Methods and plants for phytomining

Abstract

The invention relates to the field of phytomining and, particularly, to a method for collecting metal from an induced polyploid or mixoploid plant.

Description

METHODS AND PLANTS FOR PHYTOMININGFIELD OF THE INVENTION

[0001] The present invention pertains to the field of phytomining, particularly to methods and plants for phytomining and to related uses and products.BACKGROUND

[0002] Critical minerals such as copper, lithium, nickel, cobalt and rare earth elements are essential components in many of today’s rapidly growing clean energy technologies - from wind turbines and electricity networks to electric vehicles. Demand for these minerals is growing quickly as clean energy transitions gather pace. For many countries, this need for critical minerals is however hardly filled by the domestic production capacities. Of the 50 critical minerals identified in 2022 by the United States Geological Survey (USGS), the United States has a net import reliance greater than 50 percent for 31 commodities.

[0003] Innovation allowing to develop resources sustainably and affordably would help every country to secure its supply of critical minerals. Certain countries have already begun investing in mapping mineral resources, developing novel battery configurations, funding recycling initiatives, and investigating other techniques.

[0004] A promising technology for supplying alternative sources of minerals is phytomining. Some plants, called hyperaccumulators have developed an ability to accumulate high concentrations of metals into their leaves, bark, and roots. These plants can be harvested every 6 to 12 months and the metal can then be extracted, for instance through burning. Phytomining involves farming these plants above mineral deposits and harvesting them for their metal content.

[0005] Hyperaccumulators have been shown to accumulate nickel, cobalt, selenium, rare earth elements, and more. Hyperaccumulators include flowering plants (e.g., Alyssum bertolinii for nickel; Noccaea caerulescens for nickel, cadmium, and zinc), trees (e.g., Glochidon cf. sericeum for nickel and cobalt), and ferns (Dicranopteris pedata for rare earth elements). Over 700 species hyperaccumulate nickel alone.

[0006] Beyond providing sources of minerals in regions not traditionally considered as rich in mineral resources, phytomining provides several other advantages, such as avoiding the environmental damages caused by traditional mining methods and providing additional biomass energy through the burning of the plant biomass to recover metal-containing biochar. Phytomining is also a technology which is generally more acceptable to local communities across the world.

[0007] In spite of these considerable advantages, phytomining still suffers from several drawbacks which keeps it away from large-scale use. One of them is that the technology is still costly compared to traditional mining.

[0008] Hence, while phytomining is a promising technology, it still needs improvements to be more cost-effective and scaled up at a commercial level.SUMMARY OF THE INVENTION

[0009] In a first aspect, the invention relates to a method for collecting metal, comprising:(a) growing a plant having an induced polyploid or mixopioid genome, in metalcontaining soil so that the plant uptakes metal from soil; and(b) collecting plant biomass from said plant.

[0010] In a further aspect, the invention relates to a method for producing biochar and / or ashes with increased metal concentration, in particular increased nickel concentration, comprising(a) growing a plant having an induced polyploid or mixopioid genome in metal-containing soil so that the plant uptakes metal from soil, wherein said plant comprises an induced polyploid or mixopioid genome;(b) collecting plant biomass from said plant;(c) optionally drying said plant biomass;(d) processing said plant biomass by thermal treatment to obtain biochar and / or ashes, whereby said biochar and / or ashes has increased metal concentration, in particular nickel concentration in comparison to a plant from the same species having a naturally-occurring ploidy.

[0011]

[0012] In a further aspect, the invention relates to the use of a plant having an induced polyploid or mixopioid genome, for phytomining, preferably wherein said plant is a tetrapioid or mixopioid plant.

[0013] In a further aspect, the invention relates to a metal-hyperaccumulator plant having an induced polyploid or mixopioid genome.

[0014] In a further aspect, the invention relates to a plant part, cell or tissue from the plant of the invention, wherein said plant part, cell or tissue has an induced polyploid or mixopioid genome.

[0015] In a further aspect, the invention relates to a plant-derived product selected from plant biomass, plant dried biomass, biochar and / or ashes, wherein said plant-derived product is obtained by the method of the invention, and / or from the plant of the invention and / or after processing of the plant of the invention.

[0016] In a further aspect, the invention relates to a method for producing metal-hyperaccumulator plants, comprising:(a) providing one or more plant part, tissue and / or cells from a metal-hyperaccumulator plant;(b) inducing polyploidy in the plant part, tissue and / or cells; and(c) regenerating mature plants from said plant part, tissue and / or cells, wherein said mature plants have an induced polyploid or mixopioid genome.DETAILED DESCRIPTIONDefinitions

[0017] In order that the present disclosure be more readily understood, certain terms are defined. Additional definitions are set forth throughout the detailed description.

[0018] As used herein, “phytomining” refers to technologies used to collect and recover one or more metal from soil. Phytomining relies on the uptake of metal by plants grown on the soil.

[0019] As used herein, "biochar" refers to a carbon-rich solid material produced by pyrolysis (i.e., direct thermal decomposition) of biomass, e.g. pyro-gasification and pyro-oxidation, to produce oxidized or non-oxidized biochar. Pyrolysis of biomass produces a mixture of solids (biochar), liquids (biooil), and gas (biogas). Biomass includes any organic material that comes from plants or animals, for example, wood and wood processing waste, agricultural crops and waste materials, yard waste, and animal waste. Biochar can be produced with pyrolysis temperatures of at least 350 °C, in particular at least 400 °C, at least 600 °C, at least 800 °C, from 350 °C to 1000 °C.

[0020] As used herein, “bio-ore” refers to metal-rich plant biomass, from which metal can be collected and / or extracted.

[0021] As used herein, a metal “hyperaccumulator” plant is a plant which accumulates high amounts of metal, in particular heavy metals. Such accumulation generally has low or no toxicity on the plant which is able to grow in a viable manner. Certain plant species are generally considered as metal hyperaccumulator plant species. A plant species may also be considered as a hyperaccumulator for a particular metal, e.g. nickel and / or other heavy metals. As defined herein, a metal hyperaccumulator plant species can accumulate at least 0.1 wt% of a metal, in particular a heavy metal, in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots. In particular, the metal is selected from magnesium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, silver, gold, cadmium, antimony, mercury, thallium, tungsten, platinum and lead. Preferably, a metal hyperaccumulator plant species can accumulate at least 0.2wt%, 0.5 wt%, 1wt%, 2.5%, 5wt% or 10wt% of a metal, in particular a heavy metal, in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots. A nickel hyperaccumulator plant species can accumulate at least 0.1 wt% nickel in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots. Preferably, a nickel-hyperaccumulator plant species can accumulate at least 0.2wt%, 0.5 wt%, 1wt%, 2.5%, 5wt% or 10wt% nickel in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots. Metal or nickel hyperaccumulation may be measured by various ways e.g.in dried material, e.g. in the dry leaves. Such levels refer to levels in naturally-occurring plants from the plant species, i.e. plants with a naturally occurring ploidy. As discovered by the inventors, metal accumulation in plant biomass is further increased by polyploidy induction.

[0022] As used herein, a “bioconcentration factor (BCF)” corresponds to the ratio of a metal concentration in biomass, per gram of dried material, to the metal concentration in the soil, per gram of dried material. BCF reflects the metal uptake capacity of a plant.

[0023] As used herein, “ploidy” refers to the number of chromosome sets in a cell. Haploids have one set (1x) and diploids have two sets (2x) of chromosomes, and polyploids have more than two sets of chromosomes. Polyploids include triploids (3x), tetrapioids (4x), pentapioids (5x), hexapioids (6x), heptapioids (7x), octoploids (8x), nonaploids (9x), decaploid (10x) and higher ploidy levels. A mixopioid organism such as a mixopioid plant refers to an organism comprising cells with different ploidies. For instance, a mixopioid plant may comprise diploid cells and tetrapioid cells.

[0024] As used herein, the terms “naturally-occurring plant” refer to a plant which has not been subjected to an artificial polyploidy-inducing treatment, e.g. by a chromosome doubling agent or technique, or a plant which does not derive by multiplication or reproduction, from a plant which has been subjected to an artificial polyploidy-inducing treatment. As used herein, the terms “naturally- occuring ploidy” refers to the ploidy of a plant species as commonly accepted in the agricultural and / or botanical field. For instance, the naturally-occuring ploidy of Berkheya coddii is diploidy. Any comparison referred to in the present disclosure, e.g. between a plant, tissue and / or cell with an induced polyploid or mixopioid genome, and a plant, tissue and / or cell with having naturally-occuring ploidy, such as metal uptake / metal amounts or gene expression comparisons or different biomasses comparisons, is herein made in comparable conditions, i.e. on plants grown in the same conditions, wherein the biomass is collected at the same growth stage and the plants are from the same accession or cultivar.

[0025] As used herein, any reference to a plant genus followed by spp. refers to any species within the named genus.Legend of the Figures

[0026] Figure 1 shows a flow cytometry of the wild type plant and polyploid mutants. Standard is Barley with 2C and 4CDNA content A. Wild type. B. M2 showing peaks at position same as wild type as well a higher size peak for tetrapioid content. C. M38, D. M13, E. M40, and F. M3 are showing peak only at the tetrapioid size. Figure 2 shows a chromosome doubled mutant karyotyping. Pictures representing B. coddii root tips cells of wild type diploid (2n), M2 mixopioid mutant (2n, 4n), and M3 tetrapioid mutant (4n). Black arrows represent 2C and 4C cells in Telophase II, and white scales represent 100 pm magnification.

[0027] Figure 3 shows: A. a plant phenotype comparison of WT plant to M2 mutant at the same stage of growth (Scale bar =10cm). B. a plant phenotype comparison of WT plant to M12 mutant at the same stage of growth.

[0028] Figure 4 shows a plant phenotype comparison of polyploid mutants M2 to WT. A- Comparison of leaf sizes (Scale bar =2 cm); B- Comparison of leaf length and width; C- Comparison of leaf area; D. average leaf size (cm2) in control plants and in mutants M12, M13, M25, M27, M33, M34, M38 and M40.

[0029] Figure 5 shows a microscopic phenotypic analysis showing the cell size and stomata size of polyploid mutants. A- comparison of the cell sizes of WT vs M2. Scale bar is 50pm. B - Stomata length C- Stomata width. D. average stomata size (pm) of control plants and in mutants M2, M12, M22, M33, M34, M28, M27, M23, M19 and M11. E. Comparison of the cell sizes of WT vs mutant M33.

[0030] Figure 6 shows the expression level of Ubiquitin-5 (UBQ5), Actin depolymerizing factor 3 (ADF3), and Tubulin alpha chain (TUB-a) genes in the leaf tissue of wild type (WT) and M2 mutant.

[0031] Figure 7 A. shows a Nickel uptake analysis by ICP-MS. BCF fold represented as ratio of nickel concentration per gram of dry material between the leaf and soil samples, in comparison to the wild-type control. B. Analysis of further mutants. BCF fold represented as ratio of nickel concentration per gram of dry material between the leaf and soil samples, in comparison to the wildtype control. C. Analysis of further mutants. BCF represented as ratio of nickel concentration per gram of dry material between the leaf and soil samples.

[0032] Figure 8 shows (A) a pollen germination assay showing WT pollen showing pollen tube germination (red arrows). M3 mutant pollen are small deformed and do not germinate. (B) pollen of polyploid mutant is larger in size compared to the wild-type

[0033] Figure 9 shows a flower fertility assessment of polyploid vs wild type. Scale bar represents 1 cm. A- Control flower B- Polyploid mutant M3 flower C- Wild type flower showing seed dehiscence after drying D- Polyploid mutant M3 flower showing inflorescence that were not fertilized.

[0034] Figure 10 showsa plant phenotype comparison of WT plant to M11 , M12 and M27 mutants at the same stage of growth

[0035] The inventors have identified key factors to improve the metal-extraction yield in phytomining technologies. In particular, the inventors have discovered that plants which are polyploids, including plants at least partially polyploid (i.e. polyploid plants or mixopioid plants) exhibit increased metal uptake capacities, allowing to critically improve the effectiveness of phytomining.

[0036] In one aspect, the invention relates to a method for collecting metal.

[0037] According to one aspect of the invention, the method for collecting metal comprises:(a) growing a plant having an induced polyploid or mixopioid genome, in metal-containing soil so that the plant uptakes metal from soil; and(b) collecting plant biomass from said plant.

[0038] The inventors have generated plants which have a genome that is at least partly polyploid generated by artificial polyploidy induction. The plants include fully polyploid plants and mixopioid plants, wherein these plants contain cells with an induced polyploid genome. The inventors have evidenced that such polyploid or mixopioid plants surprisingly exhibit increased metal uptake capacities, when grown on a contaminated soil.

[0039] The invention relies on planting and growing plants which can uptake and accumulate, in particular hyperaccumulate, metal in their tissues. The plant biomass collected according to the method contains metal accumulated by the plant. Certain plants such as metal-hyperaccumulating plants uptake metal through low-selectivity cation transporters in the roots and, rather than pumping it out, store metals from the soil inside the plant biomass at concentrations higher than some commercially mined ores. The collected biomass can then be dried and burnt, e.g. incinerated to further concentrate the metal; the plant biomass also lacks high concentrations of major metal impurities found in conventional ore.

[0040] In some embodiments, soil is a contaminated soil. In some embodiments, the contaminated soil has a concentration of a contaminant, e.g. a metal such as heavy metal, that exceeds the level defined by applicable regulations. In some embodiments, contaminated soil has more than 50 mg / kg dry weight nickel. Contamination thresholds are described, e.g., in Toth, Gergely, et al. "Heavy metals in agricultural soils of the European Union with implications for food safety." Environment international 88 (2016): 299-309.

[0041] In some embodiments, the metal is or includes one or more heavy metals. By definition, a heavy metal is a metal with a density greater than 5 g / cm3and includes, inter alia, chromium, cobalt, nickel, copper, zinc, arsenic, silver, gold, cadmium, antimony, mercury, thallium, tungsten, platinum and lead. Heavy metals are persistent and generally toxic in the environment, and they can accumulate in the soil over time. This contamination can seriously affect the environment and human health. Heavy metal soil remediation is thus a critical process to help protect the environment and human health.

[0042] In some embodiments, the metal is or includes nickel. In other terms, in such embodiment, a plant of the invention has the capacity to uptake and accumulate, in particular hyperaccumulate, at least nickel from soil. As used herein, nickel refers to any form of nickel, including ionic forms such as Ni2+.

[0043] In some embodiments, the invention thus relates to a method for collecting nickel, wherein the method comprises:(a) growing a plant having an induced polyploid or mixopioid genome in nickel-containing soil so that the plant uptakes nickel from soil; and(b) collecting plant biomass from said plant.

[0044] The plant biomass collected from the plant contains nickel extracted by the plant from soil. The plant may also, in addition, collect any further metal also extracted from soil, such as any further heavy metal.

[0045] A plant as used in the methods of the invention is at least partly polyploid. In some embodiments, the plant is a polyploid plant, i.e. all of its cells are polyploid, for instance tetrapioid. In some embodiments, the plant is a mixopioid plant, i.e. only part of its cells are polyploid, i.e. their ploidy level exceeds the normal ploidy level of the plant species. In such mixopioid plant, the rest of the cells has the normal ploidy level of the plant species. For instance, a mixopioid plant may comprise tetrapioid cells and diploid cells, e.g. where the plant species is a naturally occurring diploid plant species.

[0046] In some embodiments, the polyploid plants and cells are selected from triploid, tetrapioid, pentapioid, hexapioid, heptapioid and octoploid plants and cells. In particular, the polyploid plants and / or cells are tetrapioid plants and / or cells.

[0047] In some embodiments, the polyploid plants are selected from triploid, tetrapioid, pentapioid, hexapioid, heptapioid and octoploid plants. In particular, the polyploid plants are tetrapioid plants.

[0048] The plants of the invention are typically obtained by induced polyploidy. By “induced polyploid”, it is referred to a cell or plant with a polyploidy which has been induced by treatment, such as a chemical, physical and / or physico-chemical treatment, e.g. a chromosome doubling treatment. Polyploidy is for instance induced by applying a polyploidy-inducing treatment selected from a physical, chemical and / or physico-chemical treatment. In some embodiments, the polyploidyinducing treatment comprises the application of an antimitotic treatment, preferably selected from colchicine, oryzalin, nitrous oxide gas, heat, amiprophos methyl, trifluralin, pronamide and any of their combinations or mixtures. As a result, the genome of the cell or plant comprises a number of chromosome sets that is higher than the number of chromosome sets in a naturally-occurring cell or plant of the same species, in particular in a same accession, variety or cultivar. As referred to in the present disclosure, any comparison between an induced polyploid cell and a naturally-occurring cell is made between cells of the same type. In particular, the compared cells are non-gamete cells.

[0049] Polyploidy induction may be direct or indirect, namely the subject cell or plant may be polyploid due to a polyploidy-inducing treatment applied to the subject cell or plant, or the subject cell or plant may be derived, e.g. by reproduction or multiplication, from another cell or plant, to which a polyploidy-inducing treatment has been applied.

[0050] In some embodiments, the cell and / or plant of the invention comprises, in its genome, a number of chromosome sets which is multiplied, e.g. doubled, in comparison to a naturally-occurring cell or plant of the same species, in particular same subspecies, same accession, variety or cultivar, irrespective of the natural ploidy of the plants. Hence, the invention concerns plants with induced artificial polyploidy or mixoploidy, whatever may be the natural ploidy of the plant. For instance, if the cell and / or plant of the invention is from a plant species which is a naturally-occurring diploid plant species, the cell and / or plant of the invention is preferably at least partly tetrapioid, i.e. eithertetrapioid or mixopioid with mixed tetrapioid and diploid cells. Likewise, if the plant species is a naturally-occurring tetrapioid plant species, the cell and / or plant of the invention has a ploidy higher than 4n, e.g. octoploid. The plant may also be mixopioid with tetrapioid and octoploid cells.

[0051] In some embodiments, the plant belongs to a hyperaccumulator plant species. Some plants, known as hyperaccumulators, have the ability to accumulate high concentrations of metals, such as heavy metals, in their tissues with minimal or no toxic effects.

[0052] Hyperaccumulator plant species include any metal hyperaccumulator plant species, in particular heavy metal hyperaccumulator plant species. Non-limiting examples of hyperaccumulator plants are listed in the hyperaccumulator database accessible online (http: / / hyperaccumulators.smi.uq.edu.au / collection / ), as also described in Reeves, Roger D., et al. “A global database for plants that hyperaccumulate metal and metalloid trace elements.” New Phyto legist 218.2 (2018): 407-411 .

[0053] In some embodiments, the plant is from a nickel hyperaccumulator plant species. In particular, the plant may belong to a family selected from Asteraceae, Brassicaceae and Violaceae. More particularly, the plant may belong to a genus selected from Alyssum, Berkheya, Brassica, Rinorea, Senecio, Thlaspi. And Noccaea. Hence, the plant may belong to a species selected from Alyssum spp., Berkheya spp., Brassica spp., Rinorea spp., Senecio spp., Thlaspi spp. and Noccaea spp. In some embodiments, the plant belongs to the genus Berkheya, i.e. a Berkheya spp. plant. In some embodiments, the plant is a Berkheya coddii plant.

[0054] At step (b), the method comprises collecting plant biomass from the plant. Any type of plant biomass may be collected at this step since the metal is absorbed in the roots and is then translocated in different tissues and organs of the plant. In particular, the collected plant biomass may comprise tissue from an explant, reproductive material, scion, cutting, fruit, root, root tip, rootstock, shoot, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole, flower, bark and / or seed.

[0055] The inventors have shown that plants of the invention display an unexpectedly increased metal uptake capacity in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally occurring plant from the same species. In some embodiments, the amounts of a metal, in particular a heavy metal, in the collected biomass, are higher by at least 1 % in comparison to a plant from the same species having a naturally-occurring ploidy. Preferably, the amounts of a metal, in particular a heavy metal, in the collected biomass, are higher by at least 5%, 10%, 25%, 50% or, more preferably at least 70%, 80%, 90 or most preferably at least 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally- occurring plant from the same species. In some embodiments, the amounts of nickel in the collected biomass, are higher by at least 1 % in comparison to a plant from the same species having a naturally- occurring ploidy. Preferably, the amounts of nickel in the collected biomass, are higher by at least 10%, more preferably at least 25%, even more preferably at least 50%, still more preferably at least 70%, 80%, 90% or most preferably at least 100%, in comparison to a plant from the same specieshaving a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. The plant metal uptake capacity is reflected by the amount of a given metal, in plant biomass, as measured, e.g., as a concentration, more particularly as a bioconcentration factor. Such amount can be measured, for instance, on dried biomass, e.g. dry leaves obtained from the plant.

[0056] . In some embodiments, the concentration, in particular the bioconcentration factor, of a metal, in particular a heavy metal, in the collected biomass, is higher by at least 1 % in comparison to a plant from the same species having a naturally-occurring ploidy. Preferably, the concentration, in particular the bioconcentration factor, of a metal, in particular a heavy metal, in the collected biomass, is higher by at least 5%, 10%, 25%, 50% or, more preferably at least 70%, 80%, 90 or most preferably at least 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. In some embodiments, the concentration, in particular the bioconcentration factor, of nickel in the collected biomass, is higher by at least 1 % in comparison to a plant from the same species having a naturally-occurring ploidy. Preferably, the concentration, in particular the bioconcentration factor, of nickel in the collected biomass, is higher by at least 10%, more preferably at least 25%, even more preferably at least 50%, still more preferably at least 70%, 80%, 90% or most preferably at least 100%, in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. Through their increased metal uptake capacity, the plants of htei nvention concentrate higher amounts of metal, in particular heavy metal, more particularly nickel, in their tissues, in comparison to wild-type plants having a naturally-occuring ploidy.

[0057] In some embodiments, the plant of the invention accumulates at least 0.15 wt% of a metal, in particular a heavy metal, in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots. In particular, the plant species accumulates at least 0.2wt%, preferably at least 0.5 wt%, more preferably at least 1wt%, still more preferably at least 2.5% even more preferably at least 5wt%, even more preferably at least 10wt% of a metal, in particular a heavy metal, in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots . Metal hyperaccumulation may be measured by various ways e.g. in the dry leaves.

[0058] In some embodiments, the plant of the invention accumulates at least 0.15 wt% of nickel in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots . In particular, the plant species accumulates at least 0.2wt%, preferably at least 0.5 wt%, more preferably at least 1wt%, still more preferably at least 2.5% even more preferably at least 5wt%, even more preferably at least 10wt% nickel in its biomass, in particular in one or more plant parts, such as the leaves, the shoot, the stem and / or the roots. Nickel hyperaccumulation may be measured by various ways e.g. in dry material, in particular in the dry leaves.

[0059] In some embodiments, the plant of the invention has increased size in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. in comparison to a naturally-occurring plant from the same species. The inventors have indeed shown that the plants of the invention, withan induced polyploidy or mixoploidy, generally exhibit a bigger and higher biomass. Increased size may be reflected by an increase of the biomass weight. In some embodiments the plant has a biomass weight which is higher by at least 1 %, preferably 5%, 10%, 25%, 50% or 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally- occurring plant from the same species. The plants may also exhibit an increased height. In some embodiments the plant has a height which is higher by at least 1%, preferably 5%, 10%, 25%, 50% or 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. The plants may also have increased leaf size, e.g. as reflected by the leaf length and / or leaf width, in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. In some embodiments the plant has leaf size, e.g. leaf length and / or leaf width which is higher by at least 1 %, preferably 5%, 10%, 25%, 50% or 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species.

[0060] In some embodiments, the plant of the invention has increased stomata size, e.g. as reflected by the stomata length and / or width, in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. In some embodiments the plant has stomata size, in particular stomata length and / or width which is higher by at least 1%, preferably 5%, 10%, 25%, 50% or 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species.

[0061] The inventors have also discovered that the expression of housekeeping genes was increased in the plants of the invention. In some embodiments, the plant of the invention thus has increased housekeeping gene expression, in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. In some embodiments, the plant of the invention has increased expression of one or more genes selected from UBQ5 (NM_116090), ADF3 (NM_180896) and TUB-a (KJ634803.1), in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. In some embodiments, the expression of one or more genes selected from UBQ5, ADF3 and TUB-a is increased by at least 1 %, preferably 5%, 10%, 25%, 50% or 100% in comparison to a plant from the same species having a naturally-occurring ploidy, e.g. a naturally-occurring plant from the same species. Gene expression is measured, for instance, in leaf tissue.

[0062] The method may further comprise further steps, for instance to extract metal from the plant biomass. Metal-rich plant biomass can also be used without further treatment steps.

[0063] In some embodiments, the method further comprises a step of drying said plant biomass. The method thus produces dried biomass. Accordingly, in some embodiments, the invention relates to a method for collecting metal, wherein the method comprises:(a) growing a plant having an induced polyploid or mixopioid genome in metal-containing soil so that the plant uptakes metal from soil;(b) collecting plant biomass from said plant; and(c) drying said plant biomass.

[0064] Various techniques of plant biomass drying known from the skilled person can be used, e.g. drying in the field, or air drying by forced heating, as non-limiting examples

[0065] The method may further comprise a step of processing said plant biomass by thermal treatment to obtain biochar and / or ashes. Accordingly, in some embodiments, the invention relates to a method for collecting metal, wherein the method comprises:(a) growing a plant having an induced polyploid or mixopioid genome in metal-containing soil so that the plant uptakes metal from soil;(b) collecting plant biomass from said plant;(c) optionally drying said plant biomass; and(d) processing said plant biomass by thermal treatment to obtain biochar and / or ashes.

[0066] In some embodiments, the metal amounts per unity of biochar and / or ashes amount (e.g. per g of biochar and / or ashes), e.g. the metal concentration, in particular the nickel concentration in said biochar and / or ashes is increased in comparison to biochar and / or ashes obtained from a plant from the same species, having a naturally-occurring ploidy. In some embodiments, said thermal treatment comprises pyrolysis and / or incineration. For instance, after harvesting and drying, the shredded biomass is incinerated in an incinerator. This ensures the removal of organic material as gaseous products leaving an inorganic incineration ash behind. Because the metal, e.g. nickel is not lost to the off gas and the significant volume shrinkage, the nickel concentration is multiplied by a factor of 10 to 20 by this operation (e.g., for some plants, the nickel content increases from 0.5-1 wt% in biomass to 10-15 wt% in ash). The variations are due to the plant type and its nickel capacity, soil concentration, degree of oxidation during incineration and the amount of (any) leftover organics. Alternative to incineration, more complex but more environmentally-friendly pretreatments can be adapted to the current flowsheet which includes pyrolysis and pyro-gasification.

[0067] Temperatures from about 260°C to about 1400°C can be used to combust the dried plant material to oxidize and vaporize the organic material present and to prevent dioxin accumulation during incineration. Preferably, the temperature is chosen to remove the organic carbon to free the ash. The process leaves a residue of the accumulated metal with few contaminants known to interfere with metal refining.

[0068] In some embodiments, the method may further comprise a step of extracting the metal from the plant biomass, from the dried plant biomass, from biochar and / or from ashes. Accordingly, in some embodiments, the invention relates to a method for collecting metal, wherein the method comprises:(a) growing a plant having an induced polyploid or mixopioid genome in metal-containing soil so that the plant uptakes metal from soil, wherein said plant comprises an induced polyploid or mixopioid genome;(b) collecting plant biomass from said plant;(c) optionally drying said plant biomass;(d) optionally processing said plant biomass by thermal treatment to obtain biochar and / or ashes; and(e) extracting the metal from the plant biomass, the dried plant biomass, biochar and / or ashes.

[0069] In some embodiments, extracting the metal comprises solubilizing the metal. Metal solubilization may comprise, for example, any, several or all of the steps of leaching the metal, purifying the metal, e.g. by ion exchange and / or solvent extraction, crystallizing the metal and / or electrowinning.

[0070] The extraction process may comprise a step of leaching. The purpose of acid leaching, e.g. sulfuric acid leaching, is to dissolve as much nickel in the ash as possible with as little co-dissolution of other metals like iron, magnesium, potassium as possible. These are impurity metals and have to be removed in the following steps as they are not wanted in the final product (crystals). In some instances, leaching is made in a H2SO4 solution. Reaction conditions for sulfuric acid leaching are described, e.g., in Houzelot, Vivian, et al. Chemical Engineering Research and Design 129 (2018): 1-11.

[0071] The Ni concentration in the leachate will depend on Ni in the dry biomass together with the microstructure of biomass and the leaching conditions (e.g. liquid to solid ratio ) which determines the leaching efficiency.

[0072] After acid leaching, most of the nickel is dissolved along with certain levels of impurity metals. To remove some of these impurities and condition the leach solution before the ion exchange stage, the pH of the solution may be increased by addition of a basic reagent, e.g. NaOH solution or other basic reagents. By doing so, an unquantified amount of trivalent iron, chromium and aluminum precipitates as their respective (oxy)hydroxides together with smaller amounts of nickel and magnesium. In this process it is crucial to minimize the nickel losses while maximizing the iron removal. In the meantime, the pH of the leachate should be optimal for selective absorption of nickel over magnesium in the subsequent ion exchange step, if performed. The leachate may also be processed by solvent extraction.

[0073] Ideally, ion exchange is used for removing impurity metals with minor concentrations from the mainstream. After neutralization and purification of the leachate, the solution may thus be passed through an ion exchange resin, which offers selectivity for the desired metal, e.g. nickel over divalent iron, magnesium, potassium and sodium. After absorption, the resin is washed with MilliQ water to remove any entrapped leachate and avoid unnecessary contamination. The washed resin is then stripped a H2SO4 solution with two to four times the volume of the resin.

[0074] The leachate may then be heated to evaporate water thereby decreasing the solubility of the nickel sulfate. Once the solubility limit is reached, nickel sulfate hexahydrate (NSH) crystals form as more water is removed. The crystal purity is more than 99.9% with a typical composition of majorcontaminants as 60-70 ppm Fe, 10-50 ppm Na, 10-30 ppm Ca, 20-50 ppm Mg and 20-50 ppm K. The NSH crystals are filtered, collected and dried while the nickel-depleted solution can be recycled back to the flowsheet.

[0075] As a non-limiting example of biomass collection and treatment, the plant may be harvested in a conventional fashion to , i.e., by cutting the plant at soil level. The harvested materials can then be left to dry in the field, or it can be dried by forced heated air drying, so as to remove most of the water present in the plant tissue by forced heated air drying. After drying, the plant tissue is collected by normal agricultural practices, and incinerated or pyrolyzed, which allows the metals in the ash or ore to be recovered according to conventional metal refining methods such as acid dissolution and electrowinning.

[0076] In another aspect, the invention relates to a method for producing biochar and / or ashes with increased metal amounts, in particular nickel amounts, comprising(a) growing and / or providing a plant having an induced polyploid or mixopioid genome in metalcontaining soil so that the plant uptakes metal from soil, wherein said plant comprises an induced polyploid or mixopioid genome;(b) collecting plant biomass from said plant;(c) optionally drying said plant biomass;(d) processing said plant biomass by thermal treatment to obtain biochar and / or ashes, whereby said biochar and / or ashes has increased metal amounts, in particular nickel amounts in comparison to a plant from the same species having a naturally-occurring ploidy..As used herein, the term metal amounts or nickel amounts refers to the amounts of metal and / or nickel per unity of biochar and / or ashes amount (e.g. per g of biochar and / or ashes), i.e. the concentration of metal and / or nickel in the biochar and / or ashes.

[0077] In another aspect, the invention relates to a method for concentrating metal and / or nickel in biochar and / or ashes, comprising(e) growing and / or providing a plant having an induced polyploid or mixopioid genome in metalcontaining soil so that the plant uptakes metal from soil, wherein said plant comprises an induced polyploid or mixopioid genome;(f) collecting plant biomass from said plant;(g) optionally drying said plant biomass;(h) processing said plant biomass by thermal treatment to obtain biochar and / or ashes, whereby metal and / or nickel in said biochar and / or ashes is concentrated in comparison to biochar obtained from a plant in comparison to biochar obtained from a plant from the same species, having a naturally-occurring ploidy.

[0078] In another aspect, the invention relates to the use of a plant having an induced polyploid or mixopioid genome, for phytomining. The invention thus relates, in one aspect, to the use of plant having an induced polyploid or mixopioid genome for collecting metal, such as heavy metal from soil. In a particular aspect, the invention relates to the use of a plant having an induced polyploid or mixopioid genome for nickel phytomining. The invention thus relates to the use of a plant having an induced polyploid or mixopioid genome for collecting nickel.

[0079] Any feature described in relation to the methods of producing metal-containing plant biomass, according to the invention is applicable to any other aspects of the invention and vice- versa. In particular, features described in relation to the methods of the invention are applicable to the above-described use as well as to the related plant, plant part, cell, tissue, plant biomass, dried biomass, biochar, bio-ore or method for producing a plant according to the invention.

[0080] In some embodiments, the plant is a polyploid or mixopioid plant. In some embodiments, the polyploid plant is selected from a triploid, tetrapioid, pentapioid, hexapioid, heptapioid and octoploid plant. In some embodiments, the mixopioid plant comprises tetrapioid cells and diploid cells.

[0081] In one aspect, the invention relates to a metal-hyperaccumulator plant having an induced polyploid or mixopioid genome. In some embodiments, the plant is a nickel-hyperaccumulator plant. In some embodiments, the plant belongs to a family selected from Asteraceae, Brassicaceae and Violaceae. More particularly, the plant may belong to a genus selected from Alyssum, Berkheya, Brassica, Rinorea, Senecio, Thlaspi. And Noccaea. Hence, the plant may belong to a species selected from Alyssum spp., Berkheya spp., Brassica spp., Rinorea spp., Senecio spp., Thlaspi spp. and Noccaea spp. In some embodiments, the plant belongs to the genus Berkheya, i.e. a Berkheya spp. plant. In some embodiments, the plant is a Berkheya coddii plant. In some embodiments, the plant is not a Brassicaceae plant. In some embodiments, the plant is not a Odontarrhena bertolinii plant.

[0082] In particular, said metal-hyperaccumulator plant is obtained by applying a polyploidyinducing treatment to a plant from a metal-hyperaccumulator plant species. In particular, the polyploid-inducing treatment is selected from a physical, chemical and / or physico-chemical treatment. More particularly, the polyploid-inducing treatment is an antimitotic treatment, as described herein.

[0083] In some aspects, the invention relates to a plant part from a plant of the invention. In particular, the invention relates to a plant part from a metal-hyperaccumulator plant having an induced polyploid or mixopioid genome. Said plant part has an induced polyploid or mixopioid genome. In some embodiments, the plant part is, or is derived from a reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole, flower, bark and / or seed.

[0084] In some aspects, the invention relates to tissue from a plant of the invention. In particular, the invention relates to a tissue from a metal-hyperaccumulator plant having an induced polyploid or mixopioid genome. Said tissue has an induced polyploid or mixopioid genome. In someembodiments, the tissue is derived from a reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole, flower, bark and / or seed.

[0085] In some aspects, the invention relates to a cell from a plant of the invention. In particular, the invention relates to a cell from a metal-hyperaccumulator plant having an induced polyploid or mixopioid genome. Said cell preferably has an induced polyploid genome. In some embodiments, the cell is derived from a reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole, flower, bark and / or seed.

[0086] In some aspects, the invention relates to plant biomass obtained from the plant of the invention and / or obtained according to the method of the invention. Any type of plant biomass is encompassed herein. In particular, the plant biomass may comprise any of the plant part(s), cell(s) and / or tissue(s) as described herein. In some embodiments, the plant biomass is fresh biomass. In other embodiments, the plant biomass is dried biomass.

[0087] In some aspects, the invention relates to biochar obtained from the plant of the invention and / or obtained according to the method of the invention. In particular, said biochar comprises more metal, in particular more nickel, in comparison to biochar obtained from a plant from the same species, having a naturally-occurring ploidy.

[0088] In some aspects, the invention relates to ashes obtained from the plant of the invention and / or obtained according to the method of the invention. In particular, said ashes comprises more metal, in particular more nickel, in comparison to ashes obtained from a plant from the same species, having a naturally-occurring ploidy.

[0089] In some aspects, the invention relates to a method for producing metal-hyperaccumulator plants, comprising:(a) providing one or more plant part, tissue and / or cells from a metal-hyperaccumulator plant;(b) inducing polyploidy in the plant part, tissue and / or cells; and(c) regenerating mature plants from said plant part, tissue and / or cells, wherein said mature plants have an induced polyploid or mixopioid genome.

[0090] In some embodiments, said plant part, tissue and / or cells is(are), or is(are) derived from, an explant, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole, flower, bark and / or seed;

[0091] In some embodiments, step (b) comprises applying a polyploidy-inducing treatment selected from a physical, chemical and / or physico-chemical treatment to the plant part, tissue and / or cells. In some embodiments, the polyploidy-inducing treatment is an antimitotic treatment, preferablyselected colchicine, oryzalin, nitrous oxide gas, heat, amiprophos methyl, triflu ralin, pronamide and any of their combinations or mixtures.

[0092] In some embodiments, the plant cells provided at step (a) are derived from a plant tissue selected from leaf, calli, seed, embryo, cotyledon, nodal cuttings, internodes, roots, flowers and pollen tissue.

[0093] In some embodiments, the invention relates to a plant obtainable or obtained by the methods of the invention, or a cell, plant part or seed thereof.

[0094] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.EXAMPLESExample 1 : Materials and methods

[0095] Plant material and Colchicine treatment

[0096] 8. coddii seeds were germinated on 1 % bactoagar plates for a period of 6 days prior to treatment. The germinated seedlings were then placed onto plates containing MS basal salts, 0.1 % PPM, 0.1 % Gamborg B5 vitamins mixture, and 1 % maltose. Different colchicine solutions (0.05%, 0.1 %, 0.2%, 0.3%, 0.5%) were prepared in 2% DMSO and were applied to each of the seedlings using a Pasteur pipette, ensuring complete submersion of the plants. The plates were then covered with aluminum foil and kept in growth chambers (25 / 21 °C Day / night temperature with 16h 8h day / night) for various time intervals (3h, 6h, 9h, 12h , 24h). After each treatment, the seedlings were rinsed with sterile milliQ water, subcultured onto new MS plates and returned to the growth chamber. The plants were continuously subcultured over the course of several months.

[0097] Oryzalin Seedling Treatment

[0098] Polyploid and mixopioid mutants of Berkheya coddii were obtained using a chemical mutagen known as Oryzalin PESTANAL. 8. coddii seeds were surface sterilized and then germinated over the course of 6 days in % MS media supplemented with 0.2% Plant Preservative Mixture (PPM). Before treatment, a 25 ppm oryzalin solution was prepared by dissolving the powder in DMSO and then very slowly adjusting the volume with deionized water so that the total DMSO concentration was equal to 5% (for example, for a 100 mL solution, 2.5 mg of oryzalin powder was dissolved in 5 mL of DMSO and then raised to 100 mL with deionized water).

[0099] For the treatment, the germinated seedlings were submerged in the prepared oryzalin solution in a sterile Erlenmeyer flask covered with aluminum foil (oryzalin is light sensitive). The flasks were then placed into a shaking incubator for 6 hours at 150 rpm and 23 °C. After treatment, the seedlings were removed from the oryzalin solution and rinsed twice with sterile water before being cultured onto % MS media with 0.2% PPM. The plants were then cultured in vitro for approximatelyone month before being transferred to a soil mixture with 2500 ppm NiSO4. Leaf samples were collected for genotyping via flow cytometry from all healthy, established plants.

[0100] Induce polyploidy in seedling

[0101] Seedlings were treated at specific concentration (0.1 % retained) and for specific incubation time (12 hours retained) by dipping on MS medium gel using sterilized seedling at 4 days after germination. After dipping, seedlings were rinsed 3 times using sterile ddH20 and placed into new MS medium until the plantlets were formed and were subculture into soil supplemented with nickel at 5000 ppm. Detailed procedure below in the detail protocol section.

[0102] Karyotyping

[0103] Using the traditional Feulgen method, the number of chromosomes was counted in the cells of the root tip derived from tiny plantlets on pots (Lillie, 1951). For 8 hours (4 at room temperature and then 4 at 4 °C), 0.002 M hydroxyguinoline was used to pre-treat the root samples. The materials were fixed for 24 hours in a 3:1 ethanol:acetic acid solution before being kept in 70% ethanol at 4 °C for chromosomal screening. The fixative was removed from the root tips, which were then given two 5-minute baths. After that, 1 N HCI was used to hydrolyze the cleaned, fixed root tips for 12 min at 60 °C. Rinse again after 10 minutes. To stain chromosomes, root tips were lightly dried on filter paper and then placed in a tiny recipient with two or three drops of the stain aceto-iron hematoxylin dye (see below for instructions on how to prepare it). For at least 30 minutes, the recipient was kept sealed and at room temperature. A root tip was added to a little drop of 45% acetic vinegar on a clean slide to prepare the slide. The terminal meristem was cut into tiny pieces and shattered. They were placed between sheets of filter paper, covered with a coverslip, and lightly tapped with a needle. To prepare aceto-iron hematoxylin staining solution, at room temperature, 4 grams of hematoxylin and 1 grams of iron alum (FeNH4(SO4)2.12H2O) were dissolved in 100 ml of 45% acetic acid to create the staining solution. With the aid of a glass stick, the mixture was homogenized before being stored in a dark flask for a week. The mixture was filtered and kept in a dark glass after that time. The solution might be put to immediate use or kept in the fridge indefinitely.

[0104] Flow cytometry for ploidy level assessment

[0105] Leaf tissues were razor blade sliced in lysis buffer (45 mM MgCh, 30 mM sodium citrate, 60 mM MOPS, 1 % (w / v), pH 7.2), and the nuclei isolation was performed using flow cytometry (Beckman sorter REF) using propidium iodide (IP) staining and excited with UV lamp (455nm). Analysis of the ploidy level was carried out using the software provided by Beckman.

[0106] Tissue clearing

[0107] Root and leaf tissues were placed in NaOH-SDS solution (NaOH: 200mM; SDS: 1 %) 2 hours at 37° (Leica VT1000S, LEICA MICROSYSTEMS SA Nanterre). Then samples were washed twice 5 min in water. After wise, washed samples were dipped in sodium hypochlorite 30 min at room temperature. Finally, samples were washed once in water and then in PBS solution. Prepared samples were stained by SCRI Renaissance 2200 optical brightener solution (SR2200: 0.1 %(v / v);DMSO: 1 %(v / v); Triton- X100: 0.05%(w / v); glycerol: 5%(w / v); para-formaldehyde: 4%(w / v), solubilized in PBS (pH:8)). For staining, samples are dipped in SCRI 2200R solution in vacuum for 30min, and then dipped 1 hour to further days. Finally, samples were washed with water and the blades were mounted in a hood. For visualization, we use the X40 objective with excitation using a 405nm laser and emission as used for DAPL

[0108] Stomata density and sizing assessment

[0109] Collected leaves were coated with a thin layer of clear nail polish on the upper and lower side and let it dry. A strip of clear stick tape was placed over the nail polish. A layer of cells is removed along with the layer of nail polish to obtain a leaf impression. Leaf impressions were observed using a bright field microscope at 100x and 400x magnification. Stomata density was performed by counting the number of stomata per FOV using 3 FOV replicates.

[0110] Epidermal cell size evaluation and Leaf area measurement

[0111] Epidermal cell size evaluation was done using Imaged software. Leaf area ratio of experiment versus wild-type was carried out by subtracting the leaf area of the wildtype of the experimental value and by normalizing by the total leaf area of the wild-type. Sampled leaf area measurement was performed using Imaged using segmentation.

[0112] Biomass estimation

[0113] One leaf was sampled and weighed per genotype and lyophilized overnight. Dry weight was measured for comparison.

[0114] Gene expression analysis of housekeeping genes

[0115] Primers were designed following gPCR recommendations. Primer’s optimization was performed for all primer couples using 6 different template cDNA concentrations from stock concentration to be diluted 64 times. Using a mesa green SYBR mix (Eurogentec, EGT group), the gene expression assessment was performed. Data analysis was performed using Bio-Rad CFX manager 3.0 software.

[0116] ICP analysis

[0117] To perform plant digestion using a digiprep machine, a combination of HNO3 and H2O2 in a 5:1 ratio was employed to break down the plant material. The digestion process was conducted at a temperature of 120 °C for 45 minutes, starting with a 0.3 g plant sample. After cooling water was added to the sample for a final volume of 20 ml. The remaining residue was subseguently filtered through filter paper, and the resulting solution was further diluted with Nitric acid 2%. The concentration of metals in the resulting solution was determined using ICP-MS.

[0118] Pollen viability and seed sterility analysis

[0119] A pollen germination media was prepared which contained Sucrose- 15%, Ca(NO3)2 -0.03% and Boric Acid - 0.01 % (pH 7.0) in milliQ water. Agarose was added at 0.2% for solidification. Themedia was poured on microscopic slides, allowed to solidify and then pollen grains were sprinkled on the slides. The slides were incubated at 25 C for 4 hours. The germination was observed under a bright field microscope to see pollen tube growth and elongation.

[0120] In order to assess the potential of the pollen or flowers to produce viable seeds, the pollen from the polyploid mutant was used for pollination of a wildtype flower and vice-versa.

[0121] Example 2: Chromosome doubling mutant lines production of the nickel hyperaccumulator B. coddii

[0122] For induction of polyploidy in plants, different concentrations of colchicine, with different treatment times, were carried out on seedlings. Stable induced polyploidy in B. coddii was most effective at a colchicine concentration of 0.1 % for 12 hours on seeds at the germination stage based on a combination of survival rate approximately 6 months after treatment and phenotype. Alternatively, oryzalin treatment were applied, as described in the material & methods section (mutants M12 and M13).

[0123] Indeed, with these parameters, we were able to induce polyploidy mutants of B. coddii in two independent experiments. Through this study, we were finally able to produce one mixopioid mutant (M2) and several tetrapioid mutants (including M3, M13, M38 and M40) of B. coddii. In orderto check the level of ploidy and its stability, we used the flow cytometry technique at different growth times, 167 and 203 day post-treatment (dpt), of the different lines (Figure 1 , 203 dpt).

[0124] We were able to confirm the stability of the genotype and ploidy at the different time points. Finally, we performed the karyotyping of root tips cells to confirm the different levels of ploidy compared to wildtype, and to count the number of chromosomes in the tetrapioids and diploid cells. We confirmed 12 chromosomes for diploid wildtype line and 24 chromosomes for the tetrapioid mutant, and a mix of both cell types for the mixopioid mutant (Figure 2). These results all together confirmed the production of stable B. coddii mutants.

[0125] Example 3: Phenotypic characterization of the chromosome-doubled B. coddii lines

[0126] In order to characterize phenotypically the mutants, we carried out a macro- and microscopic analysis of different traits such as plant size, leaf area, size and width, for macroscopic phenotypic analysis, and stomatai density and sizing of mesophyll tissue, for microscopic phenotypic study. The M2 mutant plant was more or less the same size (Figure 3A), but the leaves of the mutants were larger in size when compared to the WT (Figure 4A). The same observations were made on mutant M12 (Figure 3B and Figure 10), as well as mutants M1 1 and M27 (Figure 10).

[0127] Moreover, the leaf length x width ratio seems to show that the leaves of the mixopioid tend to be longer and wider than the wild type (Figure 4B). Area measurement of the leaf surface of the mixopioid lines shows that the m2 mutant have an average of 46% more leaf area, respectively,compared to the wild type (Figure 4C). The increase in leaf size was observed consistently throughout the different mutants obtained (Figure 4D). Microscopic phenotypic analysis focused on the stomatai density and size assessment reveals that the mutants seem to present larger mesophyll cells (data not shown) and stomata (Figure 5A), width of stomata in mixopioid mutant show to be longer and wider (Figure 5B, 5C), as observed consistently throughout the different mutants obtained (Figure 5D). In m2 mutant, the stomata count is not significantly decreased compared to the wild-type, which may be because of the particularity of the diploids and tetrapioid cell shape along the leaf as mentioned below. Overall, these results suggest that induced chromosome doubling causes increase in cell size and stomatai density due to the impact of the treatment on the cell cycle of treated plant cells.

[0128] These data contrast with published results showing that colchicine treatment may have an unfavorable effect on certain plants. Desired traits of plants can be negatively influenced through polyploidy plants induced by colchicine. For instance, colchicine treatment produced dwarf tetrapioid plants with short internodes in Vitex agnus-castus L. The Height of the plant treated with 0.1 % colchicine was four-fold smaller than untreated control plant (16 cm versus 68 cm). Seemingly, it was a dwarf plant with very short internodes and small size leaves without any flower or flower bud (Ari et al., 2015. Creation of variation through gamma irradiation and polyploidization in Vitex agnus- castus L. Scientia Horticulturae, 195, pp.74-81.) Meanwhile, severe chromosomal damage have been reported to be a result of colchicine treatment in Microsporocytes of Hard Wheat (Triticum durum Desf.). This genotoxic effect is because of various chromosomal abnormalities in gametic cells caused by colchicine treatment (Khah et al., 2022. Influence of Colchicine in Causing Severe Chromosomal Damage in Microsporocytes of Hard Wheat (Triticum durum Desf.): Possible Mechanisms and Genotoxic Relevance. Cytologia, 87(2), pp.137-143). The colchicine treatment in Katokkon pepper (Capsicum annuum L.) resulted only in mixopioid plants with reduced size and weight of the plant (Tammu et al., 2021 . Colchicine effects on the ploidy level and morphological characters of Katokkon pepper (Capsicum annuum L.) from North Toraja, Indonesia. Journal of Genetic Engineering and Biotechnology, 19(1), p.31).

[0129] Example 4: Molecular characterization of the chromosome doubled B. coddii lines

[0130] We selected 3 genes (Ubiquitin-5, Actin depolymerizing factor 3, and Tubulin alpha chain) that are constitutively expressed in plants for qRT-PCR analysis. All the genes showed significantly higher gene expression in the mutant when compared to the wild type. This indicates a positive impact of the chromosome doubling on the general gene expression in the polyploid mutant when compared to the wild type plants (Figure 6).

[0131] Example 5: Nickel hyperaccumulation assessment of the chromosome doubled B. coddii lines

[0132] In order to quantify the levels of nickel accumulated and sequestered in the leaves of the different tetrapioid, mixopioid, and diploid lines and in the soil, we used the Inductively coupled plasma mass spectrometry (ICP-MS) technique. The nickel content was normalized using BioConcentration Factor (BCF) for the soil nickel concentration. BCF corresponds to the ratio of nickel concentration per gram of dry material between the leaf and soil samples, shows an upward trend for the M2 with 0.74 fold increase compared to the wildtype (Figure 7A). New mutants were obtained, which consistently extract more nickel than control (Figure 7B and 7C). As an example, M27 mutant extract 2.1 BCF fold compared to control.

[0133] Example 6: Pollen and flower sterility

[0134] Of the two mutant lines only M3 produced flowers while the M2 remained without any flower production. In order to assess the fertility of the pollen, a pollen viability test was carried out by germination of pollen. The pollen grains from the polyploid mutant M3 were deformed, non-viable and did not produce germination tubes (Figure 8A). Moreover, the pollen grain from the polyploid mutants were larger than the pollen grains from the wild-type plants (Figure 8B).

[0135] The flowers did not produce viable seeds when the pollen from M3 was used to fertilize wild type plants or when the pollen from wild type was used to fertilize the M3 flowers. This indicates that the pollen grains and ovary of the flowers were non-viable. (Figure 9)REFERENCESThroughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS

1. A method for collecting metal, comprising:(a) growing a plant having an induced polyploid or mixopioid genome in metal-containing soil so that the plant uptakes metal from soil; and(b) collecting plant biomass from said plant, wherein the method further comprises:(c) a step of drying said plant biomass;(d) a step of processing said plant biomass by thermal treatment to obtain biochar and / or ashes; and / or(e) a step of extracting the metal from the plant biomass, the dried plant biomass, the biochar and / or ashes.

2. The method of claim 1 , wherein the metal includes one or more heavy metals.

3. The method of claim 1 or 2, wherein the metal is or includes nickel.

4. The method of any one of claims 1 to 3, wherein said plant is a tetrapioid or mixopioid plant.

5. The method of any one of claims 1 to 4, wherein said plant belongs to a metalhyperaccumulator plant species.

6. The method of any one of claims 1 to 5, wherein said plant belongs to a nickel- hyperaccumulator plant species.

7. The method of any one claims 1 to 6, wherein said plant belongs to a family selected from Asteraceae, Brassicaceae and Violaceae.

8. The method of any one of claims 1 to 7, wherein said plant belongs to a genus selected from Alyssum, Berkheya, Brassica, Rinorea, Senecio, Thlaspi and Noccaea.

9. The method of any one of claims 1 to 8, wherein said plant belongs to a species selected from Alyssum spp., Berkheya spp., Brassica spp., Rinorea spp., Senecio spp., Thlaspi spp. and Noccaea spp.

10. The method of any one of claims 1 to 9, wherein said plant is a Berkheya spp. plant, in particular a Berkheya coddii plant.

11. The method of any one of claims 1 to 10, wherein the amounts of an uptaken metal, in particular nickel, in the collected biomass, are higher by at least 70% in comparison to a plant from the same species having a naturally-occurring ploidy.

12. A method for producing biochar and / or ashes with increased metal concentration, in particular nickel concentration, comprising(a) growing and / or providing a plant having an induced polyploid or mixopioid genome in metalcontaining soil so that the plant uptakes metal from soil, wherein said plant comprises an induced polyploid or mixopioid genome;(b) collecting plant biomass from said plant;(c) optionally drying said plant biomass;(d) processing said plant biomass by thermal treatment to obtain biochar and / or ashes, whereby said biochar and / or ashes has increased metal concentration, in particular nickel concentration in comparison to a plant from the same species having a naturally-occurring ploidy.

13. The method of claim 12, further comprising extracting the metal from the plant biomass, the dried plant biomass, biochar and / or ashes.

14. Use of a plant having an induced polyploid or mixopioid genome, for phytomining, preferably wherein said plant is a tetrapioid or mixopioid plant.

15. The use of claim 14, wherein said plant belongs to a metal hyperaccumulator plant species.

16. The use of claim 14 or 15, for collecting nickel from soil.

17. A metal-hyperaccumulator plant having an induced polyploid or mixopioid genome.

18. The plant of claim 17, which is a nickel-hyperaccumulator plant.

19. The plant of claim 17 or 18, wherein said plant belongs to a family selected fromAsteraceae, Brassicaceae and Violaceae.

20. The method of any one of claims 17 to 19, wherein said plant belongs to a genus selected from Alyssum, Berkheya, Brassica, Rinorea, Senecio, Thlaspi and Noccaea.

21. The plant of any one of claims 17 to 20, wherein said plant belongs to a species selected from Alyssum spp., Berkheya spp., Brassica spp., Rinorea spp., Senecio spp., Thlaspi spp. and Noccaea spp.

22. A plant part, cell or tissue from the plant of any one of claims 17 to 21 , wherein said plant part, cell or tissue has an induced polyploid or mixopioid genome.

23. A plant-derived product selected from plant biomass, plant dried biomass, biochar and / or ashes, wherein said plant-derived product is obtained by the method of any one of claims 1 to 11 , and / or from the plant of any one of claims 17 to 21 .

24. A method for producing metal-hyperaccumulator plants, comprising:(a) providing one or more plant part, tissue and / or cells from a metal-hyperaccumulator plant, in particular wherein said plant part, tissue and / or cells is(are), or is(are) derived from, an explant, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole, flower, bark and / or seed;(b) inducing polyploidy in the plant part, tissue and / or cells, preferably by application of an antimitotic treatment, in particular selected colchicine, oryzalin, nitrous oxide gas, heat, amiprophos methyl, trifluralin, pronamide and any of their combinations or mixtures.; and(c) regenerating mature plants from said plant part, tissue and / or cells, wherein said mature plants have an induced polyploid or mixopioid genome.

25. The method of claim 24, wherein the metal-hyperaccumulator plant at step (a) is a nickel-hyperaccumulator plant species.

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