WO2023205485A1

WO2023205485A1 - Miscanthus varieties for cold geographic regions

Info

Publication number: WO2023205485A1
Application number: PCT/US2023/019503
Authority: WO
Inventors: Oliver J. Ratcliffe; Jeffrey M. Libby; Paul Carver
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-04-22
Filing date: 2023-04-22
Publication date: 2023-10-26
Anticipated expiration: 2024-10-22
Also published as: AU2023258315A1; EP4510822A1; CA3250027A1

Abstract

Low moisture content at harvest is important since green biomass material is not suitable for many applications such as for combustion in power stations. It is unusual to find a Miscanthus variety that is both cold tolerant and/or vigorous but that dries down well in years with mild winters. A mild winter may reduce the amount of usable biomass that is obtained from Miscanthus plants, including from cold hardy varieties that will stay green and fail to dry down properly at the end of a season unless there is a heavy prolonged frost. To address this issue, the present description relates to the production of increased usable biomass yield that may be obtained at harvest from Miscanthus plants that are cold tolerant, vigorous, and which also have good dry down properties during mild winters. The present methods include plants and methods for regenerating plants from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, and then selecting one or more regenerated plants that produce increased usable biomass.

Description

MISCANTHUS VARIETIES FOR COLD GEOGRAPHIC REGIONS

FIELD OF THE INVENTION

The invention pertains to methods for producing usable biomass from Miscanthus plant cultivars.

BACKGROUND

Plants with enhanced usable biomass are useful for energy production, combustion, carbon sequestration, horticulture and other applications. Miscanthus, a monocot C4 grass genus of the Saccharum complex from the family Poaceae, tribe andropogoneae, subtribe saccharinae, is a perennial grass that has the potential to produce considerable biomass with the ability to grow with little fertilizer input and on marginal land. Miscanthus varieties are often characterized by good water use and nutrient use efficiency, and general non-invasiveness. Miscanthus varieties have thus gained attention as a biofuel crop because of their ability to yield high amounts of high-quality lignocellulosic material and also to sequester significant amounts of carbon in a single year and over the lifetime of the plants. There are more than a dozen documented naturally occurring species of Miscanthus; the genus possesses a basic chromosome number of 19, with diploid and tetrapioid species being common. Common species are M. sinensis, M. sacchariflorus, M. floridulus, M. transmorrisonensis, and M. condensatus, and researchers have bred and/or selected from these to produce new varieties. Such varieties include Nagara (‘MBS 7001’; Deuter, United States Patent PP22,033, issued July 19, 2011) and Miscanthus x giganteus (M x giganteus), both of which are triploids resulting from a cross between the diploid M. sinensis and the tetrapioid M. sacchariflorus. Triploids are especially favored since their sterility reduces the potential for invasiveness.

Triploid Miscanthus plants, including 'Nagara' can be used as hardy sterile hybrid varieties with a vigorous growth habit. However, the growth rate and ultimately the amount of usable biomass produced by ‘Nagara' is affected to a large degree by its growing environment. To improve the diversity of germplasm available for growers and different end uses, new varieties are needed. New improved varieties of Miscanthus can be produced by crossing the tetrapioid Miscanthus sacchariflorus with the diploid Miscanthus sinensis and selecting the derived plants with improved characteristics. Furthermore, a means of improving sterile vegetative varieties, which are not amenable to crossing, is through mutagenesis and selection. Mutagenesis can be induced intentionally by application of mutagens such as chemicals or radiation or can occur spontaneously during cell division. The latter is well documented to occur during laboratory cell culture procedures or under field conditions where a population of plants is grown over several seasons. One approach that can be used to create new a new improved variety from an existing variety is to generate genetic variation by subjecting the existing variety to cell culture and then selecting from amongst plants that are regenerated from the cultured cells. Such new improved varieties are sometimes referred to as cultivars.

When a cell containing an induced mutation is obtained, the cell can be propagated and regenerated into a selected new plant or plant organ which is genotypically different to the original variety, and which can itself be further propagated to produce a new variety. This methodology is often followed by plant breeders to develop new varieties of sterile crops. Typically, a breeder will select from an existing plantation a “sport” or “bud sport” which is a plant part that exhibits an altered trait (often a morphological difference) from the rest of the plant and which is caused by a chance genetic mutation. Such a genetic mutation may comprise a base substitution, or deletion or rearrangement of one or more nucleotides, or in some instances may be the result of an epigenetic change, including but not restricted to methylation or histone acetylation, which in turn, results in a visible or measurable phenotype. The breeder will then propagate clonal plants from the sport and compare them in one or more additional field trials to control plants, which often comprise plants of the parental variety that yielded the sport, to demonstrate stability of the trait in the new variety.

At the end of the growing season, Miscanthus biomass may be harvested after the plants return much of their nitrogen to the plant material below the soil and have dried down above the soil. Low moisture content at harvest is an important quality for biomass material to be suitable for combustion. Some varieties of Miscanthus are cold hardy and vigorous but these varieties generally do not dry down well in years that have a mild winter, particularly if there is a wet period immediately preceding the harvest and no heavy prolonged frost. As the global climate changes and winters become milder, including in regions that have routinely experienced very cold winters in years past, there is an increased need for Miscanthus cultivars that are vigorous and also dry down effectively, irrespective of the prevailing conditions.

Lower moisture and nitrogen content influence the extent to which biomass is usable are preferable in material used for combustion. However, for some end uses of Miscanthus biomass such as fermentation, biogas, or silage production, higher moisture and/or nitrogen content at harvest would be advantageous. Varieties of Miscanthus that arc high yielding and have higher moisture and/or nitrogen content at harvest than other available triploid varieties are desirable for such alternative end uses. Features such as higher moisture and nitrogen content are often accompanied by a visible retention of chlorophyll in the aerial parts of the plant which is known as a “stay-green” phenotype.

SUMMARY OF THE INVENTION

Methods are disclosed to produce increased quantities of usable biomass and to excel at establishment and show good growth characteristics including during cold and particularly prolonged cold periods, rapid and vigorous growth, and suitable dry-down qualities after a heavy' prolonged frost. Usable biomass may be improved due to the fact that, at harvest, the Miscanthus plant has less moisture content after tiller initiation has ceased and the leaves of the plant are no longer green or have very little green color (that is, the leaves are “no longer substantially green”). A reduction in moisture content of a harvested plant, which is inversely related to improvement in dry biomass, may be observed relative to a control Miscanthus plant grown under identical conditions and harvested at the same time.

The Miscanthus plant may exhibit at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, or at least a 50% or more of a biomass yield increase aggregated over at least three seasons, and at least 5% lower moisture content after tiller initiation has ceased and leaves are no longer substantially green, relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season.

The reduction of moisture content, a process sometimes referred to as “dry down”, of the selected Miscanthus plant relative to the control plant may result in as much as least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% less moisture than that observed in the control plant.

The methods include obtaining a plant, plant cell, cutting or rhizome from a source plant that resulted from a cross of Miscanthus sacchariflorus x Miscanthus sinensis, including a source plant of the Miscanthus variety ‘MBS 7001', also known as 'Nagara', growing one or more plants from said plant, plant cell, cutting or rhizome, and selecting an improved plant relative to a control Miscanthus plant that is grown under identical conditions and harvested at the same time, wherein the selected plant has one or more traits that contribute to better biomass yield or reduced moisture content at harvest. In one iteration, the one or more of the source plants may be subjected to mutagenesis prior to selection for the improved traits. These traits include greater vigor, greater stem length, greater leaf biomass, greater leaf area, increased height, increased biomass, increased organ size, increased shoot number or density (note that “stem” and “shoot” are often used interchangeably), increased stem thickness, reduced stem thickness when combined with increased shoot number, increased canopy cover, decreased lodging, cold tolerance, increased recovery from brackling, reduced stem nitrogen content, improved water use efficiency, reduced leaf retention, increased senescence, increased carbon content (e.g. percentage of carbon per unit mass of tissue), increased energy content (note that “energy content” and “calorific content” and “caloric content” are often used interchangeably) and less ash yield following combustion. In particular, an increase in shoot number or density combined with a reduction in stem thickness may be desirable as these traits elevate overall biomass while promoting efficient dry-down, leading to an increased yield of usable biomass. Additionally, reduced stem thickness favors the production and/or combustion qualities of biomass pellets or “brickettes” which are sold as a fuel. Indeed, dry biomass is known to burn more efficiently than wet biomass in power stations and produces less ash following combustion. A low content of the element Chlorine in the biomass feedstock is also considered desirable for power generation uses. The control plant may be any of a number of suitable Miscanthus varieties and may include M x giganteus, M. sacchariflorus, M. sinensis, or ‘MBS 7001' itself before it is used as a source for progeny plants with the improved traits.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1, 2 and 3 show measurements of overall biomass yield, mature plant height, and shoot density, respectively. Each parameter taken at the end of three consecutive seasons. Light grey bars indicate year 1, dark grey bars indicate year 2 and white bars indicate year 3. “GIG” refers to M x giganteus.

Figure 1 shows yield in 5 tonnes/hectare increments on the y axis and indicates that in year 1, yields of 1.4 - 2.5 tonnes/hectare of dry yield were achieved with non-significant differences between genotypes. In year 2, GIG increased to 9.8 tonnes/hectare of dry yield, but this yield was significantly exceeded by all genotypes with Ml 17 producing 18 tonnes/hectare. In year 3 yields of dry matter were more uniform.

Figure 2 illustrates how in year 1, M 117 reached a height of 1 m. In year 2, M 116 was tallest at 2.75 m. In year 3, the tallest was GIG at 3. 11 m. All genotypes with the exception of M116 showed increasing crop height over the first 3 years of growth.

Figure 3 shows that the new genotypes M116, M117, M118 and M119 gave much higher numbers of shoots per unit area than GIG. The highest shoot number was achieved by Ml 16 in year 1. Although shoot numbers doubled on GIG in year 2, the new genotypes produced twice as many shoots. In year 3, although Ml 16, Ml 17 and Ml 18 all produced around twice the number of shoots as GIG, Ml 19 showed parity with GIG. The population dynamics between the other new genotypes showed different trends over the 3 years. Genotypes Ml 16 and Ml 19 exhibited decreasing stem populations year over year, whereas all others showed a trend towards achieving a peak population. With the possible exception of year three for Ml 19, all of the new genotypes MH 6, Mi l 7, M118 and Ml 19 produced more shoots that GIG even when compared to Year 3 when GIG produced its most shoots.

Figure 4 shows dry down progression wherein moisture content in leaf and stem tissue are displayed over the season in year 3. Moisture contents at harvest were similar for all genotypes in years 1 and 2; all achieved moistures of 20% or less. However, at the end of year 3, the early spring prior to harvest was very wet and the season did not have a strong winter frost (as is becoming increasingly common). These conditions will occur during the lifetime of a perennial crop, and it is important that harvesting can occur. It is commercial practice to monitor the post winter moisture contents to determine proximity to harvest time. During the last year (year 3) the genotypes being trialed were monitored in a comparable manner to commercial crops. Significant differences in moisture content were observed between the genotypes: GIG and Ml 16 showed different dehydration trends to other genotypes. For these genotypes a typical drying curve was observed reaching a value of recording of about 20% at harvest. All other genotypes showed constant moisture content of circa 40% until the end of the harvest window (when new stems emerge). Overall, Ml 16 showed the most advantaged dry-down characteristics.

Symbols in Figure 4 refer to:

• GIG (M x giganteus)

■ M116

A M117

■ M118

X M119

Figure 5 compares a Miscanthus plant that was asexually propagated from an initial plant selected from a population of plants descended from tissue cultured cells of Nagara (larger, dark green plant in foreground on left) and a control Miscanthus plant of a commercial variety M x giganteus “Illinois” clone (smaller, lighter plant; in foreground on right).

Figure 6 compares the appearance of rhizome buds of a Miscanthus plant that was asexually propagated from an initial plant selected from a population of plants descended from tissue cultured cells of Nagara (Fig. 6a) and a control M x giganteus “Illinois” plant (Fig. 6b). The rhizome buds of the plant derived from Nagara are generally long, pointed, heavily scaled, protrude from the below ground rhizome at an angle less than 45°, and commonly touch the rhizome. The rhizome buds of the control plant (medium length in this image) are pointed, less heavily scaled, and protrude from the below ground rhizome at an angle of 90°. Bars show the rhizome bud angle relative to the rhizome. The unique rhizome bud angle of the Nagara derived plants may be associated with improved traits described herein, and selection of plants with an angle less than 45° may be used to obtain plants with the improved traits.

The regenerated Miscanthus plant has an improved trait profile relative to control plants including more vigorous growth and cold hardiness with good dry down characteristics in years with mild winter and a period of high rainfall preceding the harvest. Other traits that are associated with Miscanthus plants having this rhizome bud appearance may include darker leaf coloration, increased tiller number, improved stand establishment (which may reduce the need for replanting), increased usable biomass of at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, wherein the increased usable biomass results in greater aggregate yield, at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% ash content at harvest, at least 0. 1% lower chlorine content, at least 0.5% lower chlorine content, at least 1% lower chlorine content, at least 5% lower chlorine content, at least 10% lower chlorine content, at least 15% lower chlorine content, at least 17% lower chlorine content, at least 25% lower chlorine content, or at least 50% lower chlorine content in stems, after tiller initiation has ceased and leaves are no longer substantially green, at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% less moisture content at harvest after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season, a height of the regenerated Miscanthus of at least 30 cm greater than the height of a M. sinensis, a M. sacchariflorus , or an M. Giganteus "Illinois" control plant, or more vigorous growth or cold hardiness with good dry down characteristics in a year with a mild winter and a period of high rainfall preceding the harvest.

DETAILED DESCRIPTION OF THE INVENTION

It will be readily apparent to the skilled artisan that various substitutions and modifications may be made in the invention disclosed herein without departing from the scope and spirit of the invention.

It is noted that as used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a plant" or "a variety" includes one or a plurality of such plants or varieties, and a reference to "a stress" is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.

DEFINITIONS

The term "plant" includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, guard cells, pollen cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the genus of Miscanthus, or may be applied more narrowly to Miscanthus species, subspecies cultivars, varieties, and/or hybrids.

A "control plant" as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against an instant Miscanthus plant for the purpose of identifying an enhanced phenotype in the instant plant. A control plant may in some cases be a parental Miscanthus plant line, or a species, subspecies, cultivar, variety, or hybrid that is an often-used or recognizable variety , for example, Miscanthus 'MBS 700 T or Miscanthus x giganteus, or the Miscanthus x giganteus ' Illinois' clone, or a different plant that has resulted from a cross between M. sinensis and M. sacchariflorus. Generally, a control Miscanthus plant or variety is grown under very similar, essentially identical, or identical conditions to an experimental plant.

A "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size or a deeper green coloration of the plant tissue, or seedling vigor, or can be measured by biochemical techniques, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or cold, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations or analysis such as reduced moisture content at harvest after tiller initiation has ceased and leaves are no longer substantially green. One or more of the source plants may be subjected to mutagenesis prior to selection for the improved traits. The traits include, but are not limited to, greater vigor, stem length, stem thickness, leaf biomass, leaf area, leaf breadth, height, biomass yield, shoot number or density, decreased lodging, cold tolerance, nitrogen content, reduced stem nitrogen content, altered chlorophyll content, increased stem deaf biomass ratio, increased c anopy cover, recovery from brackling, less ash yield following combustion, water deficit tolerance, low nutrient tolerance, hyperosmotic stress tolerance, cold tolerance, drought tolerance, salt tolerance, reduced leaf retention, increased water use efficiency, increased nutrient use efficiency, increased carbon content, increased photosynthetic capacity or reduction in percentage composition of undesirable elements such as chlorine (the latter being known to cause corrosion in power stations). Any suitable technique can be used to measure the amount of, comparative level of, or difference in the instant and control plants.

Increased or improved or enhanced "yield" or "plant yield" refers to increased plant growth, increased crop growth, increased biomass, increased usable plant matter at harvest, increased carbon content, increased calorific content of harvested material (e.g. greater energy content per unit mass of crop matter, measured in units such as KJ/Kg) and/or increased plant product production, and is dependent to some extent on temperature, plant size, organ size, planting density , light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature acclimation and water or nutrient use efficiency. For example, Miscanthus was reported to provide a yield of up to 18-20 tonnes of dry matter per hectare per year in one trial in Germany, but with significant variation in dry matter yield between sites in the first four years after planting (Jones and Walsh, cd. (2001) Miscanthus for Energy and Fibre, James & James, London, at page 62). Harvestable yields of Miscanthus in Europe have been reported to range from 10 to 40 tonnes of dry matter per hectare per year (Lewandowski et al, (2000) Biomass and Bioenergy 19: 209-227; Heaton et al. 2008b. supra). Heaton et al. have reported that fully established plants Miscanthus can provide typical autumn yields of dry' matter ranging from 10 to 30 tonnes per hectare per year, depending on local agronomic conditions (Heaton et al. (2004) Mitigation and Adaptation Strategies for Global Change 9: 433-451).

“Usable biomass yield” or “Usable biomass” refers to the amount, or quantity, of organic material that is produced or harvested that can be utilized for a desired end-use such as energy production (e g., combustion to provide heat energy or electrical energy from power generation), fermentation, silage production, animal feed, or material applications, for example, animal feed, animal bedding, construction materials, substates for paper making, or fiber for application in textiles). Usable biomass generally needs to be sufficiently low in moisture to be suitable for production of energy or the production of these materials. The yield of usable biomass may improve with a dr ing method including leaving the stand in the field at the end of a growing period or by artificial means. At the end of dry-down, the water content (also sometimes referred to as moisture content) in the biomass of the present description may be in the range of 0. 1 to 50% by mass, 0. 1 to 40% by mass, 0. 1 to 30% by mass, 0. 1 to 20% by mass, or 0. 1 to 10% by mass. A reduction in water content in a selected plant compared to a control plant may be in the range of 0. 1 to 1%, 0.1 to 2%, 0. 1 to 5%, 0. 1 to 10%, 0. 1 to 25%, or 0.1 to 50%. Generally speaking, for power generation end uses, it is preferable for biomass to have an overall water content of less than 30%. Usable biomass yield may increase due to increased carbon composition, which element usually ultimately accounts for more than 40% of the mass of the harvested crop material. An increase in carbon composition (e.g., mass of carbon per overall mass of tissue, which is also sometimes referred to as carbon content) in a selected plant compared to a control plant may be in the range of 0. 1 to 0.25%, or 0. 1 to 0.5%, 0.1 to 0.75%, 0.1 to 1%, 0.1 to 1.25%, 0.1 to 2.5% or 0.1% to 5%. Usable biomass yield may also increase because of reduced chlorine content, which element usually ultimately accounts for more than 0.01% of the mass of the harvested crop material. Typically, an overall level of not more than 0.3% chlorine is desirable for end-uses such as combustion for power generation. A reduction in chlorine content (e.g., measured as mass of chlorine per overall mass of tissue) in a selected plant compared to a control plant may be in the range of 0. 1 to 1%, or 0. 1 to 5%, 0. 1 to 10%, 0. 1 to 25%, or 0. 1% to 50%. Usable biomass yield may also increase because of an increase in the calorific content of the harvested crop material. An increase in calorific content (e.g., measured in units of KJ/Kg) in harvested material of a selected plant compared to a control plant may be in the range of 0. 1 to 0.2%, or 0. 1 to 0.5%, 0. 1 to 1%, 0. 1 to 2.5%, 0.1% to 5% or 0. 1 to 10%.

The Miscanthus variety “Nagara” or “MBS 7001 (US Plant Patent PP22,033, supra) was originally selected for its vigorous growth from a selection field which was established from seedlings. The seedlings were obtained from seeds of a polycross of tetrapioid Miscanthus sacchariflorus and diploid M. sinensis plants. 'MBS 7001 'is a sterile triploid plant; hence, it cannot be readily reproduced sexually. MBS 700 T was generated by crossing a single large-stemmed M. sacchariflorus genotype from Japan as a female parent with a population of 15 M. sinensis plants as pollen donors. From this cross, seedlings were obtained and planted in a field. Based on field observations, one triploid variety having high biomass was selected and propagated and designated as 'MBS 7001'

Miscanthus X giganteus is also a sterile triploid resulting from a cross between M. sinensis and M. sacchariflorus. Miscanthus X giganteus autumn yields in lowland areas in Europe are typically higher than 25 tonnes per hectare per year, and Miscanthus X giganteus could provide a hypothetical yield of 27-44 tonnes of dry matter per hectare per year with a mean yield of 33 tonnes of dry matter per hectare per year in 'Illinois' (Heaton et al. (2004) supra). Miscanthus X giganteus can thus yield, under various conditions of growth, biomass of at least 10, at least 15, at least 20, at least 25, at least 27, at least 30, at least 33, at least 35, at least 40, at least 44 tonnes or more of dry matter per hectare per year. It is expected that the enhanced triploid varieties of Miscanthus described herein can produce similar biomass yields, ranging from, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125% or more of the biomass yield of a control sterile triploid Miscanthus X giganteus crop at substantially the same stage of development and grown under substantially the same, or the same, environmental conditions as the enhanced triploid varieties, or, in other words, enhanced triploid varieties are expected to yield at least 75% to at least 125% or more of 10 to 44 tonnes or more of dry matter per hectare per year.

A non-naturally occurring Miscanthus plant is one that is not obtained directly from nature, but is created by human intervention by methods that may include but are not limited to breeding, selection, propagation, or regeneration from cell or tissue culture.

“Substantially green” refers to the appearance of a plant or plant part that is noticeably and primarily green in color and does not yet have substantial chlorophyll degradation due to senescence or nitrogen remobilization from above ground tissues. Carotenoids such as anthocyanin and xanthophylls do not significantly contribute to the color of the plant.

For the purposes of this description, a “mild winter” may be defined by conditions of:

(i) an average daily low temperature in the months of November, December and January inclusive of equal to or greater than -0.3° Celsius;

(ii) 43 or fewer days of air frost during the months of November through January inclusive; or

(iii) a rainfall in January of greater than 58 mm or at least 98.2 mm.

"Planting density" refers to the number of plants that can be grown per acre. For crop species, planting or population density varies from a crop to a crop, from one growing region to another, and from year to year. Using corn as an example, the average prevailing density in 2000 was in the range of 20,000-25,000 plants per acre in Missouri, USA. A desirable higher population density (a measure of yield) would be at least 22,000 plants per acre, and a more desirable higher population density would be at least 28,000 plants per acre, more preferably at least 34,000 plants per acre, and most preferably at least 40,000 plants per acre. The average prevailing densities per acre of a few other examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh et al. (2003) U.S. Patent Application No. 20030101479). For Miscanthus, a typical initial planting density is 10,000 plants per hectare (Scurlock (1999) Miscanthus: A Review of European Experience with a Novel Energy Crop, U.S. Department of Energy, Publ. ORNL/TM- 13732, at page 6). However, propagules of Miscanthus are often planted at the time of establishment of a plantation at densities in the range of 25,000 - 50,000 planting units per hectare. A desirable higher population density for each of these examples, as well as other valuable species of plants, including Miscanthus, would be at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 50%, or higher, than the average prevailing density.

Green biomass may not be suitable material, for example, for burning in power stations (a process also sometimes referred to as “co-firing” when the biomass is combusted with other substrates), but may be desirable for fermentation or silage production. “Dry-down” refers to a lowering of the moisture content of the above ground portion of the plant at the end of or after the growing season and the transport of nitrogenous compounds from the above ground to the below ground portion of tire plant. At harvest, tire Miscanthus plant has less moisture content at harvest, generally after tiller initiation has ceased and leaves of the plant are no longer green or are no longer substantially green. Reduction in moisture content of a harvested plant is inversely related to improvement in dry biomass.

“Brackling” refers to a phenomenon where the top portion of a crop, which is heavy' with leaf material, collapses over, often following a period of sustained rain. The phenomenon is undesirable as it limits the ability to harvest with farm machinery and thereby leads to reduced yield. Thus, varieties are desirable which are less susceptible to brackling and/or which show an increased recovery from brackling.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Miscanthus varieties generally do not exhibit vigorous growth or cold hardiness with good dry down characteristics in years that happen to have a mild w inter combined with a period of high rainfall preceding the harvest. Typically, hardy Miscanthus varieties that are cold tolerant or vigorous will stay green and fail to dry down properly at the end of a season unless there is a very heavy prolonged period of freezing. This problem is exacerbated in geographic regions that typically have harsh winters, but which are beginning to experience more mild winters as a result of climate change.

With regard to biomass from Miscanthus, what is particularly needed for end uses such as combustion in power stations are varieties that can be planted in cold regions, for example, in northern North America, Eastern Europe, and Asia, where the winters are still typically very cold, but where the crop will still dry down and be saleable in years where the winter is relatively warm and wet (and which are becoming more frequent).

The Miscanthus plants of the instant description may exhibit at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least 25%, or at least a 50% or more of a biomass yield increase aggregated over at least three seasons, and at least 5% lower moisture content after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season.

EXAMPLES

Example 1. Statements embodying certain aspects of the present disclosure

A. A method for increasing usable biomass yield of a Miscanthus plant by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, the method comprising selecting a Miscanthus plant obtained from a Miscanthus sacchariflorus x Miscanthus sinensis cross (for example, MBS 7001) that at harvest has less moisture content after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time.

B. The method of Statement A, wherein the selected Miscanthus plant is obtained from a cell, rhizome, or cutting of the Miscanthus sacchariflorus x Miscanthus sinensis cross that is propagated and grown into a mature Miscanthus plant.

C. The method of Statement A or Statement B, wherein the mature Miscanthus plant is selected for said less moisture content after tiller initiation has ceased and leaves are no longer substantially green relative to the control Miscanthus plant.

D. The method of Statement A or B, wherein the mature Miscanthus plant is selected for thinner stems, increased shoot density, and/or full senescence at harvest relative to the control plant.

E. The method of Statement A, wherein the selected Miscanthus plant is obtained from a Miscanthus ‘MBS 7001' plant or a cell, rhizome, or cutting of a Miscanthus ‘MBS 7001' plant.

F. The method of Statement A, wherein the selected Miscanthus plant has at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% less moisture than the control Miscanthus plant.

G. The method of Statement A, wherein the control plant is Miscanthus ‘MBS 7001' or Miscanthus x giganteus or a different plant of a Miscanthus sacchariflorus x Miscanthus sinensis cross.

H. A method for producing a Miscanthus plant that has increased yield at harvest after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant; the method comprising: growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross (for example, MBS 7001), and selecting a regenerated plant to create a selected plant that has an improved trait that results in increased yield relative to the control Miscanthus plant grown under identical conditions and harvested at the same time. I. The method of Statement H, wherein the cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross is from Miscanthus ‘MBS 700 T .

J. The method of Statement H, wherein the control Miscanthus plant is Miscanthus ‘MBS 7001' or Miscanthus x giganteus or a different plant of a Miscanthus sacchariflorus x Miscanthus sinensis cross.

K. The method of Statement H, wherein the improved trait is selected from the group including greater vigor, stem length, leaf area, leaf biomass, height, usable biomass yield, shoot density, increased stem thickness, decreased lodging, improved recovery from brackling, cold tolerance, greater canopy cover, reduced stem nitrogen content, reduced moisture content, reduced stay green, reduced nitrogen off-take, reduced stem thickness combined with increased shoot number, increased water use efficiency, increased carbon content, lower chlorine content, increased calorific content, increased stem calorific content, increased leaf calorific content, increased stemdeaf biomass ratio, and less ash yield following combustion relative to the control Miscanthus plant.

L. A method for producing a Miscanthus plant that has reduced moisture content at harvest after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant; the method comprising: growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross (for example, MBS 7001), and selecting a regenerated plant to create a selected plant that has an improved trait that results in reduced lower moisture content relative to the control Miscanthus plant grown under identical conditions and harvested at the same time.

M. The method of Statement L, wherein the cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross is from Miscanthus ‘MBS 700 T .

N. The method of Statement L, wherein the control Miscanthus plant is Miscanthus ‘MBS 700 T or M x giganteus or a different plant of a Miscanthus sacchariflorus x Miscanthus sinensis cross.

O. The method of Statement L, wherein the improved trait is selected from the group greater vigor, stem length, leaf area, height, usable biomass yield, shoot density, reduced stem thickness, reduced stem thickness combined with increased stem number, decreased lodging, improved recovery from brackling, cold tolerance, greater canopy cover, reduced stem nitrogen content, reduced moisture content, reduced stay green, reduced nitrogen off-take, increased water use efficiency, reduced leaf retention, increased stemdeaf biomass ratio, increased stem calorific content, and less ash yield following combustion relative to the control Miscanthus plant.

P. The method of Statement L, wherein said selected plant and said control plant are grown in a mild winter defined by conditions of: (i) an average daily low temperature in the months of November, December and January inclusive of equal to or greater than -0.3° Celsius;

(ii) 43 or fewer days of air frost dining the months of November through January inclusive; or

(iii) a rainfall in January of greater than 58 mm or at least 98.2 mm.

Q. The method of Statement L, wherein the selected plant displays greater vigor than the control plant.

R. The method of Statement L, wherein the selected plant is grown over one or more seasons prior to selection for the improved trait and/or lower moisture content relative to the control Miscanthus plant grown under identical conditions.

S. The method of Statement L, wherein the regenerated plant is subjected to a temperature of less than 0° C on at least 17 days during the months of November, December and January of one winter.

T. A method for decreasing moisture content at harvest of a Miscanthus plant; the method comprising: growing a regenerated plant from a cell, rhizome, or cutting of Miscanthus sacchariflorus x Miscanthus sinensis cross, and selecting a regenerated plant with an improved trait relative to a control plant to create a selected plant that has decreased moisture content at harvest when tire regenerated plant is grown under conditions of a mild winter defined by conditions of:

(ii) 43 or fewer days of air frost during the months of November through January inclusive; and/or

(iii) a rainfall in January of greater than 58 mm or at least 98.2 mm; relative to a control Miscanthus plant grown under identical conditions.

U. The method of Statement T, wherein the regenerated plant is obtained from a Miscanthus ‘MBS 7001' plant or a cell, rhizome, or cutting of a Miscanthus ‘MBS 7001' plant.

V. The method of Statement T, wherein the control plant is Miscanthus ‘MBS 7001' or M x giganteus or a different plant of a Miscanthus sacchariflorus x Miscanthus sinensis cross.

W. The method of Statement T, wherein the selected plant has an improved trait of greater vigor, stem length, leaf area, height, usable biomass yield, shoot density, reduced stem thickness, reduced stem thickness combined with increased stem number, decreased lodging, improved recovery from brackling, cold tolerance, greater canopy cover, reduced stem nitrogen content, reduced moisture content, reduced stay green, reduced nitrogen off-take, increased water use efficiency, reduced leaf retention, increased stem deaf biomass ratio, increased stem calorific content, and less ash yield following combustion relative to the control Miscanthus plant. X. The method of Statement T, wherein the selected Miscanthus plant is selected for thinner stems, increased shoot density, and/or full senescence at harvest relative to the control Miscanthus plant.

Y. The method of Statement T, wherein the selected plant displays greater vigor than the control plant.

Z. The method of Statement T, wherein said selected plant and said control plant are grown in a mild winter defined by conditions of:

(i) an average daily low temperature in the months of November, December and January inclusive of greater than -0.3° Celsius;

(ii) 43 or fewer days of air frost during the months of November through January inclusive; and

(iii) a rainfall in January of greater than 58 mm or at least 98.2 mm.

AA. The method of Statement T, wherein the selected plant is grown over one season or more than one season prior to selection for the improved trait and/or lower moisture content relative to the control Miscanthus plant grown under identical conditions.

AB. The method of Statement T, wherein the selected plant is subjected to a temperature of less than 0° C on at least 17 days during the months of November, December and January of one winter

AC. A non-naturally occurring Miscanthus plant that has reduced moisture or nitrogen content at harvest after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant, which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, and selecting a regenerated plant to create a selected plant that has an improved trait that results in reduced lower moisture or nitrogen content relative to the control Miscanthus plant grown under identical conditions and harvested at the same time.

AD. The non-naturally occurring Miscanthus plant of Statement AC, wherein the improved trait is selected from the group including greater vigor, stem length, leaf biomass, leaf area, height, usable biomass yield, shoot density, reduced stem thickness, reduced stem thickness combined with increased stem number, decreased lodging, improved recovery' from brackling, greater canopy cover, cold tolerance, reduced stem nitrogen content, reduced stay green, reduced nitrogen off-take, increased nitrogen remobilization, increased water use efficiency, reduced leaf retention, increased stemTeaf biomass ratio, increased stem calorific content, and less ash yield following combustion.

AE. A non-naturally occurring Miscanthus plant which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross (for example, MBS 7001), that has at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% less moisture and at least 0. 1% greater carbon composition, at least 0.25% greater carbon composition, at least 0.5% greater carbon composition, at least 0.75% greater carbon composition, at least 1% greater carbon composition, at least 1.5% greater carbon composition, or at least 5% greater carbon composition after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season.

AF. A non-naturally occurring Miscanthus plant which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, that has at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% lower moisture content and at least 0. 1% greater calorific content, at least 0.25% greater calorific content, at least 0.4% greater calorific content, at least 0.5% greater calorific content, at least 1% greater calorific content, at least 2.5% greater calorific content, at least 5% greater calorific content, or at least 10% greater calorific content after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season.

AG. A non-naturally occurring Miscanthus plant which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, that has at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% lower moisture content and at least 5% lower ash yield, at least 10% lower ash yield, at least 1 % lower ash yield, at least 17% lower ash yield, at least 20% lower ash yield, or at least 50% lower ash yield, after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to winter season.

AH. A non-naturally occurring Miscanthus plant which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, that has at least a 10% increase in stem density, 20% increase in stem density, 3 % increase in stem density, 40% increase in stem density, or 50% increase in stem density and at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% lower moisture content after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after tire plants have been subject to a winter season.

Al. The non-naturally occurring Miscanthus plant of Statement AH wherein the lower moisture content is associated with a smaller stem diameter and a greater aggregate yield over at least two seasons relative to the control Miscanthus plant.

AJ. A non-naturally occurring Miscanthus plant which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, that produces at least a 10% increase in biomass aggregated over at least three seasons and at least 5% lower moisture content after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season.

AK. A non-naturally occurring Miscanthus plant which is produced by growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, that has at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% lower moisture content and at least 0. 1% lower chlorine content, at least 0.5% lower chlorine content, at least 1% lower chlorine content, at least 5% lower chlorine content, at least 10% lower chlorine content, at least 15% lower chlorine content, at least 17% lower chlorine content, at least 25% lower chlorine content, or at least 50% lower chlorine content, after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season.

AL. The non-naturally occurring Miscanthus plant of any of Statements AE - AK wherein tire winter season comprises a mild winter defined by conditions of: (i) an average daily low temperature in the months of November - January inclusive of equal to or greater than -0.3 degrees Celsius, (ii) 43 or fewer days of air frost during the months of November - January inclusive or (iii) a rainfall in January of greater than 58 mm or at least 98.2 mm.

AM. The non-naturally occurring Miscanthus plant of any of Statements AE - AK wherein the reduced moisture content arises from reduced stem thickness.

AN. The non-naturally occurring Miscanthus plant of any of Statements AE - AK wherein the plant is a progeny plant of a plant selected from genotype Ml 16.

AO. A method for producing a Miscanthus plant that has increased moisture and nitrogen content at harvest after tiller initiation has ceased relative to a control Miscanthus plant; the method comprising: growing a regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, and selecting a regenerated plant to create a selected plant that has an improved trait that results in increased moisture or nitrogen content relative to the control Miscanthus plant grown under identical conditions and harvested at the same time.

AP. The method of Statement AO, wherein the improved trait is selected from the group including greater stay green, vigor, stem length, leaf area, height, usable biomass yield, shoot density, stem thickness, decreased lodging, recovery from brackling, reduced senescence, increased chlorophyll content, increased green coloration, increased photosynthesis, and cold tolerance relative to the control Miscanthus plant.

AQ. A non-naturally occurring Miscanthus plant that has increased moisture and nitrogen content at harvest after tiller initiation has ceased relative to a control Miscanthus plant, which is produced by growing regenerated plant from a cell, rhizome, or cutting of a Miscanthus sacchariflorus x Miscanthus sinensis cross, and selecting a regenerated plant to create a selected plant that has an improved trait that results in increased moisture or nitrogen content relative to the control Miscanthus plant grown under identical conditions and harvested at the same time.

AR. The non-naturally occurring Miscanthus plant of Statement AQ, wherein the improved trait is selected from the group including greater stay green, vigor, stem length, leaf biomass, height, usable biomass yield, shoot density, stem thickness, decreased lodging, recovery from brackling, reduced senescence, increased chlorophyll content, increased green coloration, increased photosynthesis, and cold tolerance.

AS. The non-naturally occurring Miscanthus plant of either of Statements AQ or AR wherein the plant is a progeny plant of a plant selected from genotype Ml 17, Ml 18, or Ml 19.

Example 2,

Plants of the variety MBS 7001 are established in a field trial site and allowed to grow for a period including at least one winter season. An individual sport is selected from a plant of the existing population which appears to be visibly more vigorous, darker green, taller, or having thicker stems than the population of MBS 7001 plants. The sport is propagated through rhizome cuttings which are established in field trials and shown on average to have a usable biomass yield of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% greater than control plants of the original MBS 7001 variety.

Example 3.

Crosses were made between the tetrapioid species Miscanthus sacchariflorus and the diploid species Miscanthus sinensis, from which four different descendant F 1 plants were selected. Material derived from each of these plants was subjected to cell culture and a population of 200 plants from each was regenerated. The cell culture derived populations were assigned the codes Ml 16, Ml 17, M118 and Ml 19 respectively, and hereafter referred to as genotypes. The population Ml 16 was derived from the same original progenitor plant that gave rise to the variety ‘MBS 7001', also known as 'Nagara' (Deuter, United States Patent PP22,033, issued July 19, 2011). Each of the populations was subjected to a blocked field trial at Taunton in the county of Somerset, United Kingdom which spanned three winters, during which growth characteristics and various phenotypes were recorded. Climatic data for the period of the trial was logged in UK Met Office records from a nearby weather station at Yeovilton, Somerset. After tire end of the trial, individual “center-plants” which sustained desirable features were selected from the blocks in the field trial site and further propagated through rhizome cuttings to establish nursery populations suitable as a source of material for propagation and bulk-up of material for establishment of high yielding commercial plantations.

MATERIALS AND METHODS

The 200 plants from each Miscanthus genotype, Mi l 6, Mil 7, M118 and Ml 19 were planted in a block trial at a site in Taunton, Somerset, UK.

M x giganteus was used as a control for the trial, using rhizomes previously held in cold store for a period of approximately three months. The trial was based on four randomized blocks; with each block containing a 25 m² plot planted at two plants/m². Plots consisted of five rows with ten plants in each row. Only the center three rows (15 m²) were recorded at harvest with the other rows acting as guards.

A seedbed, of medium clay loam soil, was worked down in such a manner that a fairly fine tilth was available at planting. Soil samples taken in October of the first year, following planting, showed a pH of 6. 1, and indices of 3 (28.4mg/l available), 2, (153 mg/1) and 4 (201mg/l) for P, K and mg respectively, indicating a satisfactory supply of these essential elements. These indices were generally not found to have changed substantially after the final harvest at the end of the third year. Levels of P and K did show a decrease of 20% and 17% over the 3 years respectively.

Plants and rhizomes were planted by hand into weed-free soil in May of the first season. Immediately following planting, watering took place. Post- planting no further watering or irrigation occurred

Plant canopy height and shoot number per plant were assessed at the end of each growing season (Spring) on 3 plants in the middle row of each plot. Harvesting took place in the first two seasons in February once stems and leaves had reached a uniform brown, dry status. Harvest dates were timed to be within the harvest window typically used for commercial Miscanthus crops in the vicinity. Due to the very slow maturation of the trial genotypes in the final season, year 3, final harvesting was delayed until March. Even at this late stage, stems of M117, M118 and M 119 were still visibly green, which was reflected in the high moisture contents.

Leaf and stem samples were taken from the third-ycar harvest and analyzed by at a UK power station for elements crucial in the combustion process. The analysis procedure followed for this was typical for assessment of any biomass fuels used at UK power stations for co-firing with coal. RESULTS Yields

In year 1, yields of 1.4 - 2.5 Tonnes per hectare were achieved (dry matter) with non-significant differences between genotypes. In year 2, M x giganteus increased to 9.8 Tonnes per hectare, but this yield was significantly exceeded by all genotypes with Ml 17 producing 18 Tonnes per hectare. In year 3 yields were more uniform. The highest yields were achieved by M x giganteus and Ml 17 at approximately, 24.5 Tonnes per hectare exceeding that produced by other genotypes. Canopy height

In year 1, Ml 17 reached a height of Im, whereas in year 2, Ml 16 was tallest at 2.75m. In year 3 tallest was M x giganteus at 3. 11m. All genotypes with the exception of Ml 16 showed increasing crop height during the first 3 years of growth.

Images of all three genotypes taken in year 2 revealed differences in the growth patterns. All new genotypes exhibited increased leafmess compared to M x giganteus, and higher stem populations.

Shoot numbers

All new genotypes exhibited much greater shoot number than M x giganteus, with the greatest shown by Ml 16. Although shoot numbers doubled on M x giganteus in year 2, the other genotypes produced twice as many shoots. In year 3 although Ml 16, 117 and Ml 18 all produced around twice the number of shoots as M x giganteus, Ml 19 showed parity' with the latter.

The population dynamics between genotypes showed different trends over the 3 years genotypes Ml 16 and Ml 19 showed decreasing stem populations, whereas all others showed a trend towards achieving a peak population.

Stem strength

It was observed in year 3 that in every replicate of Ml 16 the crop had lodged in the center of the plots, which made counting of plants in the middle row difficult. Already, by mid- June of the year following the final harvest, every replicate of this genotype has again lodged badly, presumably due to stem weakness following strong winds and rain. These effects were not observed in any other genotypes.

Moisture content at harvest

Harvest moisture contents at harvest were similar for all genotypes in years 1 and 2, all achieved moistures of 20% or less. In these years, the winters were relatively mild, but the early spring (January) was relatively dry. However, in year 3, the season once again did not have strong winter frost (as is becoming increasingly common), but there were prevailing wet conditions in the early spring (January) prior to harvest. Such conditions will occur in some seasons during the lifetime of a perennial crop, and it is important that harvesting can still occur.

In year 3 significant differences were observed between the genotypes. It is commercial practice to monitor the post winter moisture contents to determine proximity to harvest time. During the year 3 harvest the trial crops were monitored as per commercial crops. M x giganteus and Ml 16 showed different dehydration trends to other genotypes. For these genotypes a typical drying curve was observed reaching a value of recording circa 20% at harvest. All other genotypes showed constant moisture content of circa 40% until the end of the harvest window (when new stems emerge).

Samples of cane at harvest showed that the genotypes that did not dry down did so due to failure to senesce. In addition, the new genotypes all showed leaf retention which increased crop moisture content. The only new genotype that did achieve full senescence (Ml 16) had stems that were thinner than the other genotypes.

The finding that Ml 16 exhibited thinner stems associated with lower moisture content and better dry down profile compared to GIG combined with the increased shoot density and the increased aggregate biomass of Ml 16 over the three seasons, indicated that Ml 16 can provide a greater usable biomass yield for end uses such as pelleting and/or combustion than M. x giganteus and the other genotypes. Indeed, the presence of thinner stems may be advantageous for the production of biomass pellets, especially if the material is being blended with other biomass, such as biomass of other Miscanthus varieties with thicker stems.

Figures 1, 2 and 3 show measurements of overall biomass yield, mature plant height, and shoot density, respectively. Each parameter taken at the end of three consecutive seasons. Light grey bars indicate year 1, dark grey bars indicate year 2, and white bars indicate year 3.

Fuel Quality

While longer term markets exist for biomass crops, the immediate markets are in combustion and in particular co-firing with coal. It is essential for satisfactory trading that crops of Miscanthus conform to typical fuel specifications.

Results from analyses of leaves and stems of Miscanthus varieties, sampled at year 3 harvest, are shown in Table 1, Table 2, and Table 3. At harvest, all genotypes were found to have satisfactory levels of chlorine and sulfur below commercial levels. However, genotype Ml 17 was found to be within 15% of the limit for chlorine; this is important as the element poses a concern to end users due to its corrosive effects upon combustion in power stations. It is likely that this would have been caused in part due to the reduced senescence (and hence lack of leaching) during the winter months. Levels of chlorine in the stems of Ml 17 were found to be approximately double those in M x giganteus, whereas those in the stems of Ml 16 were more than 35% lower than in M x giganteus.

Table 1. Analysis of stems for combustion properties, year 3 harvest

Table 2. Analysis of leaves for combustion properties, year 3 harvest

Table 3. Analysis of total biomass material for combustion properties, year 3 harvest

For Tables 1, 2, and 3, GIG = G x giganteus

Ash levels also differed significantly between genotypes. Those that retain significant leaf material (Ml 17, Ml 18, Ml 19) had ash levels over 40% higher than M x giganteus. The levels of ash are typically higher in leaf material than in stems. Ash levels were not in excess of limits but their removal is a cost factor for power stations. Higher levels of leaf material can also pose problems in subsequent processing, as it is correlated to silica content which is abrasive. Typically, silica levels re 4-5 times higher in leaves than in stems, so it is generally undesirable if a variety has an increase in leaf: stem biomass or if a variety has enhanced leaf retention.

Only two genotypes were able to dry down sufficiently to meet commercial target moisture requirements for combustion uses in year 3 (GIG, Ml 16; of the two, Ml 16 was superior and exhibited lower moisture content combined with lower ash yield, lower chlorine content, and lower nitrogen content but a higher overall carbon percentage in stems compared to GIG). Material from the other genotypes would have been unsuitable for baling or for commercial sale for combustion uses. Energy contents were relatively similar between genotypes, with an average of 19,172 GJ/t with less than 1.5% variation. Overall leaf material produced 5% less energy than stems due to the higher ash content.

Nitrogen use

The genotypes did display substantial differences in the level of nitrogen at harvest. This is potentially due to the reduced senescence during the winter months. Ml 16 exhibited the lowest levels of stem nitrogen compared to the other genotypes and M x giganteus. Reduction in N-content of harvested biomass material can be considered as a desirable nutrient use efficiency trait and this was exhibited in genotype Ml 16 in comparison the other genotypes and M x giganteus.

DISCUSSION

Although yields increased substantially from year 1, the large increase with the genotypes over M x giganteus in year 2 was not experienced in year 3, where all types have produced a similar yield, with M x giganteus giving the highest at 24.5 Tonnes per hectare.

Shoot density was consistently greater in tire case of Mi l 6, M117 and Ml 18 versus M x giganteus, whereas with Ml 19 produced similar numbers of shoots/stool to those seen in M x giganteus. However, a general inverse effect was observed with shoot weight, with that being greater in M x giganteus and Ml 19 versus Ml 16, Ml 17 and Ml 18.

At year 3 harvest was that despite delaying harvest from February, the time proposed in the trial protocol when stems of M x giganteus are usually brown, moisture levels of stems from Ml 17, Ml 18 and Ml 19 were much higher. Moisture levels in these genotypes remained high and as a result they were over the maximum baling or supply limit at harvest in year 3. Since these samples were still visibly green, it is clear that the winter temperatures in the UK were not sufficient to produce a total winter kill of the crops. The enhanced greenness of some cultivars also influenced the nitrogen off-take which could have significant negative economic implications for certain end uses such as combustion in power stations. By contrast to M 117, M 118, and M 119, stems of M 116 and M x giganteus were brown and lacked green coloration at harvest. Figure 4 shows dry down progression wherein moisture content in leaf and stem tissue are displayed over the season in year 3 where Ml 16 showed the most favorable dry down profile.

The maximum acceptable moisture content for material used for combustion is typically around 30% moisture versus overall biomass. Following winter frosts, considerable differences were seen between cultivars in terms of the drying curves (Figure 4). This is an important component of perennial grass production, as it permits spring harvesting. For large-scale production, material needs to be baled or compacted (to produce pellets, cubes, for example). This process cannot occur if the moisture levels are too high, compaction is not efficient, but more importantly, crop material will not store for long periods, and will rot. This is especially the case when harvesting follows a mild winter and a period of high rainfall preceding the harvest, such as for example, where the high rainfall comprises a monthly average of at least 65mm in the 3 months prior to harvest, or an aggregate of at least 195mm across the 3 months prior to harvest. In a commercial situation, the very green types would have been relatively unsuitable for end uses such as combustion with the more mature M x giganteus and Ml 16 being well suited for such uses.

In general, the new genotypes, and particularly Ml 16, had lower stem diameter which would facilitate increased crop drying and pelleting therefore can be considered a beneficial trait. However, Ml 16 was also noted to exhibit brackling, but it apparently recovered from this effect.

The yield of a Miscanthus crop has to be considered in the context of the suitable characteristics for a particular targeted end use. Most markets require that, for scale, the crop has a suitable moisture content at the end of the season to allow for machine harvest, subsequent baling, storage, and in some cases compaction into cubes or pellets.

Analyses of the crop harvested in March of the final year (Table 2) revealed satis factory levels of chlorine and sulfur for co-firing with coal, although Ml 17 was near the acceptable limit. Ml 16 was the best genotype in this regard and showed an improved trait of low chlorine content of stems versus the control M. x giganteus. Ash levels were 40% higher than M x giganteus from 3 of the 4 genotypes, potentially due their ability to retain leaves longer than M x giganteus (note that leaf fall was almost complete with this control cultivar at harvest). (However, it should be noted that higher levels of ash are more of concern for smaller scale combined heat and power (CHP) projects or heating project uses, and less of an issue for fermentation, for example). Notably, ash levels were lower from Ml 16 versus GIG, which can thus be considered an improved trait relative to the control.

Data from a nearby UK meteorological office weather station at Yeovilton for the period of trial are shown in Table 4.

Table 4. Data from a UK meteorological office weather station at Yeovilton for the period of trial

CONCLUSIONS

• After 3 years all genotypes reached peak yields of between 20 to 24.5 tonnes per hectare dry matter. • The new genotypes (Ml 16-9) showed increased yield over M x giganteus in year 2 of growth.

• The new genotypes showed higher stem populations as a consistent attribute and exhibited increased retained leaf matter.

• Interesting differences were noted between the genotypes in stem density phenotype. Ml 19 showed an apparent peak stem levels being reached after year 2, followed by a decrease or even a crash in stem density in the subsequent year.

• Yield of usable biomass varies significantly between genotypes. Significant differences in usable biomass have been found based on differences in levels of ash following combustion and chlorine content. Energy content also appeared elevated in stems of Ml 16 versus M x giganteus and the other genotypes. • Harvest quality also varies due to lack of winter die back, and leaf retention, which negatively influence moisture levels. In these regards, Ml 16 has improved quality versus the other genotypes; in particular its lower stem diameter favors dry down.

• Levels of nitrogen off-take are different compared to the M x giganteus. This has potentially important implications both from a cost and from an environmental perspective, as biomass crops need to have low input requirements. In this regard. Ml 16 has improved quality versus the other genotypes.

• Increased levels of carbon content of the tissue are a potentially beneficial trait since this indicates an improvement of the ability of a genotype to act as a carbon sink. In this regard, Ml 16 has improved quality versus the other genotypes.

• The genotype Ml 16, which derived from the same original Fl plant as Miscanthus ‘MBS 7001' exhibited lower stem N levels than M. x giganteus and the other genotypes. This was not expected. Lowered N levels are often associated with a plant being visibly less green during growth but ‘MBS 700 L was reported in US patent PP22033 to be slightly greener during in the growing season than M. x giganteus.

Example 4,

Tissue culture derived plantlets of the Miscanthus variety ‘MBS 7001', also known as Nagara', which originally resulted from a cross of Miscanthus sacchariflorus x Miscanthus sinensis, were established in a field in Somerset, England and grown for three years and examined as part of a field trial during which the plants were maintained and kept weed free. Following the end of the trial, the plants were left to continue growing, during which time they formed dense clumps. It was noted that certain individual plants at the center of clumps were taller, darker, and visibly healthier and more vigorous than the surrounding plants. These more vigorous plants were selected and rhizomes were collected, and vegetatively propagated, to establish a new population of "Nagara Select" for use as source progenitor material for growing plantations of Miscanthus for the harvesting of usable biomass for applications such as biofuel pellets for combustion, packaging materials, containers, and/or construction material. Progenitor plants that were propagated from the selected rhizomes were tested by growing them alongside control plants including the commercial variety "Illinois" and several other varieties in a field plot that was subject to freezing temperatures during the winter months. Plants were phcnotypically observed in the third week of June of the second growing year and it was noted that Nagara Select plants were visibly taller (i.e., more than 30 cm), larger, darker green, and had accumulated more biomass than the comparison varieties. Additionally, the rhizomes of Nagara Select plants consistently exhibited a new phenotype that had not been previously reported for Nagara, comprising a more acute angle of rhizome bud outgrowth relative to that observed in the rhizomes of controls (e g., M. giganteus “Illinois control plants). (Figure 6).

Claims

CLAIMS What is claimed is:

1. A method of producing one or more plant propagules used for the establishment of a crop that is grown for the production of usable biomass, the method comprising:

(a) in a plot, planting one or more Miscanthus plants regenerated from tissue cultured cells resulting from a plant is the descendant of a Miscanthus sacchariflorus x Miscanthus sinensis cross and allowing said regenerated plants to grow for a period of time that includes at least one winter season;

(b) selecting from amongst the one or more regenerated Miscanthus plants one or more selected Miscanthus plants that exhibit a reduced stem thickness, greater size, greater vigor, or darker coloration than one or more other plants in the plot to produce a population of one or more selected plants;

(c) collecting rhizome material from the one or more selected Miscanthus plants;

(d) propagating one or more new Miscanthus plants from the collected rhizome material,;

(e) planting the new Miscanthus in a second plot with one or more control plants;

(f) confirming that the test plants have either larger size, darker coloration, or an altered angle of rhizome bud outgrowth a period of time that includes at least one winter season, and

(g) multiplying cells or tissue from the rhizomes collected in step (c) or step (e) to produce the propagules used for planting of the crop.

2. Use of a non-naturally-occurring first Miscanthus plant to produce a regenerated Miscanthus plant that has an improved trait relative to a control plant grown under identical conditions and harvested at the same time, wherein: the first Miscanthus plant is the result of a Miscanthus sacchariflorus x Miscanthus sinensis cross; a plurality of plants propagated from the first Miscanthus plants is grown through one or more generations; a second Miscanthus plant is selected from tire plurality of plants from tire first Miscanthus plants for the presence of an improved trait relative to a control plant; and the regenerated Miscanthus plant is obtained from the second Miscanthus plant by growing the regenerated plant from a cell, rhizome, or cutting of the second Miscanthus plant; and wherein the improved trait results in increased yield of usable biomass from the regenerated Miscanthus plant relative to a control Miscanthus plant grown under identical conditions and harvested at the same time, and wherein: the improved trait is:

(a) reduced stem thickness; (b) darker green leaf coloration;

(c) altered angle of outgrowth of rhizome buds that protrude from a main rhizome stem, wherein the angle relative to the main stem is less than 30°, less than 45°, less than 65°, or less than 85°;

(d) increased overall biomass of at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, wherein the overall increased biomass results in greater aggregate biomass yield;

(e) at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% lower ash levels following combustion of harvested biomass; or

(f) at least 0. 1% lower chlorine content, at least 0.5% lower chlorine content, at least 1% lower chlorine content, at least 5% lower chlorine content, at least 10% lower chlorine content, at least 15% lower chlorine content, at least 17% lower chlorine content, at least 25% lower chlorine content, or at least 50% lower chlorine content in stems;

(g) at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% less moisture content at harvest after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season;

(h) a height of at least 30 cm greater than the height of a M sinensis, the M. Sacchariflorus, or a M. Giganteus "Illinois" control plant; or

(i) more vigorous growth or cold hardiness with good dry down characteristics in a year with a mild winter and a period of high rainfall preceding the harvest; or

(j) lower stem nitrogen level relative to a control Miscanthus plant at harvest.

3. The method of Claim 2 wherein the plurality of plants is produced through cell culture.

4. The use of the non-naturally-occurring first Miscanthus plant of Claim 2, wherein the regenerated Miscanthus plant is produced from a cell, rhizome, or cutting of the second Miscanthus plant and the cell, rhizome, or cutting is propagated and grown into a mature Miscanthus plant.

5. The use of the non-naturally-occurring first Miscanthus plant of Claim 2, wherein the improved trait used to select the second Miscanthus plant is altered angle of outgrowth of rhizome buds, a rhizome angle of less than 65°, thinner stems, increased tiller density, increased height of 30 cm or more versus a control plant, and/or full senescence at harvest relative to the control plant.

6. The use of the non-naturally-occurring first Miscanthus plant of Claim 2, wherein the improved trait is decreased moisture content at harvest when the regenerated plant is grown under conditions of a mild winter defined by conditions of: (i) an average daily low temperature in the months of November, December and January inclusive of equal to or greater than -0.3° Celsius;

(iii) a rainfall in January of greater than 58 mm or at least 98.2 mm; relative to a control Miscanthus plant grown under identical conditions; and/or

(iv) an average monthly rainfall in the 3 months prior to harvest of at least 65 mm.

7. The use of the non-naturally-occurring first Miscanthus plant of Claim 2, wherein the regenerated plant has a smaller stem diameter, less moisture content, and a greater aggregate yield over at least two seasons relative to the control Miscanthus plant.

8. Usable plant biomass obtained from a regenerated Miscanthus plant that has an improved trait relative to a control plant grown under identical conditions and harvested at the same time, wherein: the regenerated Miscanthus plant is grown from a cell, rhizome, or cutting of a second Miscanthus plant that has been selected for the presence of the improved trait from a plurality of first Miscanthus plants that are obtained from a Miscanthus sacchariflorus x Miscanthus sinensis cross and grown through one or more generations, and wherein: the improved trait is:

(a) reduced stem diameter;

(b) darker green leaf coloration;

(c) altered angle of outgrowth of rhizome buds that protrude from the below ground rhizome wherein the angle is less than 45° or less than 65° or less than 85°;

(d) increased overall biomass of at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, wherein the increased overall biomass results in greater aggregate yield;

(g) at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 50% less moisture content at harvest after tiller initiation has ceased and leaves are no longer substantially green relative to a control Miscanthus plant grown under identical conditions and harvested at the same time after the plants have been subject to a winter season; (h) height of at least 30 cm greater than the height of a M. sinensis, the M. Sacchariflorus, or a M. Giganteus "Illinois" control plant; or

(i) more vigorous growth or cold hardiness with good dry down characteristics in a year with a mild winter and a period of high rainfall preceding the harvest; or j) lower stem nitrogen level relative to a control Miscanthus plant at harvest.

9. Use of a source Miscanthus plant to produce usable biomass where the source Miscanthus exhibits at least one improved trait as compared to a control plant, wherein the improved trait is chosen from: increased cold tolerance, increased height, increased tillering, altered angle of outgrowth of rhizome buds, altered angle of outgrowth of rhizome buds that protrude from the below ground rhizome wherein the angle is less than 65°, reduced stem thickness, darker coloration, lower ash content following combustion, reduced stem chlorine content, lower stem nitrogen content, and reduced moisture content at harvesting following a winter season; and wherein the source Miscanthus plant has been selected after one or more generations from a plurality of plants produced from a Miscanthus sacchariflorus x Miscanthus sinensis cross.

10. Use of the source Miscanthus plant of claim 9 wherein the plant is sterile and is vegetatively propagated through one or more generations from a plant selected from amongst a plurality of field grown plants of the variety “Nagara” (MBS-7001).

11. Use of the source Miscanthus plant of claim 9 wherein the biomass is used for fuel, pellet production, blending with other biomass, container production, paper production, combustion, electricity generation, construction material, packaging, animal feed, animal bedding, fermentation, or as a chemical feedstock.

12. Use of the source Miscanthus plant of claim 9 wherein the source Miscanthus plant is a non- naturally occurring plant.

13. Use of the source Miscanthus plant of claim 9 wherein the source Miscanthus plant is propagated through cell culture or rhizome cuttings.