Use of small molecule compound for improving efficiency of callus induction, transformation and regeneration in plants

Abstract

The present disclosure belongs to the field of plant genetic engineering, and specifically relates to the use of a small molecule compound for improving the efficiency of callus induction, transformation and regeneration in plants. Provided in the present disclosure is a method for callus induction in plants or for improving the efficiency of callus induction in plants. The method comprises the step of culturing plant cells in the presence of LFM-A13 and / or HDAC8-IN-1. It is found in the present disclosure that the efficiency of callus induction, transformation and regeneration in plants can be improved by culturing the plant cells in the presence of the small molecule compound(s) LFM-A13 and / or HDAC8-IN-1.

Description

Application of small molecule compounds to improve the efficiency of plant callus induction, transformation and regeneration

[0001] Citation of relevant applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202510080993.6, filed on January 17, 2025, entitled “Screening and Application of Small Molecule Compounds for Improving the Efficiency of Callus Induction, Transformation and Regeneration in Plants”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of plant genetic engineering, specifically involving the application of small molecule compounds that improve the efficiency of callus induction, transformation, and regeneration in plants. Background Technology

[0004] Plants possess high cell totipotency; the process by which differentiated cells repair damaged organs, develop new organs, and individualize through changes in cell fate is known as regeneration. Regeneration is a crucial link and bottleneck in trait improvement achieved through genetic engineering techniques such as plant genetic transformation. At the molecular level, successful regeneration requires the reactivation and reprogramming of key transcription factors regulating development. Existing research indicates that overexpression of plant development regulators can promote somatic embryogenesis or bud regeneration, thereby improving the efficiency of plant tissue culture. These factors include LEAFY COTYLEDON1 / 2, WUSCHEL, BABY BOOM, and GRF4-GIF1. Furthermore, different combinations of development regulators have been used to induce new meristematic tissues in dicotyledonous plants without tissue culture. Currently, these methods may involve operational limitations such as multi-gene delivery, and the overexpression of these genes may lead to defects in later plant growth and development. This remains a pressing issue for the engineering application of plant gene editing. Therefore, exploring new methods to avoid transgenic modifications and to artificially controllable regulate plant regeneration efficiency is crucial.

[0005] Research on screening small molecule compounds that promote cell reprogramming began in animal cells. In 2006, Professor Shinya Yamanaka proposed a group of reprogramming factors that could induce the production of totipotency in animal cells, such as Oct4, Sox2, Klf4, and c-Myc. However, all four factors are associated with tumorigenicity, especially c-Myc. This is similar to the negative effects of overexpressing developmental regulators in plants. Therefore, researchers have sought to find methods that can safely induce cell totipotency or pluripotency to avoid negative effects. In 2013, Hongkui Deng et al. pioneered the safe chemical induction of pluripotent stem cells using seven small molecule compounds.

[0006] Chemical induction methods (small molecule compound induction methods) have the advantages of being simple to operate and easy to improve. For example, the types and concentrations of compounds used, as well as the addition cycle and time, can be easily adjusted. They are highly controllable and have broad application prospects. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] To address the aforementioned problems in the prior art, this paper discloses the use of screened small molecule compounds in improving the efficiency of plant callus induction, transformation, and regeneration, as well as a method for improving the efficiency of plant callus induction, transformation, and regeneration using screened small molecule compounds.

[0009] Solution for solving the problem

[0010] [1]. A method for inducing plant callus or improving the efficiency of plant callus induction, comprising the following steps:

[0011] (i) Culture the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1.

[0012] [2]. A method for plant regeneration or improving plant regeneration efficiency, comprising the following steps:

[0013] (i) Culture the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1;

[0014] (ii) Regenerate a complete plant from the cells of the plant.

[0015] [3]. A method for plant genetic transformation or improving the efficiency of plant genetic transformation, comprising the following steps:

[0016] (i) Culture the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1;

[0017] (ii) Introducing at least one expression construct containing at least one nucleic acid sequence of interest into the cells of the plant;

[0018] (iii) Regenerate a complete plant from the cells of the plant.

[0019] [4]. The method according to [3] is characterized in that the method is a method for gene editing in plants or a method for improving the efficiency of gene editing in plants, wherein the at least one nucleic acid sequence of interest encodes a component of the gene editing system.

[0020] [5]. The method according to any one of [1] to [4] is characterized in that the plant is a non-regenerative plant;

[0021] Preferably, the plant includes at least one of rice, wheat, corn, soybean, strawberry, tomato, beet, cotton, sugarcane and tobacco;

[0022] More preferably, the plant is rice, strawberry, sugarcane, or tobacco.

[0023] [6]. The method according to any one of [1] to [5] is characterized in that, in step (i), the cells of the plant are somatic cells or embryonic cells;

[0024] Optionally, the plant cells are derived from plant explants or portions thereof, or from plant callus tissue.

[0025] [7]. The method according to any one of [1] to [6] is characterized in that, in step (i), the working concentrations of LFM-A13 and / or HDAC8-IN-1 are each independently 0.01 to 100 μmol / L.

[0026] [8]. Use of LFM-A13, HDAC8-IN-1, or a combination of LFM-A13 and HDAC8-IN-1 in any of the following (x) to (z):

[0027] (x) Plant callus induction or improving plant callus induction efficiency;

[0028] (y) Plant regeneration or improving plant regeneration efficiency;

[0029] (z) Plant genetic transformation or improving the efficiency of plant genetic transformation;

[0030] Preferably, the genetic transformation includes gene editing in the plant and / or introducing a nucleic acid sequence of interest into the plant.

[0031] [9]. A culture medium used in any one of [1] to [6], characterized in that the culture medium comprises two components as shown in (a) and (b):

[0032] (a) Plant tissue culture medium, wherein the plant tissue culture medium is a callus induction medium or a regeneration medium;

[0033] (b) A developmental regulator potentiator comprising a compound and a solvent, wherein the compound is LFM-A13 and / or HDAC8-IN-1, and the solvent is any one or a combination of dimethyl sulfoxide, dimethylformamide, and ethanol.

[0034]

[0010] . The culture medium according to [9] is characterized in that the concentration of the compound in the culture medium is 0.01 to 100 μmol / L.

[0035] The effects of the invention

[0036] This publication screened small molecule compounds that can activate the expression of developmental regulators, including LFM-A13 and HDAC8-IN-1. Experimental data showed that exogenous addition of LFM-A13 or HDAC8-IN-1 to the culture medium can effectively improve the callus induction and regeneration efficiency of different plants, and also improve the genetic transformation efficiency of plants. Attached Figure Description

[0037] Figure 1: Results of the experiment on promoting the regeneration of non-regenerating indica rice 93-11 by exogenous application of the small molecule compound LFM-A13 in Example 1 (on regeneration medium).

[0038] Figure 2: Results of the experiment on promoting the regeneration of refractory indica rice 93-11 by exogenous application of small molecule compounds LFM-A13 and HDAC8-IN-1 in Example 1 (on regeneration medium).

[0039] Figure 3A: Experimental results of promoting callus formation in Huanghuazhan and Indica rice R498 by exogenous application of small molecule compound LFM-A13 in Example 1 (on callus induction medium).

[0040] Figure 3B: Results of the experiment in Example 1 on promoting the regeneration of strawberry Red Beauty, tobacco Benedict, and sugarcane Liucheng 05-136 by exogenous application of the small molecule compound LFM-A13 (on regeneration medium).

[0041] Figure 4: In Example 1, the exogenous application of the small molecule compound LFM-A13 promoted the regeneration and callus rate of strawberry red skin. Detailed Implementation

[0042] The following describes the embodiments of this disclosure, but this disclosure is not limited thereto. Various modifications can be made within the scope of protection claimed in this disclosure, and embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of this disclosure.

[0043] In this disclosure, the terms “a”, “an”, or “the” can mean “one”, “one or more”, “at least one”, or “one or more”.

[0044] In this disclosure, the terms “comprising,” “having,” “including,” or “containing” can mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, “comprising,” “having,” “including,” or “containing” can also mean closed-ended, excluding additional, uncited elements or method steps.

[0045] In this disclosure, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0046] In this disclosure, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process.

[0047] In this disclosure, the range of values ​​referred to as “value A to value B”, “value A to value B”, “value A and above”, or “value A and below” refers to the range that includes the endpoint values ​​A and B.

[0048] In this disclosure, the term "about" is used to define that the numerical ranges and parameters of this disclosure are approximate values, and specific related values ​​have been presented as precisely as possible. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0049] In this disclosure, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., mean that a particular element (e.g., feature, structure, property, and / or characteristic) related to that embodiment is included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0050] In this disclosure, the term "plant" includes the whole plant and any offspring, plant cells, tissues, or parts. "Plant parts" include any part of the plant, including but not limited to, for example, seeds (including mature seeds, immature embryos without seed coats, and immature seeds), plant cuttings, plant cells, plant cell cultures, and plant organs (e.g., pollen, embryo, flower, fruit, bud, leaf, root, stem, and related explants). Plant tissues or plant organs can be seeds, callus tissue, or any other population of plant cells organized into structural or functional units.

[0051] In this disclosure, "callus" refers to the new tissue that grows on the surface of a wound after a localized injury to the original plant, or it can refer to a similar structure induced under artificial culture conditions. It is composed of living parenchyma cells and can originate from living cells of various tissues within any organ of the plant.

[0052] In this disclosure, “regeneration” refers to the process of growing from one or more plant cells (e.g., plant protoplasts, callus, or explants) into a complete plant.

[0053] In this disclosure, when plant somatic cells are transformed into totipotent or pluripotent cells, certain genes are specifically activated or differentially expressed, thereby reprogramming the cells and acquiring embryonic or meristem tissues. These genes typically encode transcription factors, which are collectively referred to as "developmental regulators".

[0054] In this disclosure, "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence in plants. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein. The "expression construct" of this disclosure can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or a translatable RNA (e.g., mRNA).

[0055] In this disclosure, the terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” “nucleic acid molecule,” or “nucleic acid fragment” are used interchangeably to refer to any length of nucleotide polymerization, whether it is a deoxyribonucleotide or ribonucleotide, or an analogue thereof.

[0056] In this disclosure, the terms "expression regulatory sequence" and "expression regulatory element" are used interchangeably, referring to nucleotide sequences located upstream, midway, or downstream of a coding sequence that affect transcription, RNA processing, stability, or translation of the relevant coding sequence. Expression regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0057] In this disclosure, “introducing” a nucleic acid molecule (e.g., an expression construct) into a plant cell means presenting the nucleic acid molecule to the plant cell so that the nucleic acid molecule enters the interior of the plant cell.

[0058] In this disclosure, “exogenous” in relation to a sequence means a sequence derived from an alien species, or, if derived from the same species, a sequence whose composition and / or loci have been significantly altered from its natural form through deliberate human intervention.

[0059] In this disclosure, "plant genetic transformation" or "genetic transformation" refers to the process of introducing exogenous or endogenous target genes into the genome of a recipient plant by adopting certain genetic transformation methods, or modifying specific target genes in the genome of a recipient plant by using gene editing tools.

[0060] In this disclosure, "working concentration" refers to the final concentration of a substance in a working system.

[0061] Unless otherwise defined, other technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0062] <First Aspect>

[0063] The first aspect of this disclosure provides the following method:

[0064] A method for inducing plant callus or improving the efficiency of plant callus induction, comprising the following steps: (i) culturing the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1;

[0065] A method for plant regeneration or improving plant regeneration efficiency includes the following steps: (i) culturing the cells of the plant in the presence of LFM-A13 and / or HDAC8-IN-1, and (ii) regenerating an intact plant from the cells of the plant.

[0066] A method for plant genetic transformation or improving the efficiency of plant genetic transformation, comprising the following steps: (i) culturing plant cells in the presence of LFM-A13 and / or HDAC8-IN-1, (ii) introducing at least one expression construct containing at least one nucleic acid sequence of interest into the plant cells, and (iii) regenerating an intact plant from the plant cells.

[0067] In some specific embodiments, the method for inducing or improving the efficiency of plant callus induction includes the following steps: (i) culturing the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1 to induce callus formation.

[0068] In some specific embodiments, the method for plant regeneration or improving plant regeneration efficiency includes the following steps: (i) culturing the cells of the plant in the presence of LFM-A13 and / or HDAC8-IN-1 to induce callus formation, and (ii) regenerating a complete plant from the callus obtained in step (i).

[0069] In some specific embodiments, the method for plant genetic transformation or improving the efficiency of plant genetic transformation includes the following steps: (i) culturing the cells of the plant in the presence of LFM-A13 and / or HDAC8-IN-1 to induce callus formation; (ii) introducing at least one expression construct containing at least one nucleic acid sequence of interest into the cells of the callus obtained in step (i); and (iii) regenerating a complete plant from the callus obtained in step (ii).

[0070] In step (i) of the methods described above, the plant cells are those suitable for inducing callus formation, genetic transformation (e.g., callus transformation), and / or regeneration into complete plants through tissue culture. Examples of suitable plant cells include, but are not limited to, protoplast cells, callus cells, immature embryonic cells, and explant cells.

[0071] In some implementations, plant cells suitable for inducing callus formation (and subsequent genetic transformations such as callus transformation, and the regeneration of a complete plant) include embryonic plant cells (embryonic cells) and somatic cell line plant cells (somatic cells). Plant cells can be used in isolated form or as part of plant tissue. For example, explants isolated from a plant can provide embryonic or somatic cell line plant cells. Cells are isolated from the explant or used directly for inducing callus (and subsequent genetic transformations such as callus transformation). Which part of the plant is suitable for obtaining explants depends on the specific plant species. Generally, suitable plant cells can be obtained from the hypocotyl, seedling, leaf, bud, flower, petiole, and root of the plant.

[0072] In some implementations, in step (i) of these methods, the plant cells are somatic cells or embryonic cells, preferably derived from plant explants or portions thereof, such as various explants, such as stem segments, leaves, roots, cell suspensions, etc.

[0073] This disclosure does not specifically limit the plants (or the plants targeted by the above methods) that are suitable for these methods. In some embodiments, the plants suitable for these methods (or the plants targeted by the above methods) are non-regenerating plants; non-regenerating plants refer to plants that are difficult to induce, transform, or regenerate callus tissue, such as plants with low callus induction efficiency, low transformation efficiency, or low regeneration efficiency. In some preferred embodiments, the plants include at least one of rice, wheat, corn, soybean, strawberry, tomato, sugar beet, cotton, sugarcane, and tobacco. In some more preferred embodiments, the plants are selected from rice (e.g., Indica rice 93-11, R498, Huanghuazhan), strawberry (e.g., Hongyan), sugarcane (e.g., Liucheng 05-136), and tobacco (e.g., Tobacco Benedict).

[0074] LFM-A13 is a specific Bruton's tyrosine kinase (BTK) inhibitor that inhibits the activity of BTK, JAK2, Plx1, and PLK. HDAC8-IN-1 is an HDAC8 inhibitor. This disclosure involves adding LFM-A13 and / or HDAC8-IN-1 to the culture medium of the plant cells to promote callus induction, transformation, and regeneration. Those skilled in the art can select appropriate culture media based on callus induction, transformation, and regeneration, as well as the type of plant cells; preferably, the culture medium containing LFM-A13 or HDAC8-IN-1 is a callus induction medium and / or regeneration medium.

[0075] In some specific implementations, the information for LFM-A13 and HDAC8-IN-1 is as follows:

[0076] To effectively induce plant callus, improve callus induction efficiency, promote plant regeneration, enhance plant regeneration efficiency, and facilitate plant genetic transformation, the working concentrations of LFM-A13 and / or HDAC8-IN-1 can be appropriately controlled between 0.01 and 100 μmol / L, for example, 0.01 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.15 μmol / L, 0.2 μmol / L, 0.25 μmol / L, 0.3 μmol / L, 0.35 μmol / L, 0.4 μmol / L, 0.45 μmol / L, 0.5 μmol / L, 0.55 μmol / L, 0.6 μmol / L, 0.65 μmol / L, 0.7 μmol / L, etc. The concentrations are 0.75 μmol / L, 0.8 μmol / L, 0.85 μmol / L, 0.9 μmol / L, 0.95 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L, or 100 μmol / L, etc.; preferably, the working concentration of LFM-A13 and / or HDAC8-IN-1 is controlled to be 1 to 30 μmol / L, preferably 1 to 10 μmol / L.

[0077] The plant genetic transformation described in this disclosure can be callus transformation, which refers to the process of introducing exogenous or endogenous DNA into plant callus cells using Agrobacterium infection, gene gun method or other methods.

[0078] The "at least one nucleic acid sequence of interest" can be any nucleic acid sequence that needs to be transformed into a plant. For example, the nucleic acid sequence of interest can be a nucleic acid sequence that encodes traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products. The nucleic acid sequence of interest can also include nucleic acid sequences involved in the metabolism of oil, starch, carbohydrates, or nutrients, as well as nucleic acid sequences that affect fruit size, sucrose load, etc. That is, the method (use) for plant genetic transformation or improving the genetic transformation efficiency of plants is a method (use) for transforming the nucleic acid sequence of interest into a plant or improving the transformation efficiency of the nucleic acid sequence of interest in plants.

[0079] In some embodiments, the "at least one nucleic acid sequence of interest" encodes a component of a gene editing system, thereby enabling gene editing in plants. That is, the method (use) for plant genetic transformation or improving the efficiency of plant genetic transformation is a method (use) for performing gene editing in plants or improving the efficiency of gene editing in plants, wherein the at least one nucleic acid sequence of interest encodes a component of a gene editing system.

[0080] The gene editing system can target endogenous genes or their expression regulatory sequences that are important for traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products, thereby modifying the expression or activity of the endogenous genes. For example, the gene editing system can target endogenous genes or their expression regulatory sequences involved in the metabolism of oil, starch, carbohydrates, or nutrients, or endogenous genes or their expression regulatory sequences that affect fruit size, sucrose load, etc., thereby modifying the expression or activity of the endogenous genes.

[0081] Gene editing, also known as genome editing, involves the insertion, deletion, or substitution of nucleotides in an organism's genome using sequence-specific nucleases or their derivatives. Gene editing typically works by inducing site-specific double-strand breaks (DSBs) at desired locations in the genome, followed by the introduction of the desired DNA insertion, deletion, or substitution during DSB repair. However, gene editing can also encompass base editing techniques that do not involve DSBs, transcriptional activation or repression, and epigenetic modification techniques, provided they are sequence-specific.

[0082] This disclosure does not specifically limit the gene editing system used. For example, gene editing systems suitable for use in this disclosure include, but are not limited to, zinc finger nucleases (ZFNs), large-scale nucleases (MGNs), transcription activator-like effector nucleases (TALENs), and CRISPR (Clustered regularly interspaced short palindromic repeats) systems.

[0083] Zinc finger nucleases (ZFNs) are artificial restriction enzymes prepared by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. A single ZFN typically contains 3-6 individual zinc finger repeats, each of which can recognize a unique sequence, for example, 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.

[0084] "Large-range nucleases" typically refer to homing endonucleases that can recognize nucleic acid sequences of 14–40 bases in length. The long recognition sequence gives large-range nucleases strong specificity, thus reducing their off-target effects.

[0085] "Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cleave specific DNA sequences. They are typically prepared by fusing the DNA-binding domain of a transcription activator-like effector (TALE) with its DNA-cleaving domain. Once engineered, TALEs can bind to almost any desired DNA sequence.

[0086] A “CRISPR system” typically comprises two components that can form a sequence-specific complex: a CRISPR nuclease or a variant thereof, and a corresponding guide RNA. Therefore, for a CRISPR system, the “at least one nucleic acid sequence of interest” described in this disclosure may include the nucleic acid sequence encoding a CRISPR nuclease or a variant thereof, and / or the nucleic acid sequence encoding the corresponding guide RNA.

[0087] In some preferred embodiments, the gene editing system is a CRISPR system. A large number of different CRISPR gene editing systems are known in the art, all of which can be used in this disclosure. CRISPR gene editing systems encompass systems that alter genome sequences, as well as systems used for transcriptional regulation that do not alter genome sequences.

[0088] As used herein, the term "CRISPR nuclease" generally refers to a nuclease present in the naturally occurring CRISPR system. "CRISPR nuclease variants" include modified forms of natural CRISPR nucleases, artificial mutants (including nicking enzyme mutants), catalytically active fragments, or fusions with other functional proteins / peptides. Various artificial functional variants of CRISPR nucleases are known in the art, such as highly specific variants or nicking enzyme variants, or fusion proteins of them with cytidine deaminases or adenosine deaminases. CRISPR nucleases or their variants can recognize, bind to, and / or cleave target nucleic acid structures by interacting with corresponding guide RNAs. Those skilled in the art know how to select appropriate CRISPR nucleases or their variants to achieve the purposes of this disclosure.

[0089] The CRISPR nucleases or variants thereof used in the CRISPR gene editing system disclosed herein may be selected, for example, from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1 (Cas12a), C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.

[0090] In some embodiments, the CRISPR nuclease or a variant thereof includes the Cas9 nuclease or a variant thereof. CRISPR gene editing systems based on the Cas9 nuclease or a variant thereof are also referred to herein as CRISPR Cas9 gene editing systems. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from *Streptococcus pyogenes*.

[0091] Cas9 nuclease variants may include Cas9 nickase (nCas9), in which one of the two subdomains (HNH nuclease subdomain and RuvC subdomain) of the DNA cleavage domain of the Cas9 nuclease is inactivated to form the nickase. In some embodiments, the Cas9 nickase can be combined with two gRNAs targeting upstream and downstream of the sequence to be edited to achieve deletion of the sequence to be edited, or to achieve replacement of the sequence to be edited in the presence of a donor sequence.

[0092] In some embodiments, the CRISPR nuclease or a variant thereof may further include a Cpf1 (Cas12a) nuclease or a variant thereof, such as a highly specific variant. The Cpf1 nuclease may be a Cpf1 nuclease from different species, such as Cpf1 nucleases from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006. CRISPR gene editing systems based on Cpf1 nucleases or variants thereof are also referred to herein as CRISPR-Cpf1 systems.

[0093] In some implementations, the CRISPR nuclease variant may also include a base editor. The base editor is typically a fusion protein containing a deaminase and a CRISPR nuclease variant lacking DNA cleavage activity.

[0094] In this disclosure, the "CRISPR nuclease variant lacking DNA cleavage activity" includes, but is not limited to, Cas9 cleavage nuclease (nCas9), cas9 nuclease with dead nuclease (dCas9), or Cpf1 nuclease with dead nuclease (dCpf1). Cas9 nuclease with dead nuclease (dCas9) or Cpf1 nuclease with dead nuclease (dCpf1) completely lacks DNA cleavage activity. Various CRISPR nuclease variants lacking DNA cleavage activity are known in the art.

[0095] In this disclosure, the term "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase refers to a cytosine deaminase, which accepts single-stranded DNA as a substrate and catalyzes the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase refers to an adenine deaminase, which accepts single-stranded DNA as a substrate and catalyzes the formation of inosine (I) from adenosine or deoxyadenosine (A). Various suitable cytosine deaminases or adenine deaminases that accept single-stranded DNA as a substrate are known in the art. Suitable cytosine deaminases include, but are not limited to, for example, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, and human APOBEC3A deaminase. In some preferred embodiments, the cytosine deaminase is human APOBEC3A. Examples of suitable adenine deaminases include, but are not limited to, the DNA-dependent adenine deaminases disclosed by Nicole M. Gaudelli et al. (doi: 10.1038 / nature24644, 2017).

[0096] By fusing a CRISPR nuclease variant lacking DNA cleavage activity with a deaminase (forming a so-called "base editor"), base editing of target nucleotide sequences, such as C-to-T or A-to-G conversions, can be achieved. Various base editors are known in the art, and those skilled in the art know how to select a suitable base editor to achieve the purposes of this disclosure. CRISPR gene editing systems based on base editors are also referred to as base editing systems.

[0097] In this disclosure, the terms "guide RNA" and "gRNA" are used interchangeably, referring to RNA molecules capable of forming a complex with a CRISPR nuclease or a variant thereof and targeting the target sequence by means of a certain degree of similarity to the target sequence. For example, the gRNA used by the Cas9 nuclease or a variant thereof typically consists of partially complementary crRNA and tracrRNA molecules forming a complex, wherein the crRNA contains a guide sequence that is sufficiently similar to the target sequence to hybridize with the complementary strand of the target sequence and guide the CRISPR complex (Cas9 + crRNA + tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single guide RNAs (sgRNAs) can be designed that simultaneously contain the characteristics of both crRNA and tracrRNA. The gRNA used by the Cpf1 nuclease or a variant thereof typically consists only of mature crRNA molecules, which may also be referred to as sgRNA. Designing suitable gRNAs based on the CRISPR nuclease or a variant thereof and the target sequence to be edited is within the capabilities of those skilled in the art.

[0098] The sequence-specific nucleases used for gene editing in this disclosure, such as zinc finger nucleases, transcription activator-like effector nucleases, or CRISPR nucleases or variants thereof, may also include elements such as subcellular localization signals (e.g., nuclear localization signals), peptide linkers, and detectable tags. For example, the base editor in a CRISPR base editing system typically includes one or more nuclear localization signals (NLS) to facilitate its entry into the cell nucleus and achieve editing of chromosomal DNA.

[0099] In order to achieve effective expression in plants, in some embodiments of this disclosure, the coding nucleic acid sequence or the nucleic acid sequence of interest is codon-optimized for the plant species.

[0100] Codon optimization refers to the modification of nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence). Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs within a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be tailored to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available.

[0101] In this disclosure, cells can be transformed by introducing nucleic acid molecules (e.g., expression constructs) into cells to induce the stable or transient presence of a nucleic acid sequence (preferably stable or transient expression of the nucleic acid sequence) or by introducing polypeptides into cells in a manner that induces transient presence. For example, stable presence of a DNA sequence means that the DNA sequence is stably integrated into the cell's genome. Stable expression refers to, for example, the expression of a DNA sequence stably integrated into the cell's genome. Transformation of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most suitable transformation technique will be determined by the experimenter. The choice of method varies depending on the type of plant to be transformed; those skilled in the art will recognize the applicability of a particular method to a given plant type. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG)-mediated transformation; transformation using viruses; microinjection of plant cells; micro-ballistic bombardment of plant cells (gene gun method); vacuum permeation; and Agrobacterium-mediated transformation.

[0102] In some practical embodiments, as previously described, the expression constructs of this disclosure can be introduced into plant cells by one of a variety of methods known in the art, including but not limited to PEG-mediated protoplast transformation and Agrobacterium-mediated transformation.

[0103] In some exemplary embodiments, taking Agrobacterium-mediated transformation as an example, the method for obtaining gene-edited or transgenic plants of interest, or improving the efficiency of gene-editing or transgenic transformation of interest, may include the following steps:

[0104] (i) Obtaining activated Agrobacterium, wherein the Agrobacterium contains at least one overexpression vector for introducing gene editing and / or transgenic modification of interest;

[0105] (ii) Infect the plant cells to be modified with the Agrobacterium from step (i);

[0106] (iii) Co-culture of Agrobacterium and plant cells infected in step (ii);

[0107] (iv) Induction and regeneration to obtain gene-edited or transgenic plants of interest.

[0108] <Second aspect>

[0109] This third aspect of the disclosure provides the use of the composition of LFM-A13, HDAC8-IN-1, and LFM-A13 and HDAC8-IN-1 in any of the following (x) to (z):

[0110] (x) Plant callus induction or improving plant callus induction efficiency;

[0111] (y) Plant regeneration or improving plant regeneration efficiency;

[0112] (z) Plant genetic transformation or improving the efficiency of plant genetic transformation; preferably, the genetic transformation includes gene editing in the plant and / or introducing a nucleic acid sequence of interest into the plant.

[0113] The steps of (x) plant callus induction or improving plant callus induction efficiency, (y) plant regeneration or improving plant regeneration efficiency, and (z) plant genetic transformation or improving plant genetic transformation efficiency are as described in the first aspect of this disclosure.

[0114] This disclosure reveals that adding LFM-A13 and / or HDAC8-IN-1 to the culture medium, preferably adding LFM-A13 or HDAC8-IN-1 alone, can effectively improve the callus induction efficiency and regeneration efficiency of different plants, and improve the genetic transformation efficiency of plants; preferably, the culture medium containing LFM-A13 or HDAC8-IN-1 is a callus induction culture medium.

[0115] <Third aspect>

[0116] This third aspect of the disclosure provides a culture medium used in the method described in the first aspect of the disclosure, the culture medium comprising two components as shown in (a) and (b) below:

[0117] (a) Plant tissue culture medium, wherein the plant tissue culture medium is a callus induction medium or a regeneration medium;

[0118] (b) A developmental regulator potentiator comprising a compound and a solvent, wherein the compound is LFM-A13 and / or HDAC8-IN-1, and the solvent is any one or a combination of dimethyl sulfoxide, dimethylformamide, and ethanol.

[0119] In some specific embodiments, the culture medium comprises the two components shown in (a) and (b) below:

[0120] (a) Plant tissue culture medium, wherein the plant tissue culture medium is a callus induction medium or a regeneration medium;

[0121] (b) A developmental regulator potentiator comprising a compound and a solvent, wherein the compound is LFM-A13 or HDAC8-IN-1 and the solvent is any one of dimethyl sulfoxide, dimethylformamide, and ethanol.

[0122] In some embodiments, the concentration of the compound in the culture medium is 0.01–100 μmol / L, for example, 0.01 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.15 μmol / L, 0.2 μmol / L, 0.25 μmol / L, 0.3 μmol / L, 0.35 μmol / L, 0.4 μmol / L, 0.45 μmol / L, 0.5 μmol / L, 0.55 μmol / L, 0.6 μmol / L, 0.65 μmol / L, 0.7 μmol / L, 0.75 μmol / L, 0.8 μmol / L, 0.85 μmol / L. The concentrations of the compound in the culture medium are 1–30 μmol / L, preferably 1–10 μmol / L. The concentrations are 0.9 μmol / L, 0.95 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L, or 100 μmol / L.

[0123] Example

[0124] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0125] Example 1

[0126] In this embodiment, the small molecule compounds LFM-A13 (CAS No.: 62004-35-7) and HDAC8-IN-1 (CAS No.: 1417997-93-3), which can activate the expression of developmental regulators, were screened to improve the efficiency of plant callus regeneration.

[0127] The screened small molecule compounds LFM-A13 and HDAC8-IN-1 were dissolved in DMSO and added to callus induction medium. Their effects on callus formation and subsequent regeneration in indica rice varieties 93-11, Huanghuazhan, R498, Tobacco Benedict, sugarcane Liucheng 05-136, and octoploid strawberry were observed. The specific procedures are as follows:

[0128] Callus induction in indica rice was conducted using mature embryos from mature seeds. Mature seeds of indica rice varieties 93-11, Huanghuazhan, and R498 were washed with 75% ethanol for 30 seconds, then rinsed with tap water to remove residual alcohol. Next, an appropriate amount of 3.5% sodium hypochlorite aqueous solution and 2 μl of Tween 20 were added, and the seeds were shaken and washed for 30 minutes. Afterward, the seeds were washed with sterile water in a laminar flow hood, dried, and transferred to a solvent control medium (i.e., callus induction medium containing DMSO) and a callus induction medium containing small molecule compounds for culture. Seeds were inoculated at a density of 14 seeds / plate and cultured in the dark at 28°C for 30 days. The callus induction medium was formulated as follows: N6B5 (Duchefa, DU1158) + 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 g / L sucrose + 0.5 g / L glutamine + 2.8 g / L proline + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel, pH 5.8, and autoclaved. Subculture was performed every 14 days. After approximately 4–8 weeks of induction, the bright yellow, healthy embryogenic callus tissue was transferred to a solvent control medium (i.e., a regeneration medium containing DMSO) and a regeneration medium containing small molecule compounds. The regeneration medium formula was: MS (Duchefa, MO222) + 2 mg / L 6-benzylaminopurine (6-BAP) + 0.2 mg / L kinetin + 30 g / L sucrose + 20 g / L sorbitol + 0.5 g / L acid-hydrolyzed casein + 30 mg / L hygromycin B + 3 g / L plant gel, with a pH of 5.8. After autoclaving, 200 mg / L termethin was added.

[0129] Callus induction in *Nicotiana benthamiana* was conducted using leaf discs from young leaves. Young leaves of sterile *Nicotiana benthamiana* seedlings were cut into 0.5 cm square discs using a scalpel in a clean bench. These discs were then transferred to a solvent control medium (i.e., callus induction and regeneration medium containing DMSO) and a callus induction and regeneration medium containing small molecule compounds for culture and regeneration. The callus induction and regeneration medium formulation was MS + 2 mg / L 6-benzylaminopurine (6-BA) + 30 g / L sucrose + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel, pH 5.8. After autoclaving, 200 mg / L termethin was added. Subculture was performed every 14 days.

[0130] Callus induction and regeneration in sugarcane utilizes meristems from young stems as explants. The outer skin of young sugarcane tillers is removed, and the surface is sterilized with 75% ethanol. In a clean bench, the cladding leaves are removed, and the apical meristem is harvested and sliced ​​into 0.2 cm thick pieces. These slices are then cultured in solvent control medium (i.e., callus induction medium containing DMSO) and callus induction medium containing small molecule compounds. The callus induction medium formula is: MS + 2 mg / L 2,4-D + 30 g / L sucrose + 0.5 g / L acid-hydrolyzed casein + 0.5 g inositol + 8 g / L agar, pH 5.8. After autoclaving, 200 mg / L termethin is added. Subculture is performed every 14 days. After 4–8 weeks of induction, healthy embryogenic callus tissue is transferred to solvent control medium (i.e., regeneration medium containing DMSO) and regeneration medium containing small molecule compounds for further culture. The regeneration medium formula is: MS + 1 mg / L 6-BA + 30 g / L sucrose + 0.5 mg / L indolebutyric acid + 0.5 g / L inositol + 8 g / L Agar, pH 5.8, and 200 mg / L termethin added after high temperature and high pressure sterilization.

[0131] Callus induction in strawberries was conducted using leaf discs from young leaves. Young leaves from sterile strawberry seedlings were cut into 0.5 cm square discs using a scalpel in a clean bench. These discs were then transferred to a solvent control medium (i.e., a callus induction and regeneration medium containing DMSO) and a callus induction and regeneration medium containing small molecule compounds for culture and regeneration. The callus induction and regeneration medium formulation was MS + 0.5 g / L MES + 3 g / L plant gel + 30 g / L sucrose, pH = 5.8. After autoclaving, 1–5 mg / L thiadiazole phenylurea (TDZ) + 0.2–2 mg / L indole-3-acetic acid (IAA) + 200 mg / L termethin were added. Subculture was performed every 14 days.

[0132] As shown in Figures 1 and 2, compared with the solvent control, the application of LFM-A13 and HDAC8-IN-1 both improved the regeneration efficiency of callus tissue in the difficult-to-regenerate indica rice 93-11. LFM-A13 also promoted callus induction and regeneration in other difficult-to-regenerate varieties such as indica rice Huanghuazhan, R498, Tobacco Benedict, sugarcane Liucheng 05-136, and octoploid strawberry Hongyan, as shown in Figures 3A, 3B, and 4, and Tables 1 and 2.

[0133] Table 1

[0134] Table 2

Claims

1. A method for inducing plant callus or improving the efficiency of plant callus induction, comprising the following steps: (i) Culture the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1.

2. A method for plant regeneration or improving plant regeneration efficiency, comprising the following steps: (i) Culture the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1; (ii) Regenerate a complete plant from the cells of the plant.

3. A method for plant genetic transformation or improving the efficiency of plant genetic transformation, comprising the following steps: (i) Culture the plant cells in the presence of LFM-A13 and / or HDAC8-IN-1; (ii) Introducing at least one expression construct containing at least one nucleic acid sequence of interest into the cells of the plant; (iii) Regenerate a complete plant from the cells of the plant.

4. The method according to claim 3, characterized in that, The method is a method for performing gene editing in plants or improving the efficiency of gene editing in plants, wherein the at least one nucleic acid sequence of interest encodes a component of the gene editing system.

5. The method according to any one of claims 1 to 4, characterized in that, The plant in question is a non-renewable plant; Preferably, the plant includes at least one of rice, wheat, corn, soybean, strawberry, tomato, beet, cotton, sugarcane and tobacco; More preferably, the plant is rice, strawberry, sugarcane, or tobacco.

6. The method according to any one of claims 1 to 5, characterized in that, In step (i), the plant cells are somatic cells or embryonic cells; Optionally, the plant cells are derived from plant explants or portions thereof, or from plant callus tissue.

7. The method according to any one of claims 1 to 6, characterized in that, In step (i), the working concentration of LFM-A13 and / or HDAC8-IN-1 is 0.01 to 100 μmol / L.

8. Use of LFM-A13, HDAC8-IN-1, or a combination of LFM-A13 and HDAC8-IN-1 in any of the following (x) to (z): (x) Plant callus induction or improving plant callus induction efficiency; (y) Plant regeneration or improving plant regeneration efficiency; (z) Plant genetic transformation or improving the efficiency of plant genetic transformation; Preferably, the genetic transformation includes gene editing in the plant and / or introducing a nucleic acid sequence of interest into the plant.

9. A culture medium used in the method according to any one of claims 1 to 6, characterized in that, The culture medium contains two components as shown in (a) and (b) below: (a) Plant tissue culture medium, wherein the plant tissue culture medium is a callus induction medium or a regeneration medium; (b) A developmental regulator potentiator comprising a compound and a solvent, wherein the compound is LFM-A13 and / or HDAC8-IN-1, and the solvent is any one or a combination of dimethyl sulfoxide, dimethylformamide, and ethanol.

10. The culture medium according to claim 9, characterized in that, The concentration of the compound in the culture medium is 0.01–100 μmol / L.

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