AU2018201277A1 - Plants with increased nutritional value and methods for producing same - Google Patents

Abstract

The present invention relates to methods for growing leafy green vegetables in a hydroponic system so as to provide improvements in the nutrient content of the vegetables.

Description

Plants with increased nutritional value and methods for producing same

Field of the invention

The present invention relates to methods for growing leafy green vegetables for increasing their nutritional content. The invention also relates to leafy green vegetables which have increased nutritional value.

Related application

This application claims priority from Australian provisional application AU 2017900578, the contents of which are hereby incorporated by reference in their entirety.

Background of the invention

Fortification is the practice of deliberately increasing the content of an essential micronutrient, i.e. vitamins and minerals (including trace elements) in a food, so as to improve the nutritional quality of the food supply and provide a public health benefit with minimal risk to health.

Biofortification is the process by which the nutritional quality of food crops is improved through agronomic practices, conventional plant breeding, or modern biotechnology. Biofortification differs from conventional fortification in that biofortification aims to increase nutrient levels in crops during plant growth rather than through manual means during processing of the crops (such as adding nutrients to the food during processing).

Biofortification may present a way to reach populations where supplementation and conventional fortification activities may be difficult to implement and/or limited. For example, biofortification has been identified as an upcoming strategy for dealing with deficiencies of micronutrients in the developing world.

Biofortification is also likely to have utility in the developed world, both for dealing with micronutrient deficiencies in communities with the increased consumption of processed and calorie dense/nutrient poor foods, but also in providing nutrient-dense foods to health conscious consumers.

There is a need to continue to develop biofortification strategies for improving the nutritional value of foods. Some biofortification methods attempted to date include the use of genetic modification to improve plant nutrient levels. However, such approaches can often met with consumer skeptism, presenting difficulties for the wide-spread implementation of such strategies.

The present application relates to new methods of biofortifying leafy green vegetables. The present application also relates to biofortified leafy green vegetables and products derived therefrom.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

The present invention provides a method for improving the nutritional value of a leafy green vegetable, the method comprising, contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+, thereby improving the nutritional value of the leafy green vegetable.

The present invention provides a method for improving the total iron, chlorophyll, antioxidant, vitamin and/or lutein content of a leafy green vegetable, comprising contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+, thereby improving the total iron, chlorophyll, antioxidant, vitamin and/or lutein content of the leafy green vegetable.

In a further aspect, the present invention provides a method for improving the total iron content of a leafy green vegetable, comprising contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+, thereby improving the total iron content of the leafy green vegetable.

In a further aspect, the present invention provides a method for growing a leafy green vegetable, comprising contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+, wherein the method improves the total nutritional value of the leafy green vegetable.

The present invention provides for the use of a nutrient solution for contacting a seedling of a leafy green vegetable to improve the nutrient value of the leafy green vegetable, wherein the nutrient solution contains greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+. For example, the nutrient solution may contain between about 4.2 and 8.0 ppm, or 4.5 and 8.0 ppm, or 5.0 and 8.0 ppm, or 5.5 and 8.0 ppm, or 6.0 and 8.0 ppm, or 6.5 and 8.0 ppm or 7.0 and 8.0 ppm or 7.5 and 8.0 ppm. In any aspect, method or use of the invention described herein, the nutrient solution contains greater than about 4.1 ppm and no more than about 6.0 ppm of Fe2+/Fe3+. In any further aspect, method or use, the nutrient solution contains between about 4.2 ppm and about 5.0 ppm of Fe2+/Fe3+. In still further aspects, method or uses, the nutrient solution contains between about 4.5 and about 4.9 ppm of Fe2+/Fe3+.

The present invention also provides a leafy green vegetable with increased nutrient value, wherein the leafy green vegetable is obtained by contacting a seedling of the leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+.

The present invention also provides a leafy green vegetable with increased iron content, wherein the leafy green vegetable is obtained by contacting a seedling of the leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+.

The present invention also provides a leafy green vegetable with an iron content of between about 250 ppm to about 1000 ppm by dry mass weight, preferably 300 ppm to 500 ppm by dry mass weight. The leafy green vegetable may be any leafy green vegetable that can be grown in a hydroponic system. Preferably, the leafy green vegetable is from a botanical family selected from the group consisting of: Amaranthaceae, Apiaceae/Umbelliferae, Araceae, Asteraceae/Compositae, Basellaceae, Brassicaceae/Cruciferae, Convolvulaceae, Curcubitaceae, Dryopteridaceae, Malvaceae, Moringaceae, Polygonaceae and Solanaceae. More preferably, the leafy green vegetable is a spinach, lettuce, rocket, silverbeet/swiss chard, kale, collard green, mustard green, cabbage or broccoli.

As used herein, the ‘iron content’ refers to the amount of iron found in the edible portion of the leafy green vegetable, for example, the leaves and stem. In any embodiment of the present invention, the iron content of the vegetable refers to the content found in the leaves, leaves and stem, or stem of the vegetable.

The present invention also provides a leafy green vegetable with an iron content of between about 250 ppm to about 1000 ppm by dry mass weight, wherein the leafy green vegetable is obtained by contacting a seedling of the leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+, in conditions suitable for development of the seedling into a mature plant.

The present invention also provides a spinach plant containing an amount of iron of between about 250 ppm to about 1000 ppm by dry mass weight, wherein said spinach has been produced using a hydroponic cultivation method. In one embodiment, the invention provides a spinach plant containing an amount of iron of between 300 ppm and 500 ppm, by dry weight mass.

The present invention also provides a spinach plant with an iron content of between about 250 ppm to about 1000 ppm by dry mass weight, wherein the spinach plant is obtained by contacting a seedling of the leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.00 ppm of Fe2+/Fe3+, in conditions suitable for development of the seedling into a mature plant.

The present invention also provides a spinach plant containing an amount of iron of between about 250 ppm to about 1000 ppm by dry mass weight, wherein said spinach has been produced using a hydroponic cultivation method.

The present invention provides a method of increasing the yield from a leafy green vegetable, the method comprising contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+, thereby increasing the yield from the leafy green vegetable.

The present invention also provides a freeze dried whole-plant supplement produced by freeze drying a leafy green vegetable grown according to any method described herein.

The present invention provides a nutritional supplement derived from a leafy green vegetable grown according to any method described herein.

The present invention also provides a nutritional supplement derived from a spinach plant having an iron content of between about 250 ppm to about 1000 ppm by dry mass weight, wherein said spinach has been produced using a hydroponic cultivation method.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1: Photo image of spinach seedlings 7 days after application of a nutrient solution containing 10.10 ppm Fe.

Figure 2: Photo image of a spinach plant with leaf damage following growth of the plant in a nutrient solution containing 6.10 ppm Fe.

Figure 3: Total iron (Fe2+/Fe3+) concentration in leaves of spinach grown in a nutrient flow hydroponic system using a nutrient solution containing 4.10 ppm (control), 4.12, 4.14, 4.18 or 4.26 ppm Fe for up to 18 days. Application time refers to the number of days plants were grown in nutrient solution with elevated Fe. Bars represent standard error (SE) = 8.6.

Figure 4: Total iron (Fe2+/Fe3+) concentration in leaves of spinach grown in a nutrient flow hydroponic system using a nutrient solution containing 4.10 ppm (control), 4.18, 4.26, 4.42, or 4.74 ppm Fe, for up to 35 days. Application time refers to the number of days plants were grown in nutrient solution with elevated Fe. Bars represent standard error (SE) = 16.0.

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

All of the patents and publications referred to herein are incorporated by reference in their entirety.

For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

The present invention relates to an improved method for increasing the nutrient value of a leafy green vegetable grown using a hydroponic method. The inventors have found that providing leafy greens with a nutrient solution containing a concentration of iron (Fe) falling within a particular range, is beneficial for increasing not only the total iron content of the plants, but also , surprisingly, for increasing the proportion of other valuable nutrients in the plants. Furthermore, by growing leafy green vegetables in accordance with the methods of the present invention, there is a significant increase in the overall yield from each plant.

While it is desirable to grow leafy green vegetables that have a high nutrient content and yield, it is not simply a matter of increasing the amount of the relevant micronutrients to the plant during growth. For example, while iron is an essential nutrient for plants, it is toxic when provided at high levels, acting catalytically via the Fenton reaction to generate hydroxyl radicals, which can damage lipids, proteins and DNA. Symptoms of iron toxicity in plants can include bronzing of the leaves and possibly also the formation of brown spots on leaves. Thus, the present inventors have identified an optimal subrange of Fe concentrations in nutrient solutions, to maximise the nutrient content and yield of leafy green vegetables, when grown in a hydroponic system.

The total iron content of the plants grown in accordance with the methods of the present invention are significantly higher than the levels reported for plants grown under standard hydroponic growing conditions.

Hydroponic methods for growing leafy green vegetables

The present invention provides for a method of growing leafy green vegetables using a hydroponic growing system. The skilled person will be familiar with the use of such systems for growing leafy green vegetables.

An example of a hydroponic growing system which can be used in accordance with the present invention is the nutrient film (or nutrient flow) technique. Nutrient film technique (NFT) is a hydroponic growing technique wherein a very shallow stream of water-based nutrient solution is re-circulated past the bare roots of plants in channels (also called gullies).

Examples of NFT systems for use in accordance with the present invention are well-described in the art and similar principles apply to all conventional NFT systems. In NFT, plants are supported in a gently sloping shallow gully and the roots are suspended in a shallow, flowing stream of nutrient solution. After passing down the gully, the nutrient solution is collected in a closed tank and pumped back to the top of the gully to continuously recycle the nutrient solution. Commonly used NFT systems consist of PVC channels 10-15 cm wide, with rectangular bases, and fitted with plastic covers containing plant holes. About 4-8 channels are arranged on a bench of between 4-24 metres long (less than 20 metres is recommended) and these are erected on a marginal slope (about 1.5-2.0°), to allow nutrient flow back into the recirculation tank. The movement of nutrient solution down the gullies also ensures that the nutrient solution is sufficiently aerated. The channel must not sag or pool at any point along its length to ensure that good aeration is achieved. The flow rate (1-2 L/minute) depends on the flow-rate of the pump, the size of the crop and the number of benches being used at one time. The tanks used typically range from 4000 to 10000 L, each supplying between 28-35 benches, sometimes more.

Channels with slopes ranging from 1:30 to 1:40 are typically used (although it may also be possible to increase the slope to 1:100). The slope of the growing channel may be provided by the floor, benches or racks holding the channels.

Specific examples of NFT systems which can be used in accordance with the present invention include the system described in, JP-A 8-205700. Preferably, the hydroponic system used in accordance with the present method is the system described in WO 2014/103414, the entire contents of which are herein incorporated in their entirety.

While the preferred methods described herein relate to the use of NFT systems for hydroponically growing leafy vegetables, the skilled person will appreciate that it is possible to make use of other hydroponic growing techniques such as, for example, flood and drain (ebb and flow) techniques or aeroponics.

The skilled person will also be familiar with determining and providing additional conditions for growing plants in a hydroponic system, including control of temperature and light, where appropriate. For example, the temperatures used can vary from about 18 °C to about 30 °C in daytime, and from about 10 °C to about 25 °C in night time. Preferably, the daytime growing temperatures will be between about 22 °C to about 28 °C, and night time growing temperatures will be from between about 12 °C to about 18 °C.

Vegetables grown in accordance with the methods of the present invention can be grown from seedlings that have been purchased or grown by another user. Alternatively the seedling is first grown by the user from seed, using conventional methods for germinating seeds to produce a seedling for transplantation into a hydroponic growing system as described above.

The skilled person will also be familiar with various methods for germinating seeds and cultivating seedlings (i.e., transplant production), before transplanting the seedlings into the hydroponic growing method of choice. For example, seeds can be germinated by placing seeding in rockwool or peat pellet cubes that have been soaked in distilled water. The cubes containing the seeds can then be kept in a warm environment until the seeds germinate into seedlings and are ready for transplantation into the hydroponic growing system of choice. Examples of seed germination methods used in a commercial setting include the use of a closed environment including a light source, irrigation unit and means for controlling the air humidity (see for example US 7,975,429 and US 8,327,580).

In a preferred embodiment, the system for culturing of seedlings is that described in EP 1 543 718, the entire contents of which are herein incorporated by reference in their entirety.

In yet a further preferred embodiment, the seedlings are cultivated using the system described in EP 1 543 718, and then transplanted into an NFT system such as described in WO 2014/103414 for plant growth.

Nutrient Solution

The skilled person will be familiar with various methods for providing a nutrient solution for use in a hydroponic growing system as described above. In accordance with the present invention, any standard or conventional hydroponic nutrient solution can be used, provided that it is then adapted to increase the total iron amount provided to the plants.

For example, common nutrients found in hydroponic nutrient solutions (in addition to water) include: a source of nitrogen (typically provided as ammonium and/or nitrates), a source of phosphorous (typically as dihydrogen phosphate, phosphate and monohydrogen phosphate), a source of calcium, a source of magnesium, a source of sulfur, a source of copper, a source of manganese, a source of zinc, a source of molybdenum, a source of boron, a source of borate, a source of chloride, a source of sodium and a source of iron.

Any of the compounds disclosed in Table 1 may be suitable sources for the above nutrients.

It is often practical for growers to prepare concentrated stock solutions which can then be stored before being diluted and delivered to the crop. In this case two different stock solutions (labelled A and B), are required to avoid precipitation of calcium phosphate, calcium sulphate and iron phosphate in these highly concentrated solutions. The stock solutions are 100 to 200 times stronger than the solution given to plants. Stock solutions also need to be kept out of the cold, ideally between 27-30 °C to prevent precipitates forming. Most nutrient solution formulations in use commercially are generally similar in composition. An example of a typical hydroponic nutrient solution used for growing leafy green vegetables is shown in Table 1 below:

Table 1: Example of nutrient solution for use in a hydroponic system

Equal volumes of solutions A and B would be combined (for example, to WOOL water, add 3.4 L of A and 3.4 L of B).

The final concentration of iron in conventional hydroponic nutrient solutions, such as the one described above is approximately 2.5 ppm, although concentrations ranging up to 4.0 ppm have also been described.

The electrical conductivity of a nutrient solution (EC) is a measure of the total salts dissolved in the hydroponic nutrient solution. While the EC value does not provide an indication of the nutrient content of the nutrient solution, it is a useful means for enabling the skilled person to measure the stability of the nutrient solution during application.

The skilled person will be familiar with methods for determining the electrical conductivity (EC) of the nutrient solution, including through the use of standard EC meters. Preferably the nutrient solution has a conductivity factor (electrical conductivity) of between 2 and 3.5, more preferably, between 2.5 and 3.0.

The skilled person will also be familiar with means for measuring the pH of the nutrient solution and ensuring that the pH is kept in optimal range. This is especially important as the pH of the nutrient solution can impact on the solubility of the iron present in the solution, and the ability of the iron and other nutrients to be drawn up by the plant.

In accordance with the methods of the present invention, the preferred pH range of the hydroponic nutrient solution is between about 5.8 and about 6.3.

The present inventors have found that by increasing the iron content of a nutrient solution for use in a hydroponic growing method, it is possible to improve the yield and nutrient value of leafy green vegetables. The iron may be provided in the nutrient solution in any salt or chelated form available to the skilled person, for example as Fe-diethylenetriaminepentaacetate (Fe-DTPA), Fe-Ethylenediaminetetraacetate (Fe-EDTA, for example, Fe-EDTA monosodium trihydrate), Fe-citrate, Fe-tartrate, FeCb, or FeSCU. Preferably, the iron is provided as Fe-DTPA, more preferably as Fe-DTPA disodium hydrate.

In any embodiment of the present invention, the nutrient solution that is provided to the leafy green vegetables during growth is a solution having between 4.10 ppm (parts per million) to about 8.00 ppm of total iron (i.e., Fe2+/Fe3+). For example, the nutrient solution may have 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90 or 8.00 ppm of Fe2+/Fe3+. In any embodiment of the present invention, the nutrient solution may have a total iron content which is a value between any 2 of the above listed values.

In a preferred embodiment, the nutrient solution contains greater than about 4.10 ppm and no more than about 6.00 ppm of Fe2+/Fe3+. In a particularly preferred embodiment, the nutrient solution contains between about 4.20 ppm and about 5.00 ppm of Fe2+/Fe3+, i.e., the solution can have about 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80 or 4.90 ppm Fe2+/Fe3+.

As used herein, “Fe2+/Fe3+” refers to the total concentration of iron ions. The ions may be either Fe2+ ions, Fe3+ ions, or a combination of both. The ions may be free or present in a compound, such as in complex with a chelate as described above, or bound to another ion, such as a chloride ion, sulfate ion, as described above.

As used herein, “ppm” denotes “parts per million” and is used to describe the concentration of iron or other components in a complex mixture (such as for example, a nutrient solution, or in a plant extract). The skilled person will appreciate that the units ppm can be substituted for mg/kg.

Timing of exposure to high-iron nutrient solution

The skilled person will appreciate that once the seed of a leafy green vegetable has germinated into a seedling, using the standard methods for germination applicable to hydroponic systems, it is necessary to transplant the seedling into the hydroponic growing system (such as an NFT system). Typically, the seedling will be about 5 to 10 cm in height at the time of transplantation, although transplanting of the seedling can occur at any time from 0 to 30 days from sprouting of the seedling (i.e., when the first shoots emerge from the soil surface). In certain embodiments, the seedling can be 2 to 20 cm in height at the time of transplanting, preferably 3 to 15 cm, more preferably 5 to 10 cm).

The skilled person will appreciate that once the seedling is transplanted into the hydroponic growing system, it is possible to provide the seedling with the hydroponic nutrient solution described herein, i.e., a hydroponic nutrient solution containing at least 4.20 ppm and no more than 8.00 ppm Fe2+/Fe3+. However, in alternative embodiments, it may be preferable to allow the seedlings to grow in ‘standard hydroponic nutrient solution’, i.e., a nutrient solution containing an iron level within more common usage ranges (such as 2.5 to about 3.5 ppm up to 4.10 ppm but less than 4.20 ppm Fe2+/Fe3+), for a period of time, prior to exposing the seedling/plant to a nutrient solution containing a higher iron concentration.

In certain embodiments, the seedling is contacted with ‘standard hydroponic nutrient solution’ (i.e., a solution containing less than 4.20 ppm Fe2+/Fe3+) for 1 to 30 days prior to contact with the hydroponic nutrient solution containing at least 4.20 ppm Fe2+/Fe3+. In a preferred embodiment, contact with the standard nutrient solution is for between 5 and 20 days, 10 and 15, preferably about 12 to 14 days once the seedling has been transplanted from the germination environment into the hydroponic growing system (such as NFT system).

Once the seedling is contacted with the nutrient solution containing at least 4.20 ppm and no more than 8.00 ppm Fe2+/Fe3+, contact may be maintained for a period of 1 -20 days, preferably 5-10 days, more preferably 6 to 8 days, prior to harvest.

In a particularly preferred embodiment, the present invention includes germinating seeds to seedling stage for 0-10 days (i.e., until seedlings are approximately 5-10 cm in height), growing the plants with standard levels of nutrients for approximately 14 days (e.g., 4.10 ppm Fe2+/Fe3+), and then providing the plants (which are now about 24 days old) with nutrient solution containing at least 4.20 ppm Fe2+/Fe3+, as described herein. In a preferred embodiment, the total period of exposure to the high-iron nutrient solution is about 5 to 15 days, preferably 5 to 10 days, more preferably 6 to 8 days.

In an even more preferred embodiment, the present invention includes germinating seeds to seedling stage until the seedlings are approximately 5-10 cm in height, contacting the plants in a hydroponic cultivation system with a hydroponic nutrient solution containing no more than 4.10 ppm Fe2+/Fe3+ for a period of about 12 to 14 days, then contacting the plants in a hydroponic cultivation system with a hydroponic nutrient solution containing between 4.20 ppm to about 5.20 ppm Fe2+/Fe3+ for a period of 6 to 8 days.

As used herein ‘contact’ refers to the application of the hydroponic nutrient solution to the plant, for the purposes of enabling the plant to utilise the nutrient solution for growth. Typically, contact involves providing the nutrient solution to the roots of the plant/seedling, for example, using a nutrient flow technique whereby the nutrient solution flows down a gully, coming into contact with the roots of the plants as it flows past. In certain embodiments, the nutrient solution may come into contact with the plant through other means, depending on the nature of the hydroponic growing system.

Plants grown in accordance with the methods of present invention

The present invention also provides for plants having an increasing nutritional value.

As used herein ‘increased nutritional value’ means that the plants grown in accordance with the present invention have an increased level of desirable micronutrients as compared with plants grown using alternative methods. For example, the nutrient content, as measured as a proportion of dry or fresh weight, may be greater than that found in plants grown in other systems (for example, standard hydroponic growing conditions).

The nutrients which may be increased relative to plants grown by other means may include iron, calcium, magnesium, sodium, potassium, sulphur, phosphorous, copper, zinc, manganese, aluminium and boron, antioxidants (such as vitamin C, other vitamins) lutein and chlorophyll.

The skilled person will be familiar with methods for determining levels of various nutrients in the plants of the present invention. For example, nutrient metals and minerals such as iron calcium, magnesium, sodium, potassium, sulphur, phosphorous, copper, zinc, manganese, aluminium and boron can be measured using standard inductively coupled plasma emission spectroscopy techniques (ICP-ES). The skilled person will also be familiar with standard methods for determining vitamin C levels (and other vitamin levels) in the plants of the present invention, for example using the indophenol titration method for vitamin C determination as described in Wokes et al., (1943) Biochem J, 37: 695-702. Levels of chlorophyll and carotenoids (such as lutein) can also be determined by photometric methods, for example as described in Limantaraa et al., (2015) Procedia Chemistry 14: 225 - 231 and by chromatographic methods, as described in Kidmose et al., (2005) J Chromatogr Sci, 43: 466-72).

It will be appreciated that not only do the plants grown in accordance with the present methods have desirable characteristics in terms of nutritional content, but they may also have improved characteristics from a consumer preference consideration. For example, the inventors have found that leafy green vegetables grown in accordance with the present invention may have a deeper green colour and appear more vigorous than plants grown in other hydroponic systems.

The inventors have also found that the yield obtained from leafy green vegetables grown in accordance with the present invention is greater than that obtained when the same species of plant is grown using nutrient solutions with lower or high levels of iron than those described herein. Specifically, nutrient solutions having less than 4.10 ppm or greater than 8.0 ppm of total iron content may provide for a reduced yield as compared with leafy green vegetables grown in accordance with the present invention. As used herein, the yield of a plant refers to the average (mean) fresh weight of a plant and/or size of the plant.

In one embodiment, the plants grown in accordance with the present invention have a yield (i.e., size and/or weight) per plant that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than a plant grown under identical conditions, but provided with a reduce amount of iron in the hydroponic nutrient solution.

Any number of leafy green vegetable can be grown for use in accordance with the present invention, provided that the vegetable is amenable to growth in a hydroponic system.

Examples of the types of vegetable to which the present invention applies include plants belonging to the botanical families: Amaranthaceae, Apiaceae/Umbelliferae, Araceae, Asteraceae/Compositae, Aizoaceae, Basellaceae, Brassicaceae/Cruciferae, Convolvulaceae, Curcubitaceae, Dryopteridaceae, Malvaceae, Moringaceae, Polygonaceae and Solanaceae.

Examples of plants in the family Amaranthaceae include spinach (Spinacia oleracea), Red spinach (Amaranthus dubius also called Chinese spinach, Hon-toi-moi, Yin choy, Hsien tsai), French spinach (Atriplex hortensis, also known as garden orache, red orach, mountain spinach, orache or arrach), silverbeet (Beta vulgaris, also called swiss-chard, chard or perpetual spinach), and quinoa.

Examples of plants in the family Apiaceae/Umbelliferae carrot, celery, parsley, chervil, cicely, coriander (cilantro), culantro, cumin, dill, fennel, parsnip.

Examples of plants in the family Araceae, include taro, dasheen and corm.

Examples of plants in the family Asteraceae/Compositae include lettuce and chicory.

Examples of plants in the family Aizoaceae are Tetragonia tetragonioides, also known as Botany Bay spinach, Cook's cabbage, New Zealand spinach, sea spinach, and tetragon.

Examples of plants in the family Brassicaceae/Cruciferae include collard greens, kale, mustard greens, cabbage, rocket (arugula), pak choy (bok choy), gai Ian, and broccoli.

Examples of plants in the family Convolvulaceae include water spinach (Ipomoea aquatic also called Chinese spinach, Chinese watercress, river spinach).

Plants in the family Curcubitaceae include gourds, pumpkin, zucchini, pumpkin and cucumber. Plants in the family Malvaceae include okra.

Plants in the family Moringaceae include Moringa, malunggay.

Plants in the family Polygonaceae include sorrel and rhubarb and plants in the family Solanaceae include tomatoes, eggplant, capsicum and chilli.

In a preferred embodiment, the leafy green vegetables that are grown in accordance with the methods of the present invention are spinach plants, particularly “Spinacia oleracea”. Any variety of spinach can be used. In one embodiment, the ‘Mirage” variety of spinach is used.

In a preferred embodiment, the present invention relates to a spinach plant containing an amount of iron of between about 250 ppm to about 1000 ppm by dry mass weight, wherein said spinach has been produced using a hydroponic cultivation method.

In yet a further embodiment, the present invention relates to a spinach plant containing an amount of iron between 300 ppm and about 500 ppm, by dry weight, wherein the spinach has been produced using a hydroponic cultivation method.

When referring to the nutritional content of a leafy green vegetable, it will generally be understood that this refers to the level of a given nutrient in the edible portion of the vegetable. The ‘edible portion’ may refer to the whole plant of the vegetable. Alternatively, the edible portion may include the leaves of the vegetable, the stem of the vegetable, or a combination thereof. The edible portion may also refer to the flowers, fruit, leaves, stem of the plant alone or in combination.

Products A further advantage of the present invention is that the plants of the present invention can be processed to produce a whole plant nutritional supplement that will deliver the recommended daily intake (RDI) for iron in a reasonably small size dose (eg, 5 g). This is only possible because the total iron content in the leaves of the plants is increased significantly compared to the iron levels of plants grown according to conventional methods.

In one embodiment, the nutrient supplement can be in the form of a freeze dried product, that is produced by freeze frying a leafy green vegetable grown according to any of the methods described herein.

The nutritional supplements of the present invention can be used to supplement the diet of individuals, prevent disease, restore deficiency of a particular nutrient, particularly iron.

Thus, in a further embodiment, the present invention also provides a nutrient supplement derived from a leafy green vegetable, wherein the vegetable is grown by a method comprising: - contacting a seedling of the leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.10 ppm and no more than about 8.00 ppm of Fe2+/Fe3+.

In one embodiment, the nutrient supplement is for delivery of the recommended daily intake (RDI) of Fe2+/Fe3+ to an individual in need thereof.

The RDI for individuals ranges from 10-30 mg/day. The skilled person will appreciate that the specific RDI will vary depending on the age and sex of the individual (or the particular health status of the individual). For example, the RDI for male and female infants is the same (approximately 9 mg/day). The RDI for men aged 18 and over is approximately 8 mg/day, while for women aged 19-50 years, the RDI is 18 mg/day. The RDI for pregnant women may be higher (27 mg/day).

Examples

Example 1: Biofortification trials

Summary of trials:

The aim of the biofortification trials was to investigate increasing the Fe content in spinach leaves by manipulating Fe concentration in hydroponic nutrient solution. Spinach leaves under normal nutrition (Fe content of 4.10 ppm) contained 90-140 mg/kg DW Fe, which is considered double the levels observed in commercially-grown spinach.

Table 2: Current and target Fe levels

Trial 1 tested a wide range of Fe concentrations from 4.1 ppm to 10.1 ppm, and showed that 10 day old spinach seedlings were sensitive to Fe concentrations over 6 ppm, as plants grown with nutrient containing 8.1 or 10.1 ppm Fe died within 3 days of transplanting. A leaf Fe content of over 1000 mg/kg DW was recorded in plants grown in 6.1 ppm Fe but plants were stunted and damaged by this concentration.

In Trial 2 spinach NPL8 plants were grown to a height of 5 to 10 cm (approximately 13 days) before Fe was added at 4.12, 4.14, 4.18 and 4.26 ppm. Control leaf Fe levels were lower after 18 days compared with Trial 1, possibly because uptake is reduced in older seedlings. Highest leaf Fe concentration (176 mg/kg DW) was obtained with 4.26 ppm Fe after 6 days; 217% higher than that observed in the plants

grown with the standard nutrient solution. After 18 days all treatments produced similar leaf Fe levels.

An unexpected result of supplying spinach with elevated Fe was that plants grown under 4.18 and 4.26 ppm Fe showed a significant increase in size and weight. As highest leaf Fe in Trial 2 was obtained from a nutrient solution containing 4.26 ppm Fe, a third trial was set up testing 4.10 (control), 4.18, 4.26, 4.42 and 4.74 ppm Fe for up to 35 days.

In Trial 3 highest leaf Fe content (356 mg/kg) was found in plants grown under 4.74 ppm Fe after 7 days, 324% higher than control plants. Content in plants grown under 4.74 ppm Fe declined steadily with time, with a 34% decrease by 35 days, but were consistently significantly higher than other Fe treatments. These trials indicated that Fe content can be significantly increased in spinach leaves grown in the a hydroponic nutrient flow system with elevated nutrient solution Fe concentrations between 4.74 and approximately 6.00 ppm. Leaf Fe contents between 356 and 1022 mg/kg DW were obtained, indicating that this system can provide the high Fe spinach material needed for a nutrient supplement but further research is required to optimise the timing of the application and the concentration of Fe.

Methods:

Fe was added to daughter tanks in the form of the chelate DTPA at the start of each trial with regular samples taken to ensure electrical conductivity (EC) was not significantly different between treatments during plant growth.

Three trials using different Fe nutrient solution concentrations were conducted. Plants were harvested, freeze dried and assayed for Fe using ICP chromatography. Primary trials tested nutrient solutions containing various Fe concentrations with the control solution containing 4.10 ppm Fe (which is already higher than the Fe content of most commercially available standard nutrient solutions for use in hydroponic systems).

The first trial tested a wide range of Fe concentrations in nutrient solution including 4.10 ppm (control), 6.10, 8.10 and 10.10 ppm. Plants were exposed to increased Fe concentrations (i.e., greater than 4.10 ppm) from the time the seedlings were transplanted to the nutrient flow hydroponic system.

In the second trial, spinach plants were grown for 13 days in the NFT system following transplant and to a height of 5 - 10 cm. Once plants had reached this level of maturity, nutrient solution with elevated Fe concentration was provided to the plants. Fe concentrations added were lower than in Trial 1: 4.10, 4.12, 4.14, 4.18 and 4.26 ppm. Samples were taken 6, 12 and 18 days after application of the elevated Fe concentration.

Trial 3 was set up to test the effect of nutrient solutions containing 4.10 (control), 4.18, 4.26, 4.42 and 4.74 ppm Fe for up to 35 days. As in trial 2, spinach plants were grown to a height of 5 - 10 cm before nutrient solution with higher Fe concentration (i.e., greater than 4.10 ppm) was provided to the plants. This corresponded to 14 days after transplanting.

Experimental design for Trials 1 -3: • 4-5 x Fe Treatments • 1 x crops: Spinach • 3 x Replicates • Measurements: Time to harvest, yield, visual quality

• 6 samples for analysis at harvest: Fe and other minerals by ICP • Data analysis by ANOVA using Genstat software

Results

Trial 1 tested a range of added Fe concentrations, from 4.10 ppm (current nutrient solution) to 6.10, 8.10 and 10.10 ppm:

Table 3. Mean Fe content in spinach leaves after 28 days growth in nutrient solution with different Fe concentration. (Standard error shown in brackets)

The first trial showed that 10 day old spinach seedlings transplanted straight into the NFT system were sensitive to Fe concentrations in excess of 6 ppm, as plants exposed to nutrient solution containing 8.10 or 10.10 ppm Fe died within 3 days of transplanting (Figure 1). Plants grown in nutrient solution containing 8.10 and 10.10 ppm were therefore not tested for foliar Fe2+/Fe3+ content. High Fe (>1000 mg/kg DW) was recorded in plants grown with 6.10 ppm Fe, although plants were stunted and damaged by this concentration (Figure 2). This form of spinach would still be useful for processing into a nutritional supplement or a freeze-dried product.

In the second trial, lower Fe concentrations were tested, and seedlings were allowed to become more established following transplantation into the NFT system before Fe levels were increased in the nutrient solution.

Overall, the total Fe2+/Fe3+ content of spinach leaves from plants grown in Trial 2 were lower than that observed for Trial 1. Fe2+/Fe3+ content of spinach plants exposed to control nutrient solution (i.e., 4.10 ppm Fe) was also lower in Trial 2 as compared with Trial 1. This may be because uptake is reduced in older seedlings, as plants were in the NFT system for 13 days before the Fe content of the nutrient solution was increased.

Highest leaf Fe2+/Fe3+concentration (176 mg/kg DW) was obtained in plants exposed to nutrient solution containing 4.26 ppm Fe for 6 days. This Fe level was 217% higher compared than the level observed for plants grown in the control nutrient solution (Fe 4.10 ppm; Fig. 3). As the period of exposure to the high-Fe nutrient solution increased, leaf Fe declined in plants grown with 4.26 ppm, 4.14 ppm, and 4.10 ppm Fe nutrient solution and increased in plants grown with nutrient solution containing 4.12 and 4.18 ppm Fe. After 18 days of exposure to the test nutrient solutions, all leaf Fe levels were between 60 and 95 ppm.

An unexpected result of supplying spinach with elevated Fe levels was that plants grown in solution containing 4.18 and 4.26 ppm Fe showed a significant increase in size and weight compared to plants grown with the standard level of Fe (Table 3). Plants grown in solution containing 4.14, 4.18 or 4.26 ppm Fe also appeared more vigorous and a darker green in colour (data not shown). This result indicates that a specific range of Fe levels in nutrient solution promote plant growth (since levels greater than 6.10 as in trial 1 clearly negatively impact plant growth).

Table 3: Mean spinach plant height (cm) and fresh weight (FW; g) recorded 12 days after providing plants with nutrient solution with increased Fe concentration

The results demonstrate that yield was improved by 20 - 30%. Leaves of plants grown in nutrient solution with increased levels of iron were observed to be a darker glossy green as compared to plants grown in control nutrient solution.

As highest leaf Fe in Trial 2 was obtained from a nutrient solution containing 4.26 ppm Fe, a third trial was set up testing nutrient solutions with 4.10 (control), 4.18, 4.26, 4.42 and 4.74 ppm Fe for up to 35 days. Again, spinach plants were grown to a height of 5 - 10 cm before nutrient solution with elevated Fe was applied. Application time in Figure 4 refers to number of days plants were grown in elevated Fe nutrient solutions.

Highest leaf Fe2+/Fe3+ content (356 mg/kg) was measured in plants grown in nutrient solution containing 4.74 ppm Fe after 7 days (Fig. 4). This was 324% higher than the level of Fe plants grown in control nutrient solution (4.10 ppm Fe). Fe content in plants grown in nutrient solution containing 4.74 ppm Fe declined steadily with time, with a 34% decrease after 35 days but levels were significantly higher than in plants grown using the lower Fe treatments. Fe content in plants grown in nutrient solution with lower Fe concentrations varied between approximately 100 and 200 mg/kg DW at Day 7 to 100 and 150 mg/kg DW at Day 35.

In conclusion, these trials indicate that Fe content of spinach plants can be significantly increased by growing the plants in a hydroponic system using a nutrient solution with Fe concentrations of between 4.10 ppm and approximately 6.00 ppm. Use of nutrient solution with Fe concentrations above 8.0 ppm resulted in plant damage and death (Figures 1 and 2). As such, using higher Fe in nutrient solution may not be desirable when the plants must be harvested and sold in unprocessed form to the general public (since consumer preferences will likely dictate that the plants have a particular presentation at point of sale). However, there may still be relevant applications for spinach grown in solutions with higher Fe given the high leaf Fe content obtained with nutrient solutions in excess of 6.00 ppm and despite the impact on the physical appearance of the plants. For example, even though nutrient solutions with 6.10 ppm Fe in trial 1 resulted in stunted growth of plants, these plants may still be useful in the preparation of a processed spinach product (for example, a nutritional supplement or freeze dried spinach product) for delivery of Fe to the consumer.

Example 2: Determination of iron content in plants

Principle A representative sample of leafy green vegetable is microwave digested using nitric oxide and hydrogen peroxide, or block digested in an open tube using nitric and perchloric acids and reflux funnels. The digested material is diluted and the concentration of each analyte in the digested material is determined by ICP-ES (inductively coupled plasma-emission spectroscopy).

Reagents

Analyte grade reagents are used in this method except where higher purity reagents are specified. • nitric acid, HNO3 69% (high purity and containing less than 0.01 pg/g of all analytes); • perchloric acid, 70% w/v (high purity and containing less than 0.01 pg/g of all analytes); • 4+1 nitric+perchloric acid: pour 200 mL perchloric acid into a 1 L measuring cylinder and make up to 1 L with nitric acid. Transfer to an acid dispenser bottle.

Sampling and preparation

Samples of plant tissue (edible portion: leaves and stems) are dried and ground following standard sample preparation procedures. For example, samples are dried at 65°C for 3-5 days. Once dry, the sample is finely ground in a non-metallic ring mill for 30-90 seconds according the quantity and hardness of the sample. Oily samples are freeze dried prior to grinding.

Procedure

Block digestion: 0.5 g of dried plant material is weighted into a numbered and calibrated digestion tube. 5 mL of mixed digestion acids are added and a reflux funnel is placed in the neck of the digestion tube. The digestion tube is left to pre-digest overnight (at room temperature). This may reduce foaming of the digestion mixture that occurs in the subsequent heating steps. Foaming may be eliminated completely by allowing several days of pre-digestion at room temperature.

The test tubes are placed in the heating block, with the temperature set to 80°C. The heating block is kept at this temperature until frothing has subsided (typically around 30 minutes). The temperature of the heating block is then increased to 150°C, and the samples are refluxed for 1 hour.

The temperature is increased further to 185°C for 1 hour and to allow the volume of the digested material to reduce to approximately 2.0 mL.

The heat block is kept at this temperature until white fumes of perchloric acid are seen in the test tubes. Remove each test tube from the heating block as the volume reduces to about 1.0 mL.

Samples are prepared for ICP-ES by carefully rinsing each reflux funnel with milli Q water into its tube and adding milli Q water to about 2 cm from the 25 mL mark on the tube. The contents of each tube are carefully mixed using the whirl mixer to ensure complete dissolution of any perchlorate salt crystals (this may take a few moments of mixing depending on the amount of crystal present in the sample). The crystals must be dissolved otherwise there may be low recoveries for certain analytes.

The samples are now made up to volume (25 ml_) and mixed.

Measurement'.

Measure the content of total iron, calcium, magnesium, sodium, potassium, sulphur, phosphorous, copper, zinc, manganese, aluminium and boron in the samples using the ICP-ES machine.

Example 3 - Example of cultivation of spinach using the Napperland hydroponic growing system 1. Raising seedings (i) Sowing and management pre-emergence

Seedling trays (containing 144 cells) are filled with culture medium containing rockwool granular cotton and 4 to 5 seeds are placed in each cell. Cells are hydrated with 300 ml ~ 400 ml water per tray and seedlings are allowed to germinate in the germination room (set at 20°C) over 3-4 days. (ii) Nursery management

After germination, seedling trays are placed on the nursery shelf and exposed to high temperature phase twice per day and to medium to low temperature phase once per day. 300 ml to 400 ml of culture solution is applied to the trays automatically. The nutrient solution provided to cultured seedlings is EC 1.3 dS / m at high temperature and EC 1.5 dS / m at medium to low temperature. 2. Planting seedlings

Fresh, sterile culture medium is added to each cultivation bed before planting (4 to 6 cultivation beds, 500 liter nutrient tank, water supply and drainage system constituting one block).

Seedlings are suitable for planting when the seedlings reach around 3 cm in height. Suitable seedlings are picked and dropped into the planting holes of the planting panels on the cultivation beds. The seedling roots are held on the bottom of the bed, in contact with the culture medium and the seedlings are self-supporting. Planting number is 45 seedlings per panel (1 x 0.6 m). The seedling root ball is placed in the cultivation space and immediately starts developing roots. 3. Cultivation (i) Composition of nutrient solution A standard hydroponic nutrient solution is used, supplemented with Fe-DTPA so that the final total iron concentration in the nutrient solution is at least 4.1 ppm. (ii) Concentration of nutrient solution

The EC of the nutrient solution EC should be approximately 3 dS / m. The EC decreases during cultivation, and to around 1.2 dS / m on the 2nd to 3rd day before harvest. At this time, supplementing the culture solution with nutrient solution is stopped and only water is supplied to the plants. (iii) pH of culture solution

The pH of the culture solution should ideally be between 6 and 6.5. As the plants grow, the pH of the culture solution rises gradually. Adjustment is not necessary if the pH remains in the range of 5.5 to 7.3. (iv) Temperature of culture solution

In spring/summer, the culture solution should be kept below 23 °C. In winter, the culture solution should be kept above 10 °C. The solution can be heated or cooled by flowing chilled water through the heat exchanger in the nutrient tank. (v) Water supply

Supply of culture solution per bed is 9 liters I min. The culture tank of each block is automatically refilled with the reference culture fluid from the main tank. (vi) Oxygen supply

There are no particular oxygenation requirements as the plants are able to utilise oxygen derived from the air. (vii) Environmental management on the ground

Plants are thoroughly ventilated in spring and summer. Ventilation is preferably performed at night. A shading net (providing 20 to 30% shading) is used during summer months but can be opened during cloudy weather. (viii) Crop management

Plants can be harvested when they have reached the desired size.

Characteristics of cultivation management

4. Control of diseases and pests

Regular attention is paid for evidence of wilting disease caused by Pythium fungi and the wilt disease caused by Fusarium fungi. To avoid any contamination, bed disinfection is performed after each round of cultivation. Sterilisation of the cultivation beds is performed by warming the culture solution to 60 °C with a hot water boiler and circulating this through the cultivation bed for 1 hour. This sterilizes beds, tanks, pipes and other equipment. It is simple and effective.

Aphids and caterpillars occur occasionally, but they do not cause significant damage as affected plants are easily removed. No pesticides are used. 5. Harvest

Plants are harvested with roots and culture medium attached. Early morning harvest is desirable. Because there are no dead leaves, removal of cotyledons is sufficient. Harvested plants are stored in the precooler to remove field heat.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims (25)

1. A method of improving the nutritional value of a leafy green vegetable, the method comprising: - contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.1 ppm and no more than about 8.0 ppm of Fe2+/Fe3+ thereby improving the nutritional value of the leafy green vegetable.

2. The method of claim 1 wherein improving the nutritional value comprises increasing the total iron, chlorophyll, antioxidant, vitamin or lutein content of the leafy green vegetable, or combination thereof.

3. The method of claim 2, wherein improving the nutritional value comprises increasing the total iron content of the leafy green vegetable.

4. The method of any one of claims 1 to 3, wherein the nutrient solution contains greater than about 4.2 ppm.

5. The method any one of claims 1 to 3, wherein the nutrient solution contains greater than about 4.5 ppm.

6. The method of any one of claims 1 to 3, wherein the nutrient solution contains greater than about 4.1 ppm and no more than about 6.0 ppm of Fe2+/Fe3+.

7. The method of any one of claims 1 to 3, wherein the nutrient solution contains greater than about 4.2 ppm and no more than about 5.0 ppm of Fe2+/Fe3+.

8. The method of any one of claims 1 to 3, wherein the nutrient solution contains between about 4.5 and about 4.9 ppm of Fe2+/Fe3+.

9. The method of claim any one of claims 1 to 8, wherein the seedling is contacted with the nutrient solution for a period of between about 1 and 50 days.

10. The method of any one of claims 1 to 9, wherein the seedling is contacted with the nutrient solution for a period of between 3 and 35 days.

11. The method of any one of claims 1 to 10, wherein the seedling is contacted with the nutrient solution for a period of between about 5 and 15 days.

12. The method of any one of claims 1 to 11, wherein the seedling is contacted with the nutrient solution for a period of between about 5 to 10 days.

13. The method of any one of claims 1 to 12, wherein the seedling is approximately 8 to 15 days old when it is first contacted with the nutrient solution.

14. The method of any one of claims 1 to 12, wherein the seedling has a height of between 5 to 10 cm, when it is first contacted with the nutrient solution.

15. The method of any one of claims 1 to 14, wherein the roots of the seedling are contacted with the nutrient solution.

16. The method of any one of claims 1 to 15, wherein the leafy green vegetable is harvested at the conclusion of the period of contact with the nutrient solution.

17. A leafy green vegetable, when made according to the process of any one of claims 1 to 16.

18. A method of increasing the yield from a leafy green vegetable, the method comprising: - contacting a seedling of a leafy green vegetable in a hydroponic cultivation system with a nutrient solution containing greater than about 4.10 ppm and no more than about 8.00 ppm of Fe2+/Fe3+ thereby increasing the yield from the leafy green vegetable.

19. The method of claim 18, wherein the fresh weight of the leafy green vegetable at harvest is between about 25 g to about 100 g.

20. The method of any one of the preceding claims, wherein the leafy green vegetable is from a botanical family selected from the group consisting of: Amaranthaceae, Apiaceae/Umbelliferae, Araceae, Asteraceae/Compositae, Basellaceae, Brassicaceae/Cruciferae, Convolvulaceae, Curcubitaceae, Dryopteridaceae, Malvaceae, Moringaceae, Polygonaceae and Solanaceae.

21. The method of claim 20, wherein the leafy green vegetable is selected from the group consisting of: spinach, lettuces, rocket, silverbeet or swiss chard, kale, collard greens, mustard greens, cabbage and broccoli.

22. The method of claim 21, wherein the leafy green vegetable is spinach.

23. A spinach plant containing an amount of iron of between about 250 ppm to about 1000 ppm by dry mass weight, wherein said spinach has been produced using a hydroponic cultivation method.

24. The spinach plant of claim 23, wherein the amount of iron by dry weight mass is between 300 ppm and about 500 ppm.

25. A freeze dried whole-plant supplement produced by freeze drying a leafy green vegetable grown according to the method of any one of claims 1 to 16 or 18 to 22.