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BR-102024017385-A2 - METHOD FOR OBTAINING VIABLE TRANSGENIC AGAVE SISALANA PLANTS RESISTANT TO HERBICIDE

BR102024017385A2BR 102024017385 A2BR102024017385 A2BR 102024017385A2BR-102024017385-A2

Abstract

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. 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 with Agrobacterium tumefaciens (A. tumefaciens), with increased efficiency in obtaining transgenic plants.

Inventors

  • ALINE VITÓRIA CORIM MARIM
  • CAROLINA ROSSI DE OLIVEIRA
  • CAMILA GOMES CABRAL
  • GIOVANNA RAFAELI REZENDE
  • Gonçalo Amarante Guimarães Pereira
  • Marcelo Falsarella Carazzolle
  • POLLYANA KARLA DA SILVA

Assignees

  • UNIVERSIDADE ESTADUAL DE CAMPINAS

Dates

Publication Date
20260310
Application Date
20240823

Claims (12)

  1. 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 containing 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. 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. 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. 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 dichlorophenoxyacetic acid (2,4-D) and about 8 to 10 g of agar/liter as a solidifying agent and is kept in the dark for 3 days.
  5. 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 from 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. 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. 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. 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. 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. 10. Method, according to claims 1 to 9, characterized in that in step f) transformed shoots are obtained which are transferred to a new culture medium, called elongation, comprising 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. 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. 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.

Description

FIELD OF THE INVENTION [001] 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 upon them. [002] The present invention relates, in particular, to a method of 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 with Agrobacterium tumefaciens (A. tumefaciens), with increased efficiency in obtaining viable transgenic plants. FUNDAMENTALS OF THE INVENTION [003] The following paragraphs are intended to introduce the reader to the more detailed description, but without intending to limit the subject matter claimed in this disclosure. [004] The genus Agave is endemic to the Americas, belonging to the subfamily Agavoideae and has approximately 210 species (spp.) 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 a 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). [005] 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 78 million dollars (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 amount of biomass could be used for bioenergy production. [006] Initial studies have shown the potential use of agave species in bioethanol production due to the significant biomass production, efficient physiological characteristics of energy conversion of these plants (CAM metabolism), and ability to survive in arid climates, leading to lower environmental impacts during production compared to sugarcane molasses or other bioethanol sources. [007] 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 the management and improvement of this crop have been carried out in recent years. In particular, weed competition is a significant problem in the production of these plants. [008] Among the plants, the emergence of weeds is noticeable, causing a problem of competition for nutrients between the weeds and the A. 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 in weed control 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 the herbicide 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. [009] In this sense, a necessary and promising alternative is the development of genetic transformation protocols for A. sisalana plants. Exploring this technical field makes it possible to introduce genes of interest into A. sisalana, which are capable of improving certain characteristics of these plants, such as herbicide resistance, in addition to enabling the appearance 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 genetic transformation method of A. sisalana of the present invention allows for 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 inn