LU503053B1

LU503053B1 - Somaclonal Variation

Info

Publication number: LU503053B1
Application number: LU503053A
Authority: LU
Inventors: Jerzy Paszkowski; Anna BRESTOVITSKY; Marco Catoni
Original assignee: Univ Birmingham; Cambridge Entpr Ltd
Priority date: 2022-11-14
Filing date: 2022-11-14
Publication date: 2024-05-14
Also published as: WO2024104985A1

Abstract

The invention relates generally to materials and methods of inhibiting Long Terminal Repeat Transposable Elements (LTR-TEs) during propagation of a plant by use of a reverse transcriptase inhibitor. Methods include propagation by plant tissue culture, comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to obtain the cultured plant tissue, whereby inhibiting LTR-TEs leads to reduction in somaclonal variation in cultured plant tissue. Also provided are related media adapted for such culture, and plants and tissues which have reduced genetic variation as a result of the method.

Description

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LU503053

Somaclonal Variation

Field of the Invention

The present invention relates generally to methods of reducing somaclonal variation during plant tissue culture and inhibition of genetic variation arising from Long Terminal Repeat

Transposable Elements (LTR-TEs) during in plant propagation. It further relates to materials resulting from or used in such methods.

The work leading to this invention has received funding from the European Research

Council under the European Union's Seventh Framework Programme (FP7/2007- 2013) /

ERC grant agreement number 322621.

Background

Plant tissue culture is commonly used in agriculture for micropropagation of genetically homogeneous and disease-free plant material, for genetic transformation, and for production of improved plant varieties (Hussain et al., 2012). This technique is known to induce somaclonal variation, consisting of both genetic and epigenetic unintentional alterations, which are unpredictable and therefore generally undesirable in the final regenerated plants (Larkin and Scowcroft, 1981; McClintock, 1984; Wang et al., 2013). Somaclonal variation during tissue culture can be generated by various mechanisms, however, it has been well documented that one of the most common genetic alteration observed is the uncontrolled movement of ‘Long Terminal Repeat Transposable Elements’ (‘LTR-TEs” or retrotransposons) (Azman et al., 2014; Grandbastien, 2015; Bednarek and Ortowska, 2020).

LTR-TEs are endogenous genetic elements abundant in plant genomes, which can extrachromosomally replicate their DNA copies and integrate them into new chromosomal locations.

Under normal physiological conditions LTR-TEs activity is suppressed by epigenetic mechanisms. However, tissue culture can be perceived as a genomic stress and cause TE activation and mobilisation, leading to uncontrolled mutagenic effects in regenerated plants (Hirochika et al., 1996; Takeda et al, 2001; Fukai et al., 2010; Evrensel et al., 2011; Sabot et al., 2011; Wang et al, 2013). For example, somaclonal variation has been well studied in rice (Jiang et al., 2003; Kikuchi et al., 2003; Wang et al, 2009; Wang et al., 2013), and it is known that in vitro propagation of undifferentiated callus tissue induces mobilisation of several rice LTR-TEs (Hirochika et al., 1996; Sabot et al., 2011). Consequently, each rice plant regenerated from such callus displays a variety of new retrotransposon insertions in multiple chromosomal locations (Sabot et al., 2011). Among rice LTR-TEs, 70s17 is the most active in tissue culture, and it can insert into 30 new chromosomal positions in a single regenerated plant (Hirochika et al., 1996; Lanciano et al., 2017). Most importantly, 70s17 tends to transpose into coding regions of genes, potentially interfering with their function (Miyao et al., 2003; Piffanelli et al., 2007). Considering that the locations and the precise

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LU503053 effects of a transposon insertion cannot be easily predicted, newly generated LTR-TE insertions represent a serious threat to the genetic stability and safe use of transgenic plant varieties that are obtained through in vitro regeneration processes. ltis likely that LTR-TEs mobilisation leads to phenotypic diversity that could be exploited to develop new crop varieties (Paszkowski, 2015). Nevertheless, uncontrolled TE mobilisation may also have mutagenic deleterious effects. Hence, LTR-TEs can be a valuable tool in plant breeding but only if their activity is under strict control. So far, the application of DNA methyltransferase inhibitors has been efficiently used to induce epigenetic changes and activate LTR-TEs in plants (Griffin et al., 2016; Thieme et al., 2017; Catoni and Cortijo, 2018;

Roquis et al., 2021) however, currently there are no methods to block LTR-TEs mobilisation once this has been activated in plants.

It can therefore be understood that novel methods and materials for reducing LTR-TEs mobilisation during plant propagation, and/or reducing somaclonal variation in cultured plant tissue would provide a contribution to the art.

Summary of the Invention

The present inventors have discovered that reverse transcriptase inhibitors such as

Tenofovir can be used in plant propagation and plant tissue culture, resulting in reduced

LTR-TE mobilisation and somaclonal variation.

For their extrachromosomal propagation, LTR-TEs use self-encoded Reverse Transcriptase (RT) to replicate their genome, using RNA as an intermediary template (Lisch, 2013). This process mirrors the replication strategy of mammal retroviruses, like Human

Immunodeficiency Virus (HIV), which encodes for an analogue RT enzyme (Levy, 1989).

Among possible treatments of these diseases, several drugs with RT inhibitory action have been developed and used as efficient anti-HIV agents (De Clercq, 2009). The RT inhibitors have well documented antiretroviral activity (Deeks et al., 1998; Maenza and Flexner, 1998), and Tenofovir is commonly used in therapies against HIV and hepatitis B virus (HBV) infections (De Clercq, 2009; Wassner et al., 2020). Yet, the possible effect of RT inhibitors on TE mobilisation has been poorly investigated even in animal models (Jones et al., 2008), and more recently the use of Tenofovir to suppress retrotransposon activity in human and mouse cells has been tested (Banuelos-Sanchez et al., 2019). In plants Tenofovir was previously tested only as a potential antiviral agent against pathogenic virus infections (Spak etal, 2011).

The invention relates generally to methods for inhibiting LTR-TEs during propagation of a plant by use of reverse transcriptase inhibitors.

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The invention further relates generally to a method for reducing somaclonal variation in cultured plant tissue, the method comprising a plant tissue culture step comprising a culture medium comprising a reverse transcriptase inhibitor.

There is also provided a method for reducing somaclonal variation in cultured plant tissue, the method comprising culturing the initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor.

There is also provided the use of a reverse transcriptase inhibitor in a method of plant tissue culture.

Further aspects of the invention are different media compositions (culture medium, callus induction medium, shooting medium, rooting medium, propagation medium) comprising a reverse transcriptase inhibitor.

Further aspects of the invention are cultured plant tissues or cultured or regenerated plants obtained or obtainable by the methods described herein. Such plant materials having reduced somatic variation compared to the corresponding materials obtained or obtainable using media lacking the inhibitors. They may be free or substantially free of new LTR-TE insertions with respect to the initial plant tissue prior to culture.

Some of these aspects and embodiments will now be described in more detail.

Detailed Description of the Invention

This invention relates to methods for inhibiting LTR-TEs during propagation of a plant, the method comprising propagating the plant in the presence of a reverse transcriptase inhibitor.

This can be used to reduce genetic diversity in the propagated plant. This invention further relates to methods and compositions for reducing somaclonal variation during various types of plant tissue culture.

In a first aspect of the invention there is provided a method for inhibiting LTR-TEs during propagation of a plant, the method comprising propagating the plant in the presence of a reverse transcriptase inhibitor. Such propagation can be in vitro in a propagation medium comprising the reverse transcriptase inhibitor. Alternatively, the propagation is in an environment (e.g. external environment, controlled environment) which is optionally soil, and the reverse transcriptase inhibitor is applied to the plant by root drenching or by spraying leaves.

In some embodiments the plant is propagated by plant tissue culture, comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to

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LU503053 obtain the cultured plant tissue, whereby inhibiting LTR-TEs leads to reduction in somaclonal variation in cultured plant tissue.

As used herein, “inhibiting LTR-TEs” is used broadly to encompass suppression of LTR-TE activity, such as by reducing or preventing mobilisation of LTR-TEs.

In other embodiments plant is propagated from a germinated seed.

In other embodiments the plant is propagated asexually e.g. by vegetative propagation or grafting. Such embodiments may be used to produce plants in which inhibiting LTR-TEs reduces somaclonal variation in the plant.

In a further aspect there is provided a method for reducing somaclonal variation in cultured plant tissue, the method comprising a plant tissue culture step comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue.

As explained above “somaclonal variation” consists of both genetic and epigenetic alterations. Somaclonal variation during tissue culture can be generated by various mechanisms, however, it is well documented that one of the most common genetic alterations observed is the uncontrolled movement of LTR Transposable Elements (LTR-TEs or retrotransposons). Somaclonal variation arises between clonal regenerants and their corresponding donor plants, for example in tissue culture, or different types of asexual propagation such as vegetative propagation and grafting.

Somaclonal variation may be reduced compared to control cultured or propagated plant tissue that has been cultured or propagated in corresponding (identical) media or conditions, but in which the reverse transcriptase inhibitor was not used.

The present inventors have shown that culturing initial plant tissue in media comprising a reverse transcriptase inhibitor significantly affects LTR-TE RT activity. Moreover, this may be without interfering with plant development. For example, culturing initial plant tissue in media comprising a reverse transcriptase inhibitor allows the recovery of cultured plant tissue and/or mature plants regenerated therefrom free or substantially free from new LTR-

TE insertions. This is demonstrated in the Examples below with rice callus culture.

Cultured plant tissue or mature plants derived from cultured plant tissue that has been cultured in media comprising a reverse transcriptase inhibitor may exhibit a reduced LTR-TE insertion rate compared to control cultured plant tissue or control mature plants derived from control cultured plant tissue that has been cultured in culture media which does not comprise a reverse transcriptase inhibitor.

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In any of the embodiments described herein, control media may be corresponding (identical) media, but which does not comprise the reverse transcriptase inhibitor. 5 Culturing or propagating initial plant tissue in media comprising a reverse transcriptase inhibitor may improve genetic uniformity of plant material during tissue culture or propagation and result in regeneration of genetically more uniform cultured or propagated plant tissues or plants and/or mature plants derived from cultured plant tissue.

Culturing initial plant tissue in media comprising a reverse transcriptase inhibitor may limit genetic instabilities of cultured plant tissue and/or mature plants derived from cultured plant tissue.

Cultured plant tissue and/or mature plants derived from cultured plant tissue may be more genetically uniform or have limited genetic instabilities compared to control cultured plant tissue and/or control mature plants derived from control cultured plant tissue that have been cultured in culture media which does not comprise a reverse transcriptase inhibitor.

Similarly plants propagated in the presence of a reverse transcriptase inhibitor can, in the light of the present disclosure, be expected to be more genetically uniform. This is demonstrated in the Examples below with Arabidopsis using the heat-activated LTR-TE called ONSEN.

A “reverse transcriptase inhibitor” is used herein to describe compounds that can inhibit activity of reverse transcriptase. For example, in embodiments described herein the reverse transcriptase inhibitor may be a nucleoside analogue reverse-transcriptase inhibitor, a nucleotide analogue reverse transcriptase inhibitor, a non-nucleoside reverse-transcriptase inhibitor or a nucleoside reverse transcriptase translocation inhibitor.

The term “plant tissue culture step” is used generally herein to cover any techniques used to maintain or grow plant cells, tissues or organs under physically and chemically defined conditions. For example, a plant tissue culture step may comprise or consist of micropropagation, callus culture, organ culture, meristem culture, protoplast culture, anther or pollen culture, or cell culture.

The term “initial plant tissue” in relation to the aspects and embodiments of the invention is used herein in a broad sense to cover any viable plant materials which it is desired to culture, for example to generate or regenerate calli, seedlings or plantlets from.

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The term “cultured plant tissue” in relation to the aspects and embodiments of the invention is used herein to cover plant material resulting from a plant tissue culture step. For example, cultured plant tissue may comprise calli, shooted and/or rooted calli, embryoids, embryos, seedlings or plantlets. In some embodiments, mature plants may be derived from the cultured plant tissue, for example wherein the cultured plant tissue is plantlets, these may be transplanted to soil to obtain mature plants.

The term “grafting” is used to denote a method of asexual plant propagation that joins plant parts from different plants together so they will heal and grow as one plant. This technique is used, inter alia, to maintain clonal production, for example in trees such as fruit trees.

The terms “clonal growth” and “vegetative propagation” are used to denote methods in which parental genotypes (genets) are used to produce vegetative modules (ramets) that are capable of independent growth, reproduction, and often dispersal. Examples of crop plants which propagated vegetatively include cassava, potato, sugarcane, pineapple, banana, and onion.

Another aspect of the invention is use of a reverse transcriptase inhibitor in any of the methods of plant tissue culture or propagation described herein. For example, use of a nucleotide analogue reverse transcriptase inhibitor in any of the methods of plant tissue culture described herein.

Use of a reverse transcriptase inhibitor during plant tissue culture significantly affects LTR-

TE RT activity. For example, use of a nucleotide analogue reverse transcriptase inhibitor during plant tissue culture allows the recovery of cultured plant tissue and/or mature plants derived from cultured plant tissue that are free or substantially free from new LTR-TE insertions, and genetically more uniform compared to control cultured plant tissue and/or control mature plants derived from control cultured plant tissue that have been cultured in culture media which does not comprise a reverse transcriptase inhibitor.

Also provided is use of a reverse transcriptase inhibitor in a method of suppressing LTR-TE mobilisation in plants.

Also provided is use of a reverse transcriptase inhibitor in a method of inhibiting LTR-TEs during propagation of a plant.

Also provided is use of a reverse transcriptase inhibitor for limiting genetic instabilities of cultured plant tissue and/or mature plants derived from cultured plant tissue.

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Also provided is use of a reverse transcriptase inhibitor according to any of the above embodiments, optionally wherein the reverse transcriptase inhibitor is a nucleotide analogue reverse transcriptase inhibitor, optionally wherein the reverse transcriptase inhibitor is

Tenofovir.

Reverse-transcriptase inhibitors (RTIs) inhibit activity of reverse transcriptase, a viral DNA polymerase that is required for replication retroviruses such as HIV.

RTIs include nucleoside analogue reverse-transcriptase inhibitors (NARTIs or NRTIs), nucleotide analogue reverse-transcriptase inhibitors (NtARTIs or NtRTIs), non-nucleoside reverse-transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase translocation inhibitor (NRTTIs).

In any of the embodiments described herein the reverse transcriptase inhibitor may be a nucleoside analogue reverse-transcriptase inhibitor, a nucleotide analogue reverse transcriptase inhibitor, a non-nucleoside reverse-transcriptase inhibitor or a nucleoside reverse transcriptase translocation inhibitor.

Well characterised reverse transcriptase inhibitors include the nucleoside analogue RT inhibitors Zidovudine, Stavudine and Lamivudine; the nucleotide analogue RT inhibitor

Tenofovir; and the non-nucleoside RT inhibitor Nevirapine (De Clercq, 2009).

The antiviral effect of NRTIs and NtRTIs is essentially the same; they are analogues of the naturally occurring deoxynucleotides needed to synthesize the viral DNA and they compete with the natural deoxynucleotides for incorporation into the growing viral DNA chain. NtRTIs and NRTIs are similar molecules with and without a phosphonate group, so nucleotides can also be called nucleoside phosphonates. This means that NtRTIs only need two (rather than three) phosphorylation steps to be converted to their active form (De Clercq, 2009). Unlike the natural deoxynucleotides substrates, NRTIs and NtRTIs lack a 3-hydroxyl group on the deoxyribose moiety. As a result, following incorporation of an NRTI or an NtRTI, the next incoming deoxynucleotide cannot form the next 5-3" phosphodiester bond needed to extend the DNA chain. Thus, when an NRTI or NtRTI is incorporated, viral DNA synthesis is halted, a process known as chain termination. All NRTIs and NtRTIs are classified as competitive substrate inhibitors. The phosphonate group of NtRTIs cannot be cleaved by hydrolases, thus making them harder to cleave off once incorporated.

In any of the embodiments described herein the reverse transcriptase inhibitor may be a nucleoside analogue reverse transcriptase inhibitor, for example Didanosine.

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In any of the embodiments described herein the reverse transcriptase inhibitor may preferably be a nucleotide analogue reverse transcriptase inhibitor, for example an acyclic nucleotide analogue e.g. Tenofovir or an analogue or prodrug thereof, Cidofovir and

Adefovir.

More preferably, the nucleotide analogue reverse transcriptase inhibitor is Tenofovir or an analogue or prodrug thereof.

In some embodiments the nucleotide analogue reverse transcriptase inhibitor is Tenofovir.

This is widely commercially available e.g. from Cayman Chemical (N 13874):

NH,

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Use of Tenofovir allows the recovery of plants with reduced levels of LTR-TE insertions, and are preferably free, or substantially free, of said insertions, resulting in regeneration of genetically more uniform plant material. For example, culturing initial plant tissue in media comprising Tenofovir allows the recovery of cultured plant tissue and/or mature plants derived from cultured plant tissue that are free from new LTR-TE insertions and genetically uniform.

Tenofovir is a nucleotide reverse transcriptase inhibitor approved in the United States for the treatment of HIV-1 infection in combination with other antiretroviral agents. Nucleotide analogues are very similar to nucleoside analogues but are pre-phosphorylated, and thus require less processing by the body.

Tenofovir, also known as PMPA, (R)-PMPA or (R)-9-(2-Phosphonomethoxypropyl)adenine is an analogue of adenosine monophosphate that has antiviral activity. In humans, it is converted by cellular enzymes to tenofovir diphosphate, an obligate chain terminator that inhibits the activity of HIV reverse transcriptase and hepatitis B virus polymerase. Tenofovir diphosphate is a weak inhibitor of mammalian DNA polymerases a and and mitochondrial

DNA polymerase y.

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For in vivo and cell culture use, tenofovir is supplied as a water-soluble prodrug in the form of tenofovir disoproxil, typically the fumarate salt (Cayman Chemicals No. 16922).

Vo 0 + Oo ) PN NH, © © 0 No

OX 0-p* 4, o—/ LO N=/

In mammals, the use of this prodrug increases the intracellular diphosphorylated compound >1,000-fold above the level attained with unmodified tenofovir.

Thus, in one embodiment the prodrug is tenofovir disoproxil or a salt thereof (e.g. fumarate salt).

More specifically, Tenofovir DF (disoproxil fumarate) is also known as Bis(POC)-PMPA.

Tenofovir DF (a prodrug of tenofovir) is a fumaric acid salt of bis- isopropoxycarbonyloxymethyl ester derivative of tenofovir. Tenofovir disoproxil fumarate is 9- [(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate (1:1).

Tenofovir disoproxil fumarate requires initial diester hydrolysis for conversion to tenofovir and subsequent phosphorylation by cellular enzymes to form tenofovir diphosphate.

Tenofovir diphosphate inhibits the activity of HIV reverse transcriptase by competing with the natural substrate deoxyadenosine 5'-triphosphate and, after incorporation into DNA, by DNA chain termination. Tenofovir diphosphate is a weak inhibitor of mammalian DNA polymerases alpha & beta and of mitochondrial DNA polymerase.

Tenofovir disoproxil fumarate (DF) is an analogue of adefovir and is classified as a nucleotide analogue reverse transcriptase inhibitor (NtRTI). Tenofovir DF is a competitive inhibitor of other naturally occurring nucleotides, and its ultimate biological activity is viral

DNA chain termination. Tenofovir DF is a novel nucleotide analogue with antiviral activity against both HIV and HBV. The mechanism of tenofovir DF is similar to that of nucleoside analogues, which interferes with reverse transcriptase and prevents translation of viral genetic material into viral DNA. Unlike the nucleoside analogues, the nucleotide analogue reverse transcriptase inhibitors are chemically pre-activated with the presence of phosphate group. Since the phosphorylation step is not necessary, nucleotide analogues can incorporate into viral DNA chain more rapidly than nucleoside analogues.

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Where the term “Tenofovir” is used herein in relation to aspects or embodiments of the invention, unless context demands otherwise, it will be appreciated that the term embraces analogues or prodrugs thereof, and such aspects or embodiments of the invention apply mutatis mutandis to such analogues or prodrugs.

The reverse transcriptase inhibitor for use in the present invention may be dissolved in any suitable solvent known in the art. Preferably the reverse transcriptase inhibitor may be dissolved in water or DMSO.

In some embodiments, the reverse transcriptase inhibitor is a nucleotide analogue reverse transcriptase inhibitor that is Tenofovir, and is dissolved in DMSO.

The reverse transcriptase inhibitor is added to media at any suitable concentration. For example, the reverse transcriptase inhibitor may be added to media at a concentration of 1- 100 uM. For example, the reverse transcriptase inhibitor may be added to culture medium at a concentration of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 5-50, 5-40, 10-50, 10-40 or 20-40 uM. Preferably the nucleotide analogue reverse transcriptase inhibitor is added to culture medium at a concentration of 1-50 uM.

For example, in some embodiments the reverse transcriptase inhibitor is added to culture medium at a concentration of about 1, about 10, about 20, about 30 or about 40 or about 50

MM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to culture medium at a concentration of about 1, about 10, about 20, about 30 or about 40 or about 50

MM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to culture medium at a concentration of 1-50 uM.

As is well known in the art, plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. For example, culturing plant seeds, organs, explants (e.g. leaf discs), cells, or protoplasts on a chemically defined synthetic nutrient media. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, Il and Ill,

Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and

Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

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Provided herein is method for reducing somaclonal variation in cultured plant tissue, the method comprising a plant tissue culture step comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to obtain cultured plant tissue.

As explained above the term “plant tissue culture step” is used broadly herein to cover any techniques used to maintain or grow plant seeds, tissues, organs, explants (e.g. leaf discs), cuttings, cells, protoplasts, anthers etc. under physically and chemically defined conditions.

For example, a plant tissue culture step may be for example micropropagation, callus culture, organ culture, meristem culture, protoplast culture or cell culture.

Plant tissue culture is widely used to produce clones of a plant in a method known as “micropropagation”. Micropropagation is a method of plant propagation using initial plant tissue taken from a mother plant, and growing these under laboratory conditions to produce new plants. A benefit of tissue culture or micropropagation is the generation of genetically homogeneous and disease-free plant material.

Thus, the invention provides a method for reducing somaclonal variation in plants derived from tissue culture, the method comprising a plant tissue culture step comprising culturing plant tissue in a culture medium comprising a reverse transcriptase inhibitor, wherein the plant tissue culture step is micropropagation.

Accordingly, in some embodiments, there is a method of reducing somaclonal variation during micropropagation, comprising culturing initial plant tissue in culture media comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue. In some embodiments, micropropagation excludes meristem culture. “Meristem culture” uses apical meristem to prepare clones of a plant by the vegetative propagation. For example, meristem tip culture comprises culturing the apical dome from a donor plant. In some embodiments, the tissue culture step is not meristem culture. “Organ culture” is culture of organs or plant parts. Any part of plant can serve as explants in organ culture-like shoot (for shoot tip culture), root (for root tip culture), leaf (for leaf culture), and flower (for anther, ovary, ovule cultures).

In some embodiments, organ culture excludes meristem culture. “Protoplast culture” is culture of plant protoplasts, which are plant cells without a cell wall, but bounded by a cell membrane or plasma membrane. Protoplasts can be isolated from many plant parts e.g. roots, shoots or leaves by techniques known in the art. Using protoplast culture, it is possible to regenerate whole plants from single cells.

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A further tissue culture is “anther culture”, for example for double haploid line generation.

This is used as a technique to micro propagate haploid material (e.g. anthers from flower).

Doubled haploids can be obtained by spontaneous or chemically induced chromosome doubling of the haploids during anther culture. This process has utility, for example, for highly heterozygous plants to obtain highly homozygous (and therefore more phenotypically stable) genotypes. The production of haploids through anther culture has now become an integral part of the breeding programs of several agronomically important plants. “Cell culture” or “cell suspension culture” is culture of single cells or small aggregates of cells in vitro in liquid medium.

It will be appreciated by those skilled in the art that methods described herein can be applied broadly to tissue culture methods and description in relation to any of those methods applies mutatis mutandis to the others, unless context demands otherwise. It will also be appreciated that methods of plant tissue culture may be used in combination with other methods. For example, protoplast culture may be performed prior to callus culture.

The conditions used for culture may be those which impose some kind of stress on the plant tissue e.g. an abiotic stress, for example a heat stress. However, as noted herein, tissue culture itself can be perceived as a genomic stress and cause TEs activations and their mobilisation in the absence of the present invention.

The term “initial plant tissue” in relation to the aspects and embodiments of the invention is used herein in a broad sense to cover any such viable plant materials which it is desired to culture, for example to regenerate calli, seedlings or plantlets from.

For example, the initial plant tissue may be selected from seeds, embryoids, embryos, organs, explants (e.g. segments from shoots (e.g. shoot tips), stems, tubers, roots (e.g. root tips), flowers or leaves, (e.g. leaf discs)), cells, protoplasts, seedlings, or plantlets. In some embodiments, the initial plant tissue may be callus tissue. For example calli, shooted and/or rooted calli.

In some embodiments, initial plant tissues may exclude mature adult plants. In some embodiments, initial plant tissues may exclude apical domes.

Initial plant tissue may be selected that is substantially disease or viral particle free. Thus, in some embodiments, initial plant tissue may have never been subjected to a pathogen infection step, such as a viral infection step.

The term “cultured plant tissue” in relation to the aspects and embodiments of the invention is used herein to cover plant material resulting from a plant tissue culture step. For example,

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LU503053 cultured plant tissue may comprise embryoids, embryos, explants, calli, shooted and/or rooted calli, seedlings or plantlets.

Cultured plant tissue may be genetically homogeneous and/or disease- or viral-free plant material.

In some embodiments, mature plants may be derived from the cultured plant tissue, for example wherein the cultured plant tissue is plantlets, these may be transplanted to soil or other appropriate media to obtain mature plants.

In addition to methods based on cell culture, the present invention also embraces the use of reverse transcriptase inhibitors in other forms of plant propagation e.g. from germinated seeds or via asexual reproduction (vegetative propagation, grafting etc.). In such cases the reverse transcriptase inhibitor may be applied directly to the plant or to the environment in which it is grown (whether in vitro, in controlled environments, or in external environments).

The methods may be applied to propagate plants whether genetically modified, genetically edited, or naturally occurring.

For crops which are propagated vegetatively, the ability to extrinsically control somaclonal variation may assist in breeding varieties which are less inherently stable, for example in relation to novel traits which are introduced into such varieties.

LTR-TE mobilization has been reported in tissue culture of plant species, including tobacco (Hirochika, 1993), sweet potato (Tahara et al., 2004), carrots (Kwolek et al., 2022),

Medicago (d’Erfurth et al., 2003), bamboo (Zhou et al., 2018) and date palm (Mirani et al, 2020).

However, those skilled in the art will recognise that LTR-TE activation is likely to occur in many species. Therefore those skilled in the art will appreciate that the methods described herein can be applied broadly to culture of plant tissue.

For example the plants may be a monocotyledonous plant (monocot), (e.g. as shown in

Arabidopsis in Example 2) which is a flowering plant (angiosperm) having embryos with one cotyledon or seed leaf, parallel leaf veins, and flower parts in multiples of three. Examples of monocots include, but are not limited to turfgrass, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, and palm.

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The plant may be a dicotyledonous plant (dicot), (2.9. as shown in rice in Example 5) which is a fowering plant whose embryos have two seed halves or cotyledons, branching leaf veins, and flower parts in multiples of four or five. Examples of dicots include but are not imited to, Eucalyptus, Populus, Liquid amber, Acacia, teak, mahogany, cotlon, tobacco, Arabidopsis, tomato, potato, sugar beet, hroccoii, cassava, sweet potato, pepper, poinsettia, bean, alfalfa, soyhean, carrot, strawberry, letluce, oak, maple, walnut, rose, mint, squash, daisy, geranium, avocado, and cactus.

The plant may be a gymnosperm, which is a seed producing plant that differs from angiosperms by having an exposed seed; not enclosed in an ovary or fruit. Gymnosperms include ginkgo, conifers (8.0. pines and yews), cycads, and gnetophyies

In one embodiment the plant is selected from the group consisting of acacia, alfalfa, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus,

Clementine, clover, coffee, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, sallow, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, a vine, walnut, watercress, watermelon, wheat, yams, yew, and zucchini.

Crop plants to which the invention may be applied include a maize plant, a wheat plant, a rye plant, a barley plant, an oat plant, a buckwheat plant, a sorghum plant, a rice plant, a sugarcane plant, a pigeon pea plant, a peanut plant, an onion plant, a garlic plant, a grass plant (including bent grass, fescue, brome, Timothy, orchard, Bermuda, zoysia, and the like), an Arabidopsis plant, a broccoli plant, a sunflower plant, a canola plant, a pea plant, a cowpea plant, a bean plant, a coffee plant, a soybean plant, a cotton plant, a linseed plant, a cauliflower plant, an asparagus plant, a lettuce plant, a cabbage plant, a tobacco plant, a spice plant (including curry, mustard, sage, parsley, pepper, thyme, cilantro, bay, cumin, turmeric, nutmeg, cinnamon, and the like), a sugar beet plant, a potato plant, a sweet potato plant, a carrot plant, a turnip plant, a celery plant, a tomato plant, an egg plant, a cucumber plant, a squash or melon plant and the like, a fruit tree plant (including apple, apricot, peach, pear, plum, orange, lemon, lime, and the like), a nut tree plant (including acorn, hickory,

Brazil, pecan, walnut, hazelnut, and the like), a grape plant, a berry plant (including blackberry, blueberry, strawberry, cranberry, and the like), and flower plants.

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In some embodiments there are provided a method of reducing somaclonal variation during plant tissue culture, comprising culturing initial plant tissue in culture media comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue, wherein said initial plant tissue is from Arabidopsis, rice, tobacco, sweet potato, carrots, medicago, bamboo or date palm tissue.

In one embodiment there is provided a method of reducing somaclonal variation during plant tissue culture, comprising culturing initial plant tissue in culture media comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue, wherein said initial plant tissue is rice tissue.

In some embodiments, initial plant tissue is not from Chinese cabbage (Brassica pekinensis).

Tissue culture medium, or other propagation media, may be a liquid or a solid, but for modified plants a solid culture may be preferred because a stable transformant can be produced by plating on the medium. When the medium is a liquid medium, static culture or shake culture may be performed.

The phrase “culturing initial plant tissue in a culture medium” as used herein encompasses use of both liquid and solid tissue culture medium.

When the medium is prepared as a solid medium, the medium may be converted to a solid using a solidifying agent. Non-limiting examples of the solidifying agent include agar, gellan gum (e.g. Gelrite), agarose, gelatin, and silica gel. The skilled person knows that a solidifying agent such as these may be added at any appropriate concentration for solidifying media for tissue culture or other plant propagation. For example, preferably between 0.6% and 2%. Preferably, a solidifying agent is used at a concentration of between 0.8 and 1.2%.

The medium may be prepared by adding a plant growth hormone and/or a carbon source, if necessary, to a base medium (e.g., any of the listed basal media and modified basal media obtained by altering the composition of the basal media) such as MS medium, LS medium,

B5 medium, and WP medium, among others that are well known in the art. The medium may be modified by addition of vitamins.

For example, Murashige and Skoog medium (MS) medium as used herein contains the macro- and micronutrients, and vitamins as described by Murashige and Skoog (1962) Plant

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Physiology, 15, 473-497, the contents of which are incorporated herein by reference. It is also known in the art to use half concentration MS media; “+ MS” media.

The methods of the present invention may include the active step of adding the nucleotide analogue reverse transcriptase inhibitor to a pre-existing (e.g. conventional) medium, such as those described above, or modifications of them as described below. The skilled person is aware that media can be modified, for example by the addition of sugars. Any carbon source may be used, including sugars such as sucrose, glucose, trehalose, fructose, lactose, galactose, xylose, allose, talose, gulose, altrose, mannose, idose, arabinose, apiose, and maltose.

Preferred media for use with the invention described herein are MS medium and modified

MS media obtained by altering the composition of MS medium, for example % MS media with 1% sucrose.

It is also known in the art to add plant hormones to culture media for plant tissue culture.

Examples of the plant growth hormone include auxin plant hormones and/or cytokinin plant hormones.

For further examples of conditions including plant growth hormones that may be added to plant culture media, see US10920234B2 which is incorporated herein by reference.

The skilled person will be able to select a pH for the medium that is appropriate for plant tissue culture or other methods of plant propagation. For example, the pH of the medium may be between pH 4.0 to 10.0, preferably 5.0 to 8.0, more preferably pH 5.7. As used herein, the pH of the solid medium means the pH of the medium that incorporates all the components except the solidifying agent.

In one aspect of the invention there is provided a media composition for plant tissue culture or a media composition for plant propagation, comprising a reverse transcriptase inhibitor, such as a nucleotide analogue reverse transcriptase inhibitor. As explained below, such media may be adapted for particular purposes e.g. one or more of a general plant propagation medium, a general plant tissue culture medium, a callus induction medium, a shooting medium, a rooting medium etc. The skilled person is aware that media compositions comprising a reverse transcriptase inhibitor as described herein may be adjusted so that any suitable basal media may be used, appropriate carbon source, vitamins and/or phytohormones added, and appropriate pH selected.

In some embodiments there are provided media compositions for plant tissue culture comprising a nucleotide analogue reverse transcriptase inhibitor, wherein the nucleotide analogue reverse transcriptase inhibitor is Tenofovir or an analogue or prodrug thereof.

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The skilled person will be able to select appropriate growth conditions that are appropriate for plant propagation, e.g. for culture of the relevant initial plant tissue to obtain a cultured plant tissue. For example, the propagation temperature is preferably between 4 and 40 °C.

The skilled person will know that the growth temperature is dependent on factors such as the plant species from which the initial plant tissue is derived. For example, in some embodiments the plants are propagated at 16 to 26 °C or 18 to 24 °C, preferably 19 to 23 °C. In some embodiments the plants are propagated at a temperature of 20-21 °C. In some embodiments the plants are propagated at ambient temperature e.g. between 15 and 25°C.

The skilled person will be able to select appropriate light conditions. For example, plant tissue may be cultured under 18h light conditions (18h light/6h dark), 16h light conditions (16h light/8h dark), 14h light conditions (14h light/10h dark) or 12h light conditions (12h light/12h dark).

The plant tissue culture of the present invention may include callus culture.

Thus the invention provides a method for reducing somaclonal variation in plants derived from tissue culture, the method comprising a plant tissue culture step comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue, wherein the plant tissue culture step comprises callus culture.

In one aspect there is provided a method of reducing somaclonal variation during plant callus culture, comprising culturing initial plant tissue in culture media comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue.

In some embodiments is a method of reducing somaclonal variation during plant callus culture, comprising culturing initial plant tissue in culture media comprising a nucleotide analogue reverse transcriptase inhibitor to obtain a cultured plant tissue, wherein the method comprises a callus induction step on culture media comprising the reverse transcriptase inhibitor.

A “callus” refers to undifferentiated plant cells or an undifferentiated plant cell cluster. The term “callus” as used herein may be an embryonic or pluripotent callus, or a callus in which shoots and/or roots have been induced (a shooted and/or rooted callus).

Thus, in embodiments where the plant tissue culture step comprises callus culture, the initial plant tissue may be for example a seed, embryo, explant (e.g. segments from shoots (e.g.

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LU503053 shoot tips), stems, tubers, roots (e.g. root tips), flowers or leaves, (e.g. leaf discs)), protoplast, embryonic callus, pluripotent callus, or a shooted and/or rooted callus.

Thus, in embodiments where the plant tissue culture step comprises callus culture, the cultured plant tissue may be for example an embryonic callus, pluripotent callus, a shooted and/or rooted callus, or a plantlet. In some embodiments, mature plants may be derived from the cultured plant tissue obtained by callus culture, for example by transplanting rooted calli to growth media or soil for subsequent growth.

Also provided is a method of preventing new LTR-TE insertions in cultured plant tissue, by induction of calli on culture media culture media comprising a reverse transcriptase inhibitor.

In some aspects, application of a reverse transcriptase inhibitor to callus culture media precludes or reduces new LTR-TE insertions in mature plants derived from the cultured plant tissue obtained by callus culture. In some aspects, application of a reverse transcriptase inhibitor to callus cultures limits genetic instabilities of mature plants derived from the cultured plant tissue obtained by callus culture. Methods of callus culture are well known in the art. For example, as described in Nishimura et al., 2006, and McCormack and Simon, (2006) Curr Protoc Microbiol p. 16D.1.1-16D.1.9, which are herein incorporated by reference it their entirety.

Also provided is a method according to any of the embodiments described herein, comprising a callus induction step. A callus induction step may result in formation of calli in vitro. Thus, the cultured plant tissue is callus tissue. Calli may be inducted from various sources, such as from seeds, from wounded plant tissue or from protoplasts.

In some embodiments, the callus induction step comprises culturing initial plant tissue in callus induction medium. In some embodiments, the callus induction medium comprises a reverse transcriptase inhibitor.

Suitable media for callus culture (for example for use as callus induction medium) is known inthe art. For example, Chu (N6) medium is defined to improve the formation, growth and differentiation of pollen callus in rice. It is known that the concentration of ammonium is crucial for callus development. The skilled person can select media that is appropriate for the plant species to be cultured. For example, N6D media is generally used for rice callus culture. N6D media is N6 media with the addition of casamino acids and proline, and can be further improved by adding myo-inositol (as described in more detail in Toki, S., 1997 and

Nishimura et al., 2006).

Exogenous application of auxin and cytokinin is known to induce calli in various plant species. A 2,4-Di-chlorophenoxy-acetic acid (2,4-D) is a commonly applied auxin for induction of calli.

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In some embodiments, the callus induction medium is N6D media comprising a reverse transcriptase inhibitor. In some embodiments, the callus induction medium is N6D media comprising Tenofovir or an analogue or prodrug thereof.

The skilled person is able to determine appropriate growth conditions for plant callus culture.

For example, growth temperature and light conditions may be different from tissue culture of seedlings or larger explants.

The skilled person will be able to select appropriate temperature conditions that are appropriate for plant callus culture. For example, the callus culture incubation temperature is preferably between 4 and 40° C. The skilled person will know that the growth temperature is dependent on factors such as the plant species from which the plant tissue is derived. For example, in some embodiments calli are incubated at 16 to 35 °C, 18 to 33 °C, 20 to 31 °C, 22 to 29 °C, preferably 24 to 28 °C. In some embodiments calli are incubated at ambient temperature e.g. between 15 and 25°C. In some embodiments calli are incubated at 27 °C.

When using any of the methods described herein, the skilled person will be able to select appropriate light conditions for plant callus culture. For example, calli may be cultured under 18h light conditions (18h light/6h dark), 16h light conditions (16h light/8h dark), 14h light conditions (14h light/10h dark) or 12h light conditions (12h light/12h dark). Preferably, plates for induction of calli may be incubated in the dark.

Accordingly, provided herein is a method of plant callus culture culturing initial plant tissue in callus induction medium, comprising a reverse transcriptase inhibitor, wherein calli are induced by incubation in the dark.

It may be preferred to use a different concentration of reverse transcriptase inhibitor for callus culture compared to other tissue culture methods, for example because the calli remain in tissue culture for a longer time.

In some embodiments, culture media for callus culture comprises callus induction medium, shooting media for inducing shoots and/or rooting medium for inducing roots.

In some embodiments, callus induction media comprises a reverse transcriptase inhibitor. In some embodiments, shooting media comprises a reverse transcriptase inhibitor. In some embodiments, rooting media comprises a reverse transcriptase inhibitor.

The reverse transcriptase inhibitor is added to culture media for callus culture (e.g. callus induction medium, shooting medium and/or rooting medium) at any suitable concentration.

For example, the reverse transcriptase inhibitor is added to culture media for callus culture at a concentration of 1-200 uM. For example, the reverse transcriptase inhibitor is added to

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LU503053 culture media for callus culture at a concentration of 1-200, 1-175, 1-150, 1-125, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 5-70, 5-65, 5-50, 10-50, 10-40, 20-60, 20-50 or 20-40 uM.

Preferably the reverse transcriptase inhibitor is added to culture media for callus culture at a concentration of 20-60 uM.

For example, in some embodiments the reverse transcriptase inhibitor is added to culture media for callus culture at a concentration of about 1, about 10, about 20, about 30, about 40, about 50 or about 60 uM.

In some embodiments, the reverse transcriptase inhibitor is added to callus induction media at a concentration of 1-60 uM.

In some embodiments, the reverse transcriptase inhibitor is added to shooting media at a concentration of 1-60 uM.

In some embodiments, the reverse transcriptase inhibitor is added to rooting media at a concentration of 1-60 uM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to culture media for callus culture at a concentration of about 1, about 10, about 20, about 30, about 40, about 50 or about 60 uM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to culture media for callus culture at a concentration of 1-60 uM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to callus induction media at a concentration of 1-60 uM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to shooting media at a concentration of 1-60 uM.

In some embodiments, the reverse transcriptase inhibitor is Tenofovir and is added to rooting media at a concentration of 1-60 uM.

In some embodiments there is provided a method of reducing somaclonal variation during plant callus culture, comprising a callus induction step that comprises culturing initial plant tissue on callus induction medium comprising the reverse transcriptase inhibitor, wherein the reverse transcriptase inhibitor is added to callus induction medium at a concentration of 20- 40 uM.

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It is known in the art that formation of calli may occur after several weeks. The skilled person is familiar with culturing calli for an appropriate amount of time. For example, calli may form after 3-4 weeks and are kept on plates for at least another four weeks before plant induction.

Accordingly, in some embodiments, calli are induced on callus induction media comprising the reverse transcriptase inhibitor for 7 — 8 weeks. However, it will be appreciated that this duration may be adjusted dependent on observed speed of calli formation.

A shoot inducing step is known as part of callus culture methods known in the art. A shoot inducing step may comprise culturing a callus in a shooting medium. For example, a shoot inducing step to obtain a shoot may comprise culturing the callus obtained in the callus inducing step in a shooting medium. A shooting medium may comprise appropriate phytohormones to induce formation of shoots. It is known in the art that appropriate shooting media may be optimised depending on plant species, for example carbon source, pH, and levels of phytohormones. For example, a shoot inducing step according to methods described herein may be performed by transfer of calli to MS-NK medium. Briefly, MS-NK medium is MS medium with the addition of MS-Vitamins, myo-inositol, casamino acid, NAA, kinetin, sucrose and sorbitol (as described in more detail in Nishimura et al., 2006).

A root inducing (or rooting) step is part of callus culture methods known in the art. A rooting step may comprise culturing a callus in a rooting medium. For example, after shoots have developed, shoots of 3—4 cm in length may be transferred to MS medium or any other suitable plant growth medium for use as a rooting medium. This rooting medium may comprise appropriate phytohormones to induce formation of roots. In some embodiments, the method comprises a rooting step comprising culturing a callus in a rooting medium comprising a reverse transcriptase inhibitor. For example, a rooting step to obtain a rooted callus may comprise culturing shooted calli obtained in a shoot inducing step in rooting medium. This rooting medium may comprise a reverse transcriptase inhibitor.

It will be appreciated that shooting and rooting steps can be performed in either order.

Callus tissue following shooting and rooting steps may be referred to as a shooted/rooted callus and may be encompassed by the term ‘plantlet’. In some embodiments, plantlets may be further cultivated on suitable media (e.g. MS media) or may be subsequently moved to soil pots. In some embodiments, there is a method of regenerating a plant by transferring the cultured plant tissue to growth media or soil.

Also provided herein is a plant tissue culture medium, a callus induction medium, a shooting medium, or a rooting medium (or a medium providing more than one of these functionalities) which medium comprises a reverse transcriptase inhibitor. In some embodiments, the reverse transcriptase inhibitor is a nucleotide analogue reverse transcriptase inhibitor,

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LU503053 optionally Tenofovir. Said medium may be adapted for use in any method described herein, for example choice of carbon source, pH and levels of phytohormones.

Methods for reducing somaclonal variation as described herein may be used during methods of plant transformation and plant regeneration, for example for preparation of genetically modified plants.

Accordingly, in some embodiments of the methods described herein, the initial plant tissue comprises a genetic modification. A genetic modification may be present relative to a control plant. A control plant may be one which is otherwise identical but has not been subjected to any methods of genetic modification.

Methods of genetic modification are well known in the art, and do not per se form part of the present invention. Examples of common genetic modifications include: (i) introduction of a gene or other nucleotide sequence heterologous to the plant tissue; (ii) gene editing of a native gene in the plant tissue; (iii) silencing of a native gene in the plant tissue, optionally by introduction of a silencing agent.

The term “genetic modification” is used broadly herein to indicate that a gene or other sequence of nucleotides in question has been modified in said cells or tissues using genetic engineering, i.e. by human intervention.

The term "heterologous" is used broadly herein to indicate that the gene or other sequence of nucleotides in question has been introduced into said cells or tissues using genetic engineering, i.e. by human intervention. The gene or other sequence of nucleotides in question are not necessarily foreign to the plant tissue, but may be introduced, for example, such as to be in a different genetic context. “Transformed” in this context means that the genetic modification alters one or more of the cell's genetic characteristics, and generally also phenotype. Such transformation may be transient or stable. Such transformation does not necessarily imply the presence of heterologous genes or other sequences.

Whether a genetically modified plant has been transformed can be determined by conventional methods, such as by performing DNA extraction from the plant followed by

PCR analysis of whether the target gene and the like have been introduced, or by further incorporating a reporter gene, e.g. GUS gene or GFP gene, into the vector followed by GUS or GFP observation.

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Accordingly, there is provided a method for reducing somaclonal variation in cultured plant tissue that comprises a genetic modification, comprising the step of introducing a genetic modification into the initial plant tissue prior to culture in a culture medium comprising a reverse transcriptase inhibitor.

In some embodiments, the initial plant tissue may be plant tissue comprising a desired genetic modification.

There is also provided a method of genetically modifying plants which includes tissue culture or callus culture steps of the invention described herein. In one embodiment the method may comprise a callus inducing step of culturing initial plant tissue comprising the desired genetic modification in a callus induction medium comprising a reverse transcriptase inhibitor to obtain cultured plant tissue comprising the desired genetic modification.

Thus, a method provided herein may comprise: (i) introducing a genetic modification into initial plant tissue, (ii) optionally a selection step for selecting initial plant tissue comprising the desired genetic modification, (iii) a tissue culture step comprising culturing the initial plant tissue comprising the desired genetic modification in a culture medium comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue comprising the desired genetic modification.

The step of introducing a genetic modification into the initial plant tissue may comprise: (i) introduction of a gene or other nucleotide sequence heterologous to the plant tissue; (ii) gene editing of a native gene in the plant tissue; and/or (iii) silencing of a native gene in the plant tissue, optionally by introduction of a silencing agent.

The tissue culture step may comprise a callus induction step. For example, the culture medium comprising a reverse transcriptase inhibitor may be callus induction medium, shooting medium and/or rooting medium.

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression (e.g. for expressing a heterologous nucleic acid within a host or one or more cells of a host). Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a

Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press

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LU503053 or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley &

Sons, 1992.

The use of Agrobacterium tumefaciens provides good transformation efficiency for many plant species. Accordingly, a method of reducing somaclonal variation in cultured plant tissue that comprises a genetic modification may comprise an infection step of infecting a tissue fragment from a plant with an Agrobacterium tumefaciens containing a plasmid comprising a target gene or a fragment thereof. The infection step may be performed prior to, or during, culture in the culture medium comprising a reverse transcriptase inhibitor.

The Agrobacterium tumefaciens containing a plasmid containing a target gene and the like may be prepared by any conventional method, such as by incorporating a target gene and the like into a plasmid capable of homologous recombination with the T-DNA region of the Ti plasmid of Agrobacterium tumefaciens to prepare a target gene recombinant intermediate vector, and introducing the target gene recombinant intermediate vector into Agrobacterium tumefaciens. Alternatively, it may be prepared by incorporating a target gene and the like into a binary vector, which is generally used in Agrobacterium techniques, to prepare a target gene binary vector, and introducing the vector into Agrobacterium tumefaciens.

Another method includes introducing a plasmid containing a target gene and the like into

Agrobacterium tumefaciens by electroporation.

Genome editing (gene editing) is a group of technologies that allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A well-known one is called CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9.

The CRISPR/Cas9 system has been successfully applied in various plant species. These include not only model plants, such as Arabidopsis, but also crops, such as rice, tobacco, sorghum, wheat, maize, soybean, tomato, potato, poplar, apple and banana. Calli, leaf discs, protoplasts and flowers have been used as a plant material.

Conventionally, editor genes are placed in DNA constructs and then delivered to various plant cells using Agrobacterium tumefaciens or particle bombardment-mediated transformation. Accordingly, provided herein is a method of genetically modifying plants provided herein may comprise an infection step of infecting a tissue fragment from a plant with an Agrobacterium tumefaciens containing a plasmid comprising a nucleic acid construct encoding a CRISPR-Cas nuclease.

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However, CRISPR-Cas systems can also be used in transgene-free methods of gene editing, such as using transient expression of CRISPR/Cas9 DNA, for example either by using particle bombardment or using transient expressing using Agrobacterium tumefaciens.

Another approach may be using preassembled Cas9 protein.

Gene silencing may be achieved by methods well known in the art.

For example, in using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al,(1988) Nature 334, 724-726; Zhang et al,(1992) The Plant Cell 4, 1575- 1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression. See, for example, van der Krol et al., (1990) The Plant

Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The

Plant Cell 4, 1575-1588, and US-A-5,231,020. Further refinements of the gene silencing or co-suppression technology may be found in W0O95/34668 (Biosource); Angell & Baulcombe (1997) The EMBO Journal 16,12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553.

Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire A. et al., Nature, Vol 391, (1998)). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAI) (See also

Fire (1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490, Hammond et al. (2001) Nature Rev. Genes 2: 1110-1119 and Tuschl (2001) Chem. Biochem. 2: 239-245).

RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt). The siRNAs then target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001)).

Another methodology known in the art for down-regulation of target sequences is the use of “microRNA” (miRNA) e.g. as described by Schwab et al 2006, Plant Cell 18, 1121-1133.

This technology employs artificial miRNAs, which may be encoded by stem loop precursors

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LU503053 incorporating suitable oligonucleotide sequences, which sequences can be generated using well defined rules in the light of the disclosure herein.

It will be appreciated that methods of callus culture as known in the art and described hereinabove can be used with methods of genetically modifying plants as described herein.

For example, methods of genetically modifying plants as described herein may comprise genetic modification of plant tissue, followed by propagation on media comprising a reverse transcriptase inhibitor.

For example, propagation of genetically modified material may comprise a callus inducing step of culturing the tissue fragment in a callus induction medium comprising a reverse transcriptase inhibitor, and/or may comprise a shoot inducing step to obtain a shoot, of culturing the callus obtained in the callus inducing step in a shooting medium, and/or may comprise a rooting step of culturing the shoot obtained in the shoot inducing step in a rooting medium to root the shoot. The shooting medium or rooting medium comprise a reverse transcriptase inhibitor.

Also provided is a cultured plant tissue, callus, or plant, produced by any of the methods described herein.

A plant produced by the methods described herein may have reduced somaclonal variation compared to control plants or control cultured plant tissue that has been cultured in culture media which does not comprise a reverse transcriptase inhibitor. For example, plants produced by the methods described herein may be free or substantially free from new LTR-

TE insertions. Plants or cultured plant tissue produced by the methods described herein may be more genetically uniform or have limited genetic instabilities compared to control plants or control plant tissue that have been cultured in culture media which does not comprise a reverse transcriptase inhibitor.

The invention also extends to cloned, or selfed or hybrid progeny of such plants obtained by the process, and also a leaf, stem, or edible portion of such plants.

The invention also provides a plant propagule from such plants, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.

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Also provided is use of a reverse transcriptase inhibitor as a new tool for studies of LTR-TE transposition, for example for studies of LTR-TE transposition during plant propagation or plant tissue culture.

In some embodiments is provided use of a reverse transcriptase inhibitor to discriminate the role of LTR-TEs mobilisation from other TEs or epialleles, or in relation to genome instability observed in hypomethylated plant genomes.

Thus, provided is a method for assessing LTR-TE transposition, comprising comparing the rate of LTR-TE transposition between cultured plant tissue that was cultured on media comprising a reverse transcriptase inhibitor with cultured plant tissue that was cultured on media lacking a reverse transcriptase inhibitor.

Also provided is use of a reverse transcriptase inhibitor to discriminate specific effects of

LTR-TE exDNA accumulation independently from the production of LTR-TE mRNA.

A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier’ includes mixtures of two or more such carriers, and the like.

Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

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The invention will now be further described with reference to the following non-limiting

Figures and Examples. Other embodiments of the invention will occur to those skilled in the artin the light of these.

Figures

Figure 1. Inhibition of ONSEN exDNA in Arabidopsis thaliana using Tenofovir. a) Quantification of ONSEN DNA copy number in heat stressed Arabidopsis thaliana nprd1 mutant seedlings germinated in presence of Tenofovir (1 uM and 10 pM). Unstressed mutant seedlings (marked as “Not stressed”) and heat stressed seedlings without drugs (Mock) were used as control. Each bar represents the mean of three technical replicates; +s.d. marked by error bars. b) Quantification of changes in ONSEN mRNA accumulation as consequence of heat stress in Arabidopsis nprd1 seedlings, in absence (left hand side) or in presence (right hand side) of 10 pM Tenofovir in the medium. CSO = not stressed plants. HSO = after heat stress. HS3 = three days after stress was applied.

Figure S1. Screening for chemicals inhibiting activation of retrotransposition in plants.

Arabidopsis nrpd1 seedlings have been growing in the presence of chemical inhibitors of

Reverse Transcriptase (Nevirapine, Stavudine, Lamivudine, and Zidovudine) at two concentrations in the medium (1 and 10 uM). Plants were heat stressed for 24 hours at 7 days after germination, and accumulation of exDNA of ONSEN have been evaluated by gPCR. Unstressed mutant seedlings (marked as not stressed) and heat stressed seedlings without drugs (Mock) were used as control. Each bar represents the mean of three technical replicates; +s.d. marked by error bars.

Figure S2A. Schematic representation of the heat stress experiment.

One-week old Arabidopsis seedlings were incubated at 4 °C for 24 hours and immediately transferred to optimal growth conditions (Control plants, marked as CS) or to 37 °C for 24hr (Heat Stressed, marked as HS) to induce ONSEN activation (Ito et al., 2011). Then, control and heat stressed plants grown at constant 21 °C, and samples have been collected immediately (0 days) and 3 days after heat-stress (generating the samples CS0, HSO and

HS3).

Figure S2B. Hierarchical Clustering of the full transcriptomic profiles of samples treated as described in Fig. S2a.

Samples treated with Tenofovir are marked with a T.

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Figures S2C and S2D. Relative copy number of ONSEN in samples treated with

Tenofovir

Relative copy number of ONSEN in Col-0 (S2C) and nrpd1-3 (S2D) seedlings grown in absent or in presence of Tenofovir (10 uM), calculated using the Actin 2 (ACT2) and C-

REPEAT/DRE BINDING FACTOR 2 (CBF2) genes as reference. Values displayed are relative to the ONSEN DNA levels in Col-0 collected at CS0. Each bar represents the mean of three repetitions; +s.d. marked by error bars. P-values were calculated by a one-tailed

Student's t-test. * = p < 1x10-2; ** = p < 1x10-3.

Figure 2. Tenofovir prevents ONSEN exDNA accumulation and its genome integration without significantly affect plant growth and response to heat. a) Representative pictures of mock-treated control plants (0 uM) and Tenofovir-treated plants (10 uM) grown in plate for 14 days at 21 °C, displaying absence of visible alterations induced by Tenofovir. b) Scatter plots summarizing correlation of gene expression in plant samples grown in absence or in presence of Tenofovir (10 uM). The data are from not stressed plants (CSO), plants collected after heat stress (HSO) and three days after the stress was applied (HS3).

Each dot represents a single gene. Pearson correlation was used to assess the significance of the gene expression differences (r and p-value are indicated). c) Detection (by inverse-PCR) of circular DNA forms of ONSEN in Col-0 and nrpd1-3 plants, grown in absence or presence of Tenofovir (10 uM). The amplification of ACT7 (AT5G09810) was used as loading control. The conditions tested include stressed plants (HSO0); plants collected after 1, 4, and 11 days of recovery from the stress (respectively HS1,

HS4 and HS11); and flower tissue obtained from recovering plants at flowering time, 20 days after the stress (HS-flower). d) Transposon display performed on genomic DNA from single nprd1 mutant seedlings from the seed progeny of Arabidopsis stressed plants in presence or absence of Tenofovir (10 pM). Samples from Col-0 heat stressed parents were used as control. New insertions are marked with stars (*).

Figure S3. Tenofovir inhibits Tos77 mobilisation in rice calli. a) Representative pictures of generated rice calli at the stages of 3 weeks (upper panels) and 7 weeks (lower panels) following the induction. The calli grown in media containing 0, 20 or 40 uM of Tenofovir, as indicated. b) Detection of 70s17 circular exDNA forms accumulated during rice callus induction in absence (0 uM) or in presence (20 uM and 40 uM) of Tenofovir. PCR of eEF1a gene is used as loading control. c) Accumulation of linear exDNA of Tos 77 extrachromosomal DNA from rice calli generated in absence (0 uM) and in presence (40 uM) of Tenofovir. The Tos 17 exDNA was amplified from cDNA obtained by ALE-seq using two different pairs of Tos 77 specific primers (left and

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LU503053 right panels) (Primers sequences are reported in Table 1). DNA from wild type leaves was used as negative control. d) Scheme representing the sequence of two new 70s17 insertions in coding genes, obtained by cloning and sequencing the Transposon Display product from lane#3 in the Fig. 3d. The figure displays the region joining Tos77 LTR and the genes neighbouring the new integration. The scheme was obtained with the program Geneious (Biomatter). e) Validation of two new Tos 77 insertions in plants regenerated from calli grown in absence of Tenofovir (sample #3 in the Fig. 3d). Total DNA from wild type leaves was used as negative control (marked as NC). Location of the primers used for PCR validation is shown.

Figure 3. Rice plants regenerated in presence of Tenofovir are free from Tos77 new insertions. a) Quantification by qPCR of circular exDNA of Tos 17 accumulating in rice calli induced in media supplemented with 0 uM, 20 uM and 40 uM Tenofovir. The relative copy number of exDNA was calculated using Ubiquitin gene as reference. The values displayed are relative to the 70s17 DNA levels in samples from Tenofovir-untreated callus. The DNA from leaves was used as negative control. Two biological replicates are displayed. Each bar represents the mean of three technical replicates; +s.d. marked by error bars. P-values were calculated by a one-tailed Student's t-test. b) Read coverage plots obtained from the ALE-seq of rice callus induced in the absence (0

MM) or presence (40 uM) of Tenofovir, mapped at Tos77 and Tos79 genomic loci. DNA extracted from leaves was used as negative control. The coverage range is indicated in square brackets. c) Two representative examples of rice plants regenerated from calli propagated in absence orin presence of Tenofovir (0 uM and 20 uM) displaying similar development and architecture. d) Transposon display of 70s17 in plants regenerated from calli in absence or in presence of

Tenofovir (0 uM, 20 uM and 40 uM). The most evident band common to all plants corresponds to the original Tos77 copy in the reference genome. Bands marked with stars (*) correspond to the new 70s17 insertions. The number of new 70s17 insertions in

Tenofovir-treated and control plants has been found significantly different (P-value = 0.0067,

Mann-Whitney U test). DNA extracted from leaves of naturally propagated plants was used as negative control.

Examples

EXAMPLE 1: Materials and Methods

Screening for chemical inhibition of retrotransposition using Arabidopsis ONSEN activation by heat.

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All Arabidopsis thaliana plants used were in Col-0 background. The nrpd1a-3 mutant line used was described in Herr et al, 2005. Seedlings were grown on % MS media (1% sucrose and 0.8% agar, pH5.7) at 20 °C under 12h light conditions (12h light/12h dark). In drug treated plants, Tenofovir (Cayman Chemical, N 13874), Nevirapine (Merck, SML0097),

Stavudine (Merck, Y0000408), Lamivudine (Merck, L1295) or Zidovudine (Stratech Scientific

Ltd, B2221) were dissolved in DMSO and added to the medium at the final concentration specified. The same volume of DMSO was added to the control untreated medium. Seven days after germination, plants were treated to activate ONSEN, as described previously (Ito et al, 2011) (Fig. S2a). Briefly, one-week old seedlings were primed by cold treatment for 24 °C and immediately transferred to normal growth conditions (Control plants or CS) or to 37 °C for 24hr (Heat Stressed or HS) plants. Plants were collected 24h after heat application (HSO0) and accumulation of linear extra-chromosomal DNA (ecDNA) of ONSEN were evaluated by qPCR as described before (Ito et al., 2011).

Tenofovir-treated and control Arabidopsis plants were subsequently grown at 21 °C for the remaining time, and samples were collected 0, 3, 11 and 14 days after heat-stress depending on the experiment (condition defined CSO, CS3, CS11, CS14 and HSO, HS3,

HS11, HS14 for control and heat stressed plants respectively). Pools of plants or individual samples were harvested, flash-frozen in liquid nitrogen. Individual seedlings were transferred on soil (Levington F2) and grown until the flowering time. Progeny of these plants were used to test for the presence of new insertions by Transposon Display.

Genome wide expression analysis

Arabidopsis plants were grown and treated as described above. Pools of plants were harvested, flash-frozen in liquid nitrogen at specific time points (CS0, CSO+T, HSO, HSO+T,

HS3, HS3+T). Three replicates were collected from each condition. Total RNA was isolated from seedlings using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer's instructions. The RNA quality and integrity were assessed on the Agilent 2200 Tape Station.

Library preparation was performed using 1 mg of high-integrity total RNA (RIN>8) using the

TrueSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). Libraries were sequenced in-house on an Illumina NextGen 500 sequencer using paired-end sequencing of 150 bp in length.

RNA-Seq Mapping and Differential Expression Analysis

The raw reads obtained from sequencing were analysed using a combination of publicly available software and in-house scripts. Trimmomatic (Bolger et al., 2014) was used to remove adapters and discard low-quality reads. The cleaned reads were mapped to the

Arabidopsis reference genome (TAIR10 version) by TopHat2 (Kim et al., 2013). Picard tools (available from http://broadinstitute.github.io/picard/) was used to discard duplicated reads.

The processed data was visualised in SeqMonk (version 0.32.1 available from www. bioinformatics.babraham.ac.uk). The reads were quantitated and clustered following

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RNA-seq Quantitation pipeline integrated into SeqMonk. Mapped reads from TopHat2 analysis were counted using htseq-count (Anders et al., 2015), and the raw counts per gene was used to determine differentially expressed genes between the control and Tenofovir- treated samples using DESeq (Anders and Huber, 2010) using the default parameters.

Genes with a significance p-value below 0.05 after Benjamini and Hochberg correction were considered to be differentially expressed and were used for analysis of Gene Ontology (GO) enrichment using agriGO, a GO analysis toolkit (Du et al., 2010). Hierarchical Clustering of the full transcriptomics of the samples was undertaken using the SeqMonk tool. The correlation between transcriptomic profiles of samples untreated and treated with Tenofovir was visualised as scatter plots in an R programming environment. Pearson correlation coefficient was calculated using R/Bioconductor (v3.8). Sequencing data have been deposited in Gene Expression Omnibus under the accession numbers GSE196868 and

GSE196869.

Procedure of induction of Rice calli and regeneration of the plants

N6D medium (for callus induction) was prepared by dissolving 3.98g CHU (N6) basal salt mixture, 10 ml of 100 N6-vitamin, 0.1g myo-inositol 0.3g casamino acid, 2.878g proline, 10 ml of 100 2,4-D and 30g sucrose in 800 ml distilled water. The pH was adjusted to 5.8 and 3g gellan gum was added. The volume was adjusted to 1 litre with distilled water and autoclaved.

MS-NK medium (for plant regeneration) was prepared by dissolving 4.6g MS plant salt mixture, 10 ml of 100 x MS-Vitamins, 0.1g myo-inositol, 2g casamino acid, 1ml of 1,000x

NAA, 20 ml of 50x kinetin, 30g sucrose and 30g sorbitol in 800 ml distilled water. The pH was adjusted to 5.8 and 3g gellan gum was added. The volume was adjusted to 1 litre with distilled water and autoclaved. Appropriate selection (1000 x Carb) was added after cooling.

Rice callus were induced by the method used for rice transformation as previously described (Nishimura et al., 2006). Briefly, seeds of Oryza sativa ssp. japonica cv. Nipponbare were surface-sterilised in 20% bleach for 15 minutes, rinsed three times with sterile water and placed on N6D media in presence of Tenofovir. Compared to the Arabidopsis experiments,

Tenofovir concentration in the media was increased, and Tenofovir was supplemented at 0

MM, 20 uM or 40 uM in the plates. This was done because the rice calli were planned to remain in tissue culture for a longer time, and also because we assumed that the assimilation of the drug in the calli was less efficient than in Arabidopsis seedlings. Plates have been incubated at 27 °C in the dark. The calli formed after 3-4 weeks and were kept on plates for at least another four weeks before plant induction. For shoot induction, calli were transferred to MS-NK medium and were kept in plates for around 8 weeks. Shoots of 3—4 cm in length were transferred in MS medium in plant boxes. Transplant rooted plantlets were then moved into soil pots, with one plantlet per pot (Nishimura et al., 2006). We managed to

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LU503053 regenerate eight, five and one plant from 36 initial calli induced in presence of respectively 0 pM, 20 uM and 40 pM of Tenofovir (12 calli initialised per treatment) (Fig. S3a).

Detection of exDNA of ONSEN and Tos17

Total DNA was extracted from Col-0 and nrpd1-3 Arabidopsis plants or from rice calli grown with or without Tenofovir, using the plant DNeasy kit (Qiagen) according to the manufacturer’s instructions. The presence of ONSEN and Tos 77 circular exDNA was tested using inverse PCR with primers designed inside LTRs in opposite directions (Table 1).

Table 1. List of primers/oligos used for RNA-seq validations

IE 8 _2 ACCAGCCC- 3'-Amino . 8

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The PCR reaction was performed using Go Tag polymerase (Promega). The PCR conditions were as followed: (i) ONSEN — 95 °C 2 min, 35 cycles 95 °C 30 s, 58 °C 30 s, 72 °C 2 min; (il) 70s17 — 95 °C 2 min, 35 cycles 95 °C 30 s, 55 °C 30 s, 72 °C 30 s. PCR of actin and eEF-la were used as loading control for ONSEN and 70s17, respectively.

Analysis of LTR-TE DNA by qPCR ran on a LightCycler480 (Roche) using LightCycler 480

SYBR Green Master (Roche) with three technical replicates for each sample. For ONSEN, the relative copy number was calculated using the actin 2 gene (ACT2) and C-REPEAT/DRE

BINDING FACTOR 2 (CBF2) as reference and normalised by DNA levels in Col-0 collected atCSO0. For 70s17, the relative copy number of circular extrachromosomal DNA was calculated using the Ubiquitin (UBS) as a reference and normalised by DNA levels measured in the samples collected from untreated calli. Primers used are listed in Table 1.

Transposon Display

Transposon Display was applied as described previously with minor modifications (Griffiths et al., 2018). GenomeWalker (GW) adaptor oligoes were prepared at 25 uM (Table 1) and then heated at 95°C for 10 minutes and allowed to cool to room temperature. Genomic DNA was extracted from seedlings using the CTAB method; 300 ng DNA was digested using the

Dra1 restriction enzyme (New England Biolabs), which produced blunt-ended fragments compatible for ligation with the GW adaptor (300 ng DNA, 5 pL cut smart buffer, 2.5 pl Dra and H20 to 50 pl). Digestion was carried out at 37°C for 16 h, cleaned using a QIAGEN Gel

Extraction Kit and eluted in 2x15 pl EB buffer. Adaptor ligation was carried out at 16°C for 24 h (digested DNA 5 ul, GW adaptors (25 uM) 2 ul, 10x ligation buffer 1.6 pl, T4 Ligase 1 pl and H0 to 16 pl). Ligated DNA was diluted 1:20. To detect new insertions of ONSEN, PCR was carried out using a primer matching the adaptor sequence (GenomeWalker_AP1) and a primer matching the LTR sequence (TD_ONSEN_LTR). In the case of 70s17, nested PCR was carried out using two primers matching the adaptor sequence (GenomeWalker_AP1 and Genome\Walker_AP2) paired with two primers matching 70s17 LTR (TD_TOS17_LTR_1 and TD_TOS17_LTR_2). All primer sequences are reported in Table 1. PCR was carried out using GoTaq polymerase (Promega) in the following conditions: primary PCR 95°C 2 min,

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LU503053 33 cycles 95°C 30 s, 58°C 30 s, 72°C 1 min; secondary PCR on 1:100 dilution of primary

PCR 95°C 2 min, 35 cycles 95°C 30 s, 58°C 30 s, 72°C 1 min. PCR products were cut from the gel and ligated into pGEM-T for sequencing. The fragment sequences are reported in

Fig. S3d.

ALE-seq and targeted LTR detection

Genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instruction, and 100 ng were used for ALE-seq following the previously described protocol (Cho et al., 2019), with minor modifications. An adapter, containing T7 promoter sequence at its 5’ end, was ligated to the end of extrachromosomal DNA, following by in vitro transcription with T7 RNA polymerase. The synthesised RNA was then reversed transcribed using the primer binding the transcripts at the LTR-TE conserved PBS site. At this stage, to specifically amplify Tos77 LTR, the cDNA was PCR-amplified using PBS and

Tos 17 specific primers (Table 1) and the PCR product was loaded onto agarose gel. PCR was carried out using GoTaq polymerase (Promega) and with the following conditions: 95°C 2 min, 30 cycles 95°C 30 s, 55°C 30 s, 72°C 30 s. The remaining cDNA was amplified following the ALE-seq procedure (Cho et al., 2019), and strand specific sequencing was performed using lllumina MiSeq platform and 300bp reads.

Insertion confirmations in rice

To confirm the presence of new Tos 77 insertions in regenerated rice plants (plant #3, Fig. 3d), the genomic DNA extracted as described above was used for PCR performed using primer pairs including a transposon-specific primer (Tos 77) and a primer located in the flanking region of the new insertion (the loci LOC_0Os07g35610 and LOC_Os11g10510).

Primer sequences are reported in Table 1. The PCR products were then run and extracted from the gel, and then ligated into pPGEMT-easy (PROMEGA) to confirm their DNA sequence by Sanger sequencing.

EXAMPLE 2: Tenofovir efficiently suppresses ONSEN extrachromosomal DNA (exDNA) accumulation and its retrotransposition in Arabidopsis without affecting plant growth

Here we examined RT inhibitors for their possible activity to suppress LTR-TEs mobilisation in plants, employing well documented conditions provoking retrotransposition

To screen for the most effective drug to inhibit the activity of retrotransposon encoded RT in plants, we used a system based on the environmentally controlled mobilisation of the LTR transposon ONSEN in Arabidopsis thaliana. ONSEN is activated in response to heat stress and mobilises in plants mutated in NPRD1, which is the main subunit of the plant specific

RNA polymerase IV (PollV) (Ito et al., 2011). In heat-stressed nprd1 seedlings, ONSEN activation can be easily detected by measuring its increased copy number, which is induced by the accumulation of its reverse transcribed exDNA and by integrations into new chromosomal loci. We germinated nprd1 seedlings in media containing two concentrations

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LU503053 (1uM and 10uM) of well-characterised RT inhibitors. This included the nucleoside RT inhibitors Zidovudine, Stavudine and Lamivudine; the nucleotide RT inhibitor Tenofovir; and the non-nucleoside RT inhibitor Nevirapine (De Clercq, 2009). Using quantitative PCR, we measured accumulation of ONSEN exDNA induced by heat stress in planta for each drug concentration, compared to the levels accumulated in seedling grown without RT inhibitors.

We observed that Tenofovir at 1 uM in the medium was able to reduce ONSEN exDNA to approximatively 50%, while exDNA accumulation was barely detectable when 10 uM concentration of Tenofovir was used (Fig. 1a).

In this screening attempt, we did not observe significant effects in reducing ONSEN exDNA levels for the other tested drugs (Fig. S1), and therefore we focused on Tenofovir in all subsequent experiments.

This could reflect different molecular proprieties of Tenofovir (a nucleotide analogue) compared to nucleoside and non-nucleoside RT inhibitors. For example, it is known that nucleotide RT inhibitors cannot be cleaved by esterases and are therefore more resistant to

DNA repair mechanisms (De Clercq, 2009). Nonetheless, we surmise that that Tenofovir may be assimilated into roots and systemically mobilised in plant tissue more efficiently compared to the other chemicals, which therefore may also have utility in suppressing LTR-

TE mobilization using different methods or conditions.

EXAMPLE 3: Determining specificity of Tenofovir activity

To determine the specificity of Tenofovir activity, we investigated the possible effects induced by this drug on Arabidopsis gene expression by genome wide RNA-seq. We compared the expression profiles of wild type seedlings grown in control media and in media supplemented with 10 uM Tenofovir. Experiments were performed under standard growth conditions, with or without heat stress, and after three days of recovery following the heat stress (Fig. S2a). First, we focused our attention on ONSEN mRNA level, which is known to accumulate during heat stress (Ito et al., 2011; Cavrak et al., 2014). We observed that the presence of Tenofovir in the media did not alter the levels of ONSEN transcripts induced in response to elevated temperatures (Fig. 1b). Therefore, while Tenofovir inhibited accumulation of ONSEN exDNA (Fig. 1a), it did not affect the increase in ONSEN's mRNA levels after application of heat stress (Fig. 1b).

In contrast to DNA methyltransferase inhibitors that have been efficiently used to induce epigenetic changes and activate LTR-TEs in plants, Tenofovir is the first drug with a specific inhibitory effect on LTR-TE mobilisation, acting with a mechanism which is independent on the alteration of RNA silencing or associated epigenetic mechanisms. Because the Tenofovir action is independent from the production of LTR-TE mRNA (Fig. 1), this drug could be used to discriminate specific effects of LTR-TE exDNA accumulation directly. Therefore, Tenofovir has also the potential to be a valuable tool to study LTR-TE regulation in various biological models.

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The seedlings grown in the presence of Tenofovir have not shown significant alterations in term of their growth and development for all life cycle (Fig. 2a), all plants flowered and produced seeds at comparable time, and their progeny developed normally. To determine possible hidden effects of Tenofovir on gene expression, we applied hierarchical clustering of the whole plant transcriptomics obtained from our various experimental samples. We observed that regardless of the presence of Tenofovir, the replicates of all the samples clustered only in accordance with their heat treatments, indicating that the effect of Tenofovir is negligible in relation to the effect of heat stress (Fig. S2b). Additionally, the expression profiles of seedlings grown in absence or in presence of Tenofovir under control conditions, heat stress and after recovery correlate respectively at 99%, 99% and 97% (Fig. 2b), indicating very similar mRNA profiles.

Our differential expression analysis identified only 80 genes with more than two times difference in the level of MRNA that can be attributed to the Tenofovir treatment. Of these, 44 differentially expressed genes (DEGs) were found in control conditions, 13 DEGs were found in the heat stress condition and 26 DEGs after recovery from heat stress, with only three DEGs identified in more than one condition. Within the DEGs identified in the control samples grown in the presence and absence of Tenofovir (CSO+/-T), there was an enrichment of “Plant-type cell wall organisation” (7 genes, FDR=0.00054) and “Root morphogenesis” (5 genes, FDR=0.038) genes. There was no significant GO enrichment in differentially expressed genes due to Tenofovir treatment after heat stress (HSO+/-T), while 14 differentially expressed genes in recovery samples (HS3+/-T) had GO related to “Response to stress, heat and bacterium” (FDR= 0.00028).

EXAMPLE 4: Tenofovir inhibits exDNA formation and transposition

The production of exDNA is a necessary step for transposition of LTR elements. New

ONSEN insertions were observed in the progeny of heat stressed nprd1 in previous studies (Ito et al., 2011), indicating that ONSEN exDNA persists during plant development and can integrate in flower germ cells of mutant plants. Therefore, we measured by qPCR the exDNA accumulation in both wild type and nprd7 mutant heat-stressed seedlings at 0, 1, 4, and 11 days following the heat stress, as well as in the flowers of fully developed plants.

We observed that the reduction in ONSEN exDNA induced by Tenofovir application was still significant at 11 days after the heat stress (Fig. S2c). Considering that qPCR detects exDNA formation by measuring the number of all ONSEN DNA copies (including the original copies integrated in the genome), this approach seems to be not sensitive enough to detect low levels of residual exDNA persisting in flowers of plants stressed at the seedling stage.

Therefore, to detect ONSEN exDNA in plants at flowering we used inverse primers to amplify selectively ONSEN circular DNA molecules. This circular DNA is considered to be a by-product of LTR-TE transposition with exclusively extrachromosomal nature (Lanciano et al., 2017). We observed that Tenofovir application can abolish or strongly reduce the production of circular exDNA in Col-0 and nprd1 mutant seedlings (Fig. 2c). Interestingly, the circular exDNA was also not detectable in flowers of Tenofovir-treated nord? seedlings,

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LU503053 potentially suggesting that the drug application could limit ONSEN transposition in the progeny of treated plants. To test this hypothesis directly, we investigated the presence of new ONSEN insertions in the progeny of heat-stressed nprd1 plants by Transposon Display.

While the progeny of control plants displayed several new ONSEN insertions, these were not detected in the progeny of plants treated with Tenofovir (Fig. 2d).

Collectively, these results indicate that the application of Tenofovir can efficiently suppress

ONSEN RT activity, inhibiting both ONSEN exDNA formation and subsequent transposition, with negligible effects on gene expression and Arabidopsis development.

EXAMPLE 5: Rice plants regenerated from Tenofovir-treated calli are free from new

LTR-TE insertions

To directly test if the application of Tenofovir in tissue culture of crop plants prevents LTR transposon mobilisation, we tested the use of this drug during propagation of rice calli, which is a condition where multiple LTR retrotransposons can be mobilised and lead to genetic variation in the regenerated plants (Hirochika et al., 1996). We induced rice calli in vitro from scutellum tissue of mature seeds (Nishimura et al., 2006), using either a standard medium, or the medium containing Tenofovir at 20 uM and 40 uM (Fig. S3a). We observed that the presence of Tenofovir in the medium strongly decreased the accumulation of Tos 77 circular exDNAs, measured by semi-quantitative PCR (Fig. S3b). Consistently, Tos77 copy number (estimated by gPCR) was reduced by about 50% and 80% in calli generated in presence of 20 uM and 40 uM Tenofovir, respectively, compared to calli generated in the absence of the drug (Fig. 3a).

Circular exDNA of LTR-TEs, a by-product of LTR transposons activity, can be generated either by recombination of LTR-TE copies integrated in the genome or from linear exDNA, and itis generally not reintegrated into the genome (Gaubatz, 1990). In contrast, linear LTR-

TE exDNA is a direct product of reverse transcription and can be inserted into the genome.

Therefore, we used the de-novo sequencing approach called ALE-seq (Cho et al., 2019) to detect LTR sequence of linear exDNA. With this method we detected a significant number of reads corresponding to the Tos 77 linear LTR sequence in calli propagated on standard media, but virtually no reads in samples propagated in presence of 40 uM Tenofovir (Fig. 3b). This result was validated by semi-quantitative PCR with two sets of Tos 17 specific primers applied on the same cDNA used for sequencing (Fig. S3c, Materials and Methods).

We also applied this approach to Tos 19, a different LTR transposon which is also active in rice calli (Sabot et al., 2011), and we obtained a pattern very similar to what was observed for 70s17 (Fig. 3b). These results indicate that Tenofovir application to rice calli can efficiently reduce levels of linear exDNA of the two most active LTR-TEs mobilised by in vitro tissue culture.

Finally, to directly evaluate the efficiency of Tenofovir to restrict LTR-TE transposition during the entire process of plant regeneration from tissue culture, we used an established protocol (Nishimura et al, 2006) to induce shoots and regenerate plants from Tenofovir-treated and control rice calli. We regenerated eight, five and one plants from 36 initial calli induced

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LU503053 respectively in presence of 0 uM, 20 uM and 40 uM Tenofovir (12 calli initialised per treatment) (Fig. S3a). Although the highest Tenofovir concentration used (40 uM) might have reduced the regeneration rate, all regenerated plants developed normally and did not display alterations until the reproductive stage, independently of the concentration of

Tenofovir in the original media (Fig. 3c).

Transposon Display showed that none of the six plants we regenerated from calli exposed to

Tenofovir had new Tos 77 insertions (Fig. 3d). In contrast, we observed an average of 3.25 of new Tos 77 insertions per genome in the eight plants regenerated from calli propagated in standard medium, with all plants but one showing new Tos 77 integrations (Fig. 3d). By cloning and sequencing of new junction fragments we verified that at least in one plant (plant #3, Fig. 3d) 70s17 had been inserted at new chromosomal locations, disrupting the coding sequences of two genes (Fig. S3d and S3e). These results indicate that the application of 20 uM Tenofovir in the culture medium is likely to allow the regeneration of rice plants free from new LTR-TE insertions.

The tissue culture passage necessary for generation of transgenic plants induces genome instability predominantly involving uncontrolled mobilisation of LTR retrotransposons (LTR-

TEs), which are the most abundant class of mobile genetic elements in plant genomes. Here we demonstrate that in conditions inductive for high LTR-TE mobilisation, like abiotic stress in Arabidopsis and rice callus culture, the application of the Reverse Transcriptase inhibitor known as Tenofovir significantly affects LTR-TE RT activity, without interfering with plant development.

Specifically, these results show that the application of Tenofovir, a nucleotide RT inhibitor, prevents mobilisation of the heat-inducible LTR-TE known as ONSEN in Arabidopsis, without significant impact to gene expression and plant development. Moreover, that the application of Tenofovir to rice callus cultures precludes new LTR-TE insertions in regenerated plants. These results show that Tenofovir could be widely used in tissue culture procedures, resulting in regeneration of genetically more uniform plant material.

EXAMPLE 6: Discussion

In Arabidopsis, the mutations of epigenetic factors such as the main DNA methyltransferase

MET1 or the chromatin remodeler DDM1 produce genome-wide DNA demethylation and bursts of TE mobilisation. This leads to phenotypic abnormalities and transgenerational decrease of fithess (Mirouze et al., 2009; Tsukahara et al., 2009; Griffiths et al., 2018).

Interestingly, these effects remain mostly evident in crosses of MET1 and DDM1 mutants with isogenic wild type lines, and in their segregating wild type progenies (Catoni and Cortijo, 2018). Specifically, several TEs were found to remain mobile for up to eight generations following the re-establishment of wild type alleles in epigenetic recombinant inbred lines obtained from one met1 or ddm1 original parent (Catoni et al., 2019; Quadrana et al., 2019).

Therefore, Tenofovir could be also used to discriminate the role of LTR-TEs mobilisation from other TEs or epialleles, in relation to genome instability observed in hypomethylated plant genomes.

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Although several protocols for crop transformation and genome editing are available, they all require growing plant tissue /n vitro as a necessary step in the regeneration of plants starting from genetically modified cells. Therefore, the uncontrolled LTR-TE mobilization induced during tissue culture makes it challenging to demonstrate “substantial equivalence” of new varieties obtained in this way with the natural antecedent cultivars, a process which is necessary to commercialise a new crop variety (Millstone et al., 1999; Gao et al., 2009; Li et al., 2019). In this context, the application of Tenofovir in the tissue culture media could become a very valuable approach to reduce the genetic diversity in propagated plant material, by inhibiting LTR-TE mobilisation and facilitating the regeneration of plants genetically more uniform.

Tenofovir suppression is expected to occur only for TEs that use RT enzymes for their transposition, while it will not affect somaclonal variation caused by other transposons (i.e.

DNA transposons), sequence rearrangements or changes in epigenetic state. Although rice is virtually the only major crop where LTR-TE activation during micropropagation has been extensively described, there are reports of LTR-TE mobilization occurring in tissue culture of other plant species, including tobacco (Hirochika, 1993), sweetpotato (Tahara et al., 2004), carrots (Kwolek et al., 2022), Medicago (d’Erfurth et al., 2003), bamboo (Zhou et al, 2018) and date palm (Mirani et al., 2020). Considering that for many crops the genetic resources are still limited, it could be possible that LTR-TE mobilization during plant regeneration has been mostly overlooked. However, considering the increasing availability of plant genome reference assemblies and TE detection tools (Satheesh et al., 2021), it is likely that LTR-TE activation will be documented in even more species, and Tenofovir will have application going beyond the stable in vitro propagation of Arabidopsis and rice.

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Claims

BL-5585 46 LU503053 CLAIMS 1 A method for inhibiting Long Terminal Repeat Transposable Elements (LTR-TEs) during propagation of a plant, the method comprising propagating the plant in the presence of areverse transcriptase inhibitor.
2 The method of claim 1 wherein the plant is propagated by plant tissue culture, comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to obtain the cultured plant tissue, whereby inhibiting LTR-TEs leads to reduction in somaclonal variation in cultured plant tissue
3 A method for reducing somaclonal variation in cultured plant tissue, the method comprising a plant tissue culture propagation step comprising culturing an initial plant tissue in a culture medium comprising a reverse transcriptase inhibitor to obtain the cultured plant tissue.
4. The method of claim 2 or claim 3, wherein the plant tissue culture step is micropropagation.
5. The method of any one of claims 2 to 4, wherein the initial plant tissue is selected from a seed, an embryoid, an embryo, an organ, an explant, pollen, or an anther which is optionally selected from material from shoots, stems, tubers, roots, flowers or leaves, a cell, a protoplast, a seedling, a plantlet, callus, which is optionally shooted and/or rooted callus.
6. The method of any one of claims 2 to 5, wherein the initial plant tissue comprises a genetic modification.
7 The method of claim 6, wherein the method comprises the step of introducing a genetic modification into the initial plant tissue prior to the plant tissue culture step.
8. The method of claim 6 or claim 7 wherein the genetic modification is selected from the list consisting of: (i) introduction of a gene heterologous to the initial plant tissue; (ii) gene editing of a native gene in the initial plant tissue; (iii) silencing of a native gene in the initial plant tissue, optionally by introduction of a silencing agent.
9. The method of claim 7 or claim 8, wherein the method comprises: (i) introducing a genetic modification into initial plant tissue, (ii) optionally a selection step for selecting initial plant tissue comprising the desired genetic modification,

BL-5585 47 LU503053 (iii) a tissue culture step comprising culturing the initial plant tissue comprising the desired genetic modification in a culture medium comprising a reverse transcriptase inhibitor to obtain a cultured plant tissue comprising the desired genetic modification. xxx
10. The method of any one of claims 2 to 9, wherein the nucleotide analogue reverse transcriptase inhibitor is present in the culture medium at a concentration of 1-50 uM.
11. The method of any one of claims 2 to 10, wherein the culture medium is % MS medium comprising 1% sucrose.
12. The method of any one of claims 2 to 11, wherein the tissue culture step is performed at a temperature of 19-23 °C.
13. The method of any one of claims 2 to 12, wherein the plant tissue culture step is a callus induction step, and the culture medium is a callus induction medium, and the cultured plant tissue is callus, and optionally the initial plant tissue is a seed, which is optionally rice.
14. The method of any one of claims 2 to 13, wherein the method further comprises a callus induction step prior to the plant tissue culture step, comprising culturing plant material in callus induction medium to provide initial plant tissue which is callus.
15. The method of claim 14, wherein the callus induction medium comprises a reverse transcriptase inhibitor.
16. The method of any one of claims 13 to 15, wherein the callus induction medium is N6D medium.
17. The method of any one of claims 13 to 16, wherein the reverse transcriptase inhibitor is present in the callus induction medium at a concentration of 20-40 uM.
18. The method of any one of claims 13 to 17, wherein the callus induction step comprises: (i) culturing in the callus induction medium for 7 — 8 weeks, and/or (ii) incubation of the callus induction medium at 24 to 28 °C, and/or (iii) incubation of the callus induction medium in the dark.

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19. The method of any one of claims 13 to 18, wherein the method comprises a shoot inducing step comprising culturing the callus in a shooting medium.
20. The method of claim 19, wherein either: (i) cultured plant tissue is a callus, and the method further comprises the shoot inducing step, and the shooting medium optionally comprises a reverse transcriptase inhibitor, or (ii) the initial plant tissue is a callus, and the plant tissue culture step is a shoot inducing step, and the culture medium is a shooting medium. kkk
21. The method of any one of claims 13 to 20, wherein the method comprises a rooting step comprising culturing the callus in a rooting medium.
22. The method of claim 21, wherein either: (i) cultured plant tissue is a callus, and the method further comprises the rooting step, and the rooting medium optionally comprises a reverse transcriptase inhibitor, or (ii) the initial plant tissue is a callus, and the plant tissue culture step is a rooting step, and the culture medium is a rooting medium.
23. A method for producing a plant, which method comprises the steps of: (a) performing a method as claimed in any one of claims 2 to 22, and (b) regenerating a plant from the cultured plant tissue, optionally by transferring the cultured plant tissue to growth media or soil for propagation.
24 The method of claim 1 wherein the plant is propagated from a germinated seed.
25 The method of claim 1 wherein the plant is propagated asexually
26 The method of claim 25 wherein the plant is propagated by vegetative propagation
27 The method of claim 25 wherein the plant is propagated by grafting
28 The method of any one of claims 25 to 27 wherein inhibiting LTR-TEs reduces somaclonal variation in the plant.
29 The method of claim 24 or claim 25 wherein the propagation is in vitro in a propagation medium comprising the reverse transcriptase inhibitor.

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30 The method of any one of claims 24 to 28 wherein the propagation is in an environment, which is optionally soil, and the reverse transcriptase inhibitor is applied to the plant by root drenching or by spraying leaves.
31. The method of any one of claims 1 to 30, wherein said reverse transcriptase inhibitor is a nucleotide analogue reverse transcriptase inhibitor.
32. The method of claim 31, wherein said nucleotide analogue reverse transcriptase inhibitor is tenofovir or an analogue or prodrug thereof.
33. The method of claim 32, wherein said nucleotide analogue reverse transcriptase inhibitor is tenofovir disoproxil or a salt thereof.
34. The method of any one of claims 1 to 33 wherein the plant or plant tissue is Arabidopsis, rice, tobacco, sweet potato, carrot, medicago, bamboo or date palm.
35. A cultured plant tissue or propagated plant obtained or obtainable by the method of any one of claims 1 to 34.
36. A plant propagation medium, a plant tissue culture medium, a callus induction medium, a shooting medium, or a rooting medium, which medium comprises a reverse transcriptase inhibitor, and which is optionally adapted for use in a method of any one of claims 1 to 34, or which is as defined in a method of any one of claims 1 to 34.
37. Use of a reverse transcriptase inhibitor in a method of plant tissue culture plant propagation, which is optionally a method according to any one of claims 1 to 34.
38. A method for assessing LTR-TE transposition, comprising comparing the rate of LTR-TE transposition between cultured plant tissue that has been cultured on media comprising a reverse transcriptase inhibitor with cultured plant tissue that has been cultured on media that does not comprise a reverse transcriptase inhibitor.