, to maximize the production of this compound and increase the virulence of the infection, triangular sections of explants, preferably approximately 1 cm in diameter, were made from the meristematic region of the plant using a scalpel. Triangular sections were not made in part of the material, which was considered the control group (Figure 1).

[0047] Next, the type of explant section was evaluated according to the number of shoots formed after the genetic transformation method, which occurred from the exposure of the explant to A. tumefaciens, containing the commercial expression cassette 2x35S+RR / genR, which confers resistance to the herbicide glyphosate, containing the RR gene, whose sole function was to generate a rapid screening strategy through the detection of the BAR protein, and also from explants exposed to the control treatment, i.e., without the presence of A. tumefaciens, using culture medium containing MS salts (Murshigue & Skoog, 1962).

[0048] Figure 2 shows the number of shoots obtained after 60 days of co-cultivation, using different sections, without the use of plant growth regulators, to evaluate only the effect of the sections after inoculation in MS culture medium (control) and with A. tumefaciens.

[0049] As can be seen in Figure 2, the use of explants sectioned in a triangular shape (T440) indicates a 64% increase in the number of responsive explants (shoots obtained), compared to shoots obtained using meristematic discs (D440), with 11 shoots obtained. sprouts from triangular tissue and 7 sprouts from discal tissues. In this respect, it is noteworthy that the use of the triangular explant shape in the method for obtaining viable transgenic plants of A. si salana is one of the differentiating characteristics of the present invention.

[0050] Specifically, it is noteworthy that the choice of a triangular explant shape for *A. si salana* resulted in an increased number of wounds in the explant relative to the meristematic disc, promoting an increase in *Agrobacterium* infection in the plant. On the other hand, although other strategies were tested to further increase the number of wounds in the explant, such as tissue scraping, these strategies caused irreversible damage to the explants and were not as efficient as the triangular cuts performed. This evidence corroborates the fact that the use of triangular explant sections in the method for obtaining viable transgenic *A. si salana* plants, as described herein, is a differentiating and surprising characteristic achieved by the present invention. Evaluating the effect of exposure time of explants in co-culture medium with A. tumefaci ens

[0051] With the objective of evaluating the effects of different exposure times of explants to the agrobacterium *A. tumefaciens*, this stage of the work explored different exposure times to agroinfection, since the specificity of the interaction between the bacterium and the plant can vary greatly, even within the same species (Aricelli et al., 2011; Gao et al., 2013). Therefore, determining the best incubation time was essential for the... Establishment of genetic transfer, using the A. tumefaciens system (Brasileiro et al., 1997). In this way, to maximize the positive interaction effects, different exposure time analyses were performed, considering five different times: 0 min, 5 min, 10 min, 15 min, and 20 min of exposure, without the presence of light.

[0052] A greater number of sprouts was obtained after 60 days of co-cultivation, using different exposure times during agroinfection. In short, the 15-minute exposure time was considered the most relevant. This experiment was carried out in 5 different biological replicates, which provided quite reliable information. Thus, the exposure time during agroinfection was crucial for the success of the invention method and differs significantly from the data reported in the Agave transgenesis literature (Bautista-Montes, et al., 2022). Evaluating co-culture methods in resting culture medium of explants with plant growth regulators

[0053] Due to the positive effect of the triangular sections performed on the explants and the incubation time with Agrobacterium, the effect of using plant growth regulators during the regeneration method of the transformed explants was evaluated. It was observed that after the infection stage, the explants presented a dark coloration and, over time, became necrotic, possibly due to the prolonged action of Agrobacterium. Therefore, a co-culture stage of the explants using the bactericidal agent Timentin was implemented. added to the method of the invention.

[0054] A. sisalana explants were cultivated in a culture medium called resting medium, which was prepared from the same components as the co-culture medium (i.e., about 4 to 8 g / L of MS salts (Murashigue & Skoog, 1962), about 20 to 50 g / L of sucrose, preferably added to about 3 mg / ml of 2,4-D, about 100 mg / L of proline, about 0.5 mg / L of casein and about 1 to 5 mg / L of timentin).

[0055] Next, the A. sisalana explants were kept in the dark for a period of about 5 days. The culture medium was prepared and placed in Petri dishes with the solidifying agent agar in a proportion of about 8 to 10 g of agar / liter.

[0056] The resting stage, disclosed in the present invention, refers to a differentiating step in the method of obtaining genetically modified A. sisalana plants, considering mainly the transgenesis methods currently available for the Agave genus. This stage was justified due to the appearance of darkened explants observed in the culture medium, a fact related to the presence of remaining Agrobacterium in the medium, causing apoptosis in the explants. This strategy, that is, the inclusion of a resting stage in the method of obtaining A. sisalana plants, prevented necrosis of the explants.

[0057] Following the resting period, the explants were subjected to a new subculture for approximately 15 days in a culture medium called selection, which included the selection agent derived from gene construction. This is exemplified by genetics. At this stage, only the transformed explants containing the gene insertion survived, to the detriment of the others, as will be detailed below. Evaluating co-culture methods in explant regeneration culture medium with the addition of a plant growth regulator.

[0058] Finally, for the last stage, after the explant selection period, these were co-cultured in a culture medium called regeneration, which was prepared from the same components as the culture medium called resting (i.e., MS salts (Murashigue & Skoog, 1962)) and sucrales, but without the addition of 2,4-D, proline, and casein, and supplemented with approximately 1 mg / L of BAP, and maintained under these conditions for about 5 subcultures, each of about 15 days. The culture medium was prepared and placed in Petri dishes with the presence of the solidifying agent agar and with the addition of a selective agent, geneticin.

[0059] Figure 3 shows the regenerated shoots obtained from the use of a single supplement with approximately 1 mg / L of BAP, where Figure 3A shows two green shoots regenerating, while Figure 3B shows a shoot in the process of regeneration. It is important to note that values ​​of 0, 1, 3, 5, and 10 mg / L of BAP were tested in this stage, and only in experiments using approximately 1 mg / L of BAP was it possible to observe shoot regeneration in a selective medium. Assessing shoot formation and elongation.

[0060] After obtaining the shoots that survived the selection and regeneration stages, these were subcultured in a culture medium called elongation. For this, it was The elongation culture medium used comprised approximately 4 to 8 g / L of MS salts (Murshigue & Skoog, 1962), approximately 20 to 50 g / L of sucrose, preferably approximately 1 mg / mL of indolebutyric acid (IBA), and agar as a solidifying agent, in a proportion of approximately 8 to 10 g of agar / liter. The buds were maintained in 2-3 subcultures, each for approximately 15 days. In the present invention, said subculture means that every 15 days the explants are transferred to a fresh culture medium, comprising the same components as the main stage in which it is carried out.

[0061] Figure 4 indicates the elongation of transgenic shoots in culture medium, properly termed elongation. Usually, in the literature, high amounts of BAP associated with IBA are used for elongation and development of plants in vitro. In the literature on the genus Agave, high values ​​of BAP are also observed in tissue regeneration (Gutiérrez-Aguilar et al., 2014); however, from the experiments carried out, it was observed that only the use of approximately 1 mg / mL of IBA, without the addition of BAP, was sufficient for the development of the shoot stage (Figure 4A) for whole plants (Figure 4B). Supplementation tests using the BAP and IBA combination did not show an elongation effect on the shoots obtained in the previous stage.

[0062] Considering the genotype-specific characteristic reported for the genus Agave (Aracelli et al., 2011; Gao et al., 2013) and also very common in other plant genera with a long history of transgenesis, such as sugarcane (Budeguer et al., 2021), the method revealed by The present invention proved to be an efficient method, as it enabled the production of transgenic A. sisalana plants, mediated by A. tumefaciens. r via organogenesis. Results Examples of implementation

[0063] As previously highlighted, the Agave genus is genotype-dependent, meaning that specific protocols are needed for each Agave species (Gao et al., 2014). This makes evident the importance of the technology described in this patent application, which allowed the production of transgenic A. sisalana plants resistant to the herbicide glyphosate. Therefore, seeking to develop a safe and effective method for obtaining transgenic A. sisalana plants, the method of the present invention is based on the organogenesis method, including several innovative steps, as described in Tables 1 and 2.

[0064] Through Tables 1 and 2, it is possible to observe that 15 experiments were carried out involving more than 6,700 explants, which are equivalent to approximately 1,300 bulbils. These explants were subjected to and evaluated under various experimental conditions, such as: 1) type of explant cut (circle and triangle), 2) Agrobacterium infection time, 3) composition and subculture time of the co-culture medium, 4) establishment, composition and subculture time of the resting medium, 5) composition and subculture time of the selection medium, 6) composition and subculture time of the regeneration medium, and 7) composition and subculture time of the elongation medium. Table 1 - Summary of experiments performed for the development of the method described by the present invention. Legend: In bold, we highlight the experiments that demonstrated positive results and were considered for the next stage. We also highlight in bold the superior results obtained from the use of our technology. Table 2 - Summary of experiments performed for the development of the method described by the present invention. Legend: In bold, we highlight the experiments that demonstrated positive results and were considered for the next stage. We also highlight in bold the superior results obtained from the use of our technology.

[0065] Table 1, column B, indicates that two types of meristematic cuts (circular and triangular) were explored, with circular cuts being the most commonly used. Before the start of the experiments, the bulbils were disinfected with bleach and 70% ethanol. This is a mandatory step for all in vitro methods and experiments and was performed preliminarily in all experiments evaluated.

[0066] During the disinfection stage of the bulbils, bleach degrades the edges of the tissue, and this damage can cause inefficiency in the infection method with Agrobacterium. Therefore, a triangular cut was made in the circular segments after the disinfection stage, in order to expose an internal region of the tissue that was not degraded by the bleach, seeking a more preserved tissue for infection. The comparison of lines 11 and 12 of Table 1 allows us to evaluate the effect of the circular and triangular cuts, since it was the only variable modified. Figure 5 indicates that the triangular cut was more efficient (0.91%) than the circular cut (0.57%) in obtaining the transformation efficiency rate using 440 and 350 explants, respectively.

[0067] Table 1, column D, indicates the infection times of Agrobacterium in contact with the explants. Due to the type of explants (circular and triangular), the contact surface of the explant with Agrobacterium was different. Thus, the incubation time was directly influenced by the type of cut and drying time of the explants in the subsequent resting stage. The inclusion of the resting stage was fundamental for defining the infection time with Agrobacterium, which This differs from previous studies that did not explore the resting phase. Figure 6 indicates that the 15-minute infection time was the most efficient compared to the others (5 and 10 minutes) in terms of transformation efficiency rate, which reached 2.7%.

[0068] Table 1, column E, indicates the components, hormonal balance, and incubation time of the co-culture medium after the Agrobacterium infection method. Again, the hormonal balance used in this co-culture step differs from the hormonal balance commonly used in the technique, for example, MS + 5 pM BAP + 2.7 pM NAA + 100 pM acetosyringone (Flores-Benitez et al., 2007) and SH + 13.2 pM BAP + 2.68 pM NAA + 0.45 pM 2,4-D + 6.5 g / L carrageenan (Gao et al., 2014) (Table 1). Furthermore, the type of explants used, i.e., circular and triangular, and the contact surface of the explants with Agrobacterium were determining factors in defining this step. All post-infection steps were performed in the dark, and therefore the composition of the culture medium also prevented oxidation of the explants.The incubation time for this stage (3 days) was also adjusted in relation to the Agrobacterium infection time, defined in the previous stage, since the explants still show traces of Agrobacterium contact at this stage.

[0069] In turn, Table 1, column G, indicates the components, hormonal balance, and incubation time of the resting culture medium. This resting stage is a novel and distinctive step in the Agaves genetic transformation method. This is because prior art methods do not utilize a resting stage. described in the method disclosed in this patent application. Instead, available prior art methods suggest only the infection step, followed by co-culture and, furthermore, a selection step immediately afterward.

[0070] Regarding the resting phase, it was observed that the triangular cut of the explants caused the tissue to be even more exposed to Agrobacterium infection and, consequently, that this caused greater tissue necrosis, leading to the death of the explants. This evidence prompted the establishment of an extra step to "dry and pause" the Agrobacterium infection before the explants came into contact with the selective agent in the selection culture medium.

[0071] The incubation time for this stage (5 days) was also adjusted taking into account the bactericidal agent. The resting stage was carried out in the dark and aimed to provide a pause for the possibly transformed cells to organize themselves before the selective necrosis of the non-transformed explants. In this respect, Figure 7 indicates a significant reduction in the number of necrotic explants when subjected to this stage, confirming its relevance in the method disclosed in this patent application. The increase in the number of viable explants (40%) increases the chances of obtaining transformed explants at the end of the regeneration process, since they remain viable before the regenerative process.

[0072] As can be observed in column L, row 10 of Table 2, the frequency of generated shoots increased as a function of protocol optimization, and made it possible to obtain two transgenic plants under the experimental conditions of Experiment 15, due to the higher probability of obtaining plants.

[0073] With the method disclosed in this patent application, it was possible to obtain the first two transgenic plants of A. sisalana, which express the cp4-epsps gene, responsible for causing resistance to the herbicide glyphosate. Protein expression experiments carried out show the efficient insertion of the gene into the plant genome (Figure 4A) and the transgenic plant acclimatized in a greenhouse (Figure 4B).

[0074] In addition to improving agronomic traits, genetic transformation offers opportunities for several other studies of interest. The development of efficient strategies, such as the method of the present invention, presents several advantages for optimizing the production of A. sisalana in the field and implementing coordinated genetic improvement programs.

[0075] The present invention is defined herein in terms of its preferred embodiment. Nevertheless, a person skilled in the art is perfectly capable of observing that modifications may be made to the information described herein, such modifications still being covered by the same scope of the subject matter described and claimed.

Claims

CLAIMS 1. Method for obtaining genetically modified herbicide-resistant A. sisalana plants characterized by comprising the following steps: a) Cutting triangular sections from the A. sisalana explant; b) Exposing the A. sisalana explants in co-culture medium with A. tumefaciens; c) Co-cultivating the explants in resting culture medium with growth regulators and a bactericidal agent; d) Co-cultivating the explants in selection culture medium comprising the gene construct selection agent; e) Co-cultivating the explants in regeneration culture medium with the addition of a growth regulator; f) Obtaining shoots and elongation of the explants.

2. Method according to claim 1, characterized in that the explants of step (a) are obtained from meristematic regions of bulbils and have a size ranging from 1 to 3 cm, preferably 1 cm.

3. Method, according to claims 1 and 2, characterized in that in step b) the A. sisalana explants are exposed in a co-culture medium comprising A. tumefaciens for a period of about 10 to 25 min, preferably 15 min.

4. Method according to claims 1 to 3, characterized in that the culture medium of step b) comprises about 4 to 8 g / L of MS salts, about 20 to 50 g / L of sucrose, about 3 mg / mL of acid dichlorophenoxyacetic acid (2,4-D) and approximately 8 to 10 g of agar / liter as a solidifying agent and should be kept in the dark for 3 days.

5. Method according to claims 1 to 4, characterized in that in step c) the A. sisalana explants are cultivated in a resting culture medium comprising the components of the co-culture medium of step b), adding 3 mg / ml of 2,4-D, 100 mg / L of proline and 0.5 mg / L of casein, and immediately kept in the dark for 5 days.

6. Method according to claims 1 to 5, characterized in that the bactericidal agent of step c) is at a concentration of 1 to 5 mg / L.

7. Method according to claims 1 to 6, characterized in that in step d) the explants are exposed to the selection agent selected from geneticin, kanamycin and other related selective agents at a concentration of 1 mg / ml.

8. Method according to claims 1 to 7, characterized in that the culture medium of step e) comprises 4 to 8 g / L of MS salts, 20 to 50 g / L of sucrose, 1 mg / mL of 6-Benzylaminopurine (BAP) and 8 to 10 g of agar / liter as a solidifying agent.

9. Method, according to claim 8, characterized in that the A. sisalana explants from step e) are maintained for 5 subcultures, each for 15 days.

10. Method, according to claims 1 to 9, characterized in that in step f) transformed sprouts are obtained which are transferred to a new medium of culture, called elongation, which comprises 4 to 8 g / L of MS salts, 20 to 50 g / L of sucrose, 1 mg / mL of indolebutyric acid (IBA) and 8 to 10 g of agar / liter as a solidifying agent.

11. Method, according to claim 10, characterized in that the shoots obtained from step f) are maintained for 3 subcultures, each for 15 days.

12. Method, according to claims 1 to 11, characterized in that the Agrobacterium of step b) comprises the 2x35S+RR / genR expression cassette, which confers resistance to the herbicide glyphosate.

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