Note: Descriptions are shown in the official language in which they were submitted.
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ALTERATION OF PLANT NITRATE AND OXALIC ACID
CONCENTRATION
BACKGROUND OF THE IN~'ENTIO1V
Oxalic acid is an organic acid present in algae, Fungi, lichens, higher
plants, and animals including humans (Olce, 1909). The role of oxalic acid is
unclear and it is usually thought to be a useless end product of carbohydrate
metabolism (Hodgkinson, 1977). Oxalic acid forms crystals with several
minerals including (but not limited to) calcium, magnesium, potassium, sodium,
iron, and zinc. The oxalic acid in spinach is primarily bound to calcium or
potassium salts. Oxalic acid levels as high as 13°% of the dry weight
ofspinach
have been reported for some cultivars (Hodgkinson, 1977).
Plants, animals and humans form oxalic acid as a secondary metabolite of
vitamin C (Hodglcinson, 1977). Human urine always contains small levels of
calcium oxalate (Olce, 1949). Deposits of calcium oxalate crystals in the
kidneys
are a common form of kidney stone (Massey et al, 1993). Cn healthy adults,
with U.S. or European type diets, 90°~0 of oxalic acid excreted in the
urine comes
from endogenous synthesis (Massey et al., 1993) and is a secondary metabolite
of ascorbic acid (vitamin C) in both plants and animals. However, reduction of
foods known to increase urinary oxalate is recommended as a dietary change for
kidney stone-fonners (Massey et crl, 1993). High dietary oxalate is associated
with reduced calcium absorption from the gut, as oxalate has the ability to
bind
calcium. Calcium oxalate crystals formed in the gut are not absorbed, but are
can-ied out with the feces. Thus sufficient dietary calcium reduces absorption
of
oxalate from foods (Massey et al., 1993).
The top 8 foods or plants known to increase urinary oxalate are identified
by Nlassey c~t ul ( 1993) as rhubarb, spinach, beets, nuts, chocolate>
strawberries,
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wheat bran, and tea. Other foods or plants containing oxalate include leafy
green
vegetables, asparagus, runner beans, beetroot, brussel sprouts, cabbage,
carrots,
cauliflower, celery, chives, lettuce, marrow, mushrooms, onions, parsley,
green
peas, potatoes, radishes, rhubarb, spinach, tomatoes, turnips, apples,
apricots,
ripe bananas, gooseberries, grapefruits, melons, oranges, peaches, pears,
pineapples, plums, blueberries, raspben-ies, strawberries, arugula, beet
greens,
collard greens, kale, endive, bok choy, dandelion greens, escarole, cole,
macho,
mustard greens, radicchio, rapini, Swiss chard, and watercress.
There have been cases of oxalate-poisoning, especially from the species
rhubarb (Rhezzrn rluzpontiezzm, L.) and sorrel grass (Rztrnex czeetoscz. L.).
In high
levels, oxalic acid can be con-osive to the gastrointestinal tract
(Hodglcinson,
1977). Cattle have been poisoned by high oxalate content of Setczria found in
grazing areas. However, workers in Australia found a cultivar of Setaricr
cattle
can graze safely (Hodgkinson, 1977).
Oxalic acid oxidase is an enzyme that catalyses the breakdown of oxalic
acid into carbon dioxide and hydrogen peroxide. Oxygen is required for the
reaction. The formula for the reaction is:
C~O,~H~ + O~ ---~ (oxalic acid oxidase)--~ 2C0~ + H~O
The enzyme is present in spinach leaves, but is usually not active.
Through studies on beet root extracts it was determined that the sole factor
inhibiting oxalic acid oxidase was nitrate. Low concentration of nitrate in
oittw
(50 yM) is sufficient to inhibit the enzyme (Oke, 1969).
Varietal differences in spinach oxalate concentration have been reported.
Differences in oxalate concentration have been reported with respect to
plant age. The oxalate concentration, in general, increases with plant age and
lid
to the suggestion that earlier harvesting may be beneficial. However, some
investigators found oxalate increased with plant age (Hodgkinson, 1977),
others
reported spinach oxalate concentration decreases with age.
An enzyme, oxalic acid oxidase, catalyses the break down of oxalic acid
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into carbon dioxide and hydrogen peroxide in the presence of oxygen. Oxalic
acid oxidase has been studied in spinach leaves (Oke, 1969). Although present
in the leaves, the oxidase is inhibited by even low levels of nitrate (~- 50
M)
(Oke, 1969). Nitrate may also play another role in increasing leaf oxalate.
The evolutionary advantage of spinach possessing higher oxalate than
most ofher vegetables has not been determined. Some have suggested it may be
protective against foraging animals (Hodgkinson, 1977).
Nitrate is ubiquitous in most fresh vegetables and accounts for
approximately 80% of nitrate in the diet. Nitrate itself is not particularly
toxic.
Most nitrate is excreted from the body through the urine {Lee, 1970). However
approximately 5% of consumed nitrate is converted to nitrite in the oral
cavity,
gastrointestinal tract, and liver. It is the reduced form of nitrate, nitrite,
that is
the primary cause for concern. High levels of nitrate consumed by infants can
lead to methemoglobinaemia, or blue baby syndrome. The primary concern For
healthy adults is the potential for post-consumption formation of carcinogenic
N-
nitrosamine compounds. Fresh spinach may contain up to 740 ppm nitrate, and
reducing soluble nitrate and oxalate levels in vegetables would improve the
nutritional value of these crops to humans or other animals.
There is a need for altering certain compounds in plants in order to
improve the nutritional quality of the plant for human consumption.
SUMI<~IARY OF THE INVENTION
~fhe present invention provides methods for altering the concentration
or level of soluble oxalic acid or oxalate in a plant, comprising growing the
plant in a hydroponic nutrient medium which contains a nitrogen source, and
then altering the content of the hydroponic medium prior to harvest of the
plant
so that the medium does not contain a nitrogen source, so that the
concentration
ofoxalic acid or oxalate in the plant is altered.
The methods of the present invention may also be used to alter the
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concentration or level of soluble nitrate in a plant, and to alter the
concentration
or level of nitrate and soluble oxalic acid or oxalate in a plant. Further,
the
methods of the present invention may be used to alter the concentration or
level
of one or more nutrient quality indicators in a plant.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 Oxalate and Nitrate concentration in spinach leaves treated
with six pre-harvest draw-dowm method treatments.
Figure 2 Time-course pre-harvest draw down of nitrate and oxalate
in spinach, cv Whitney transferred to RO water for five days.
Figure 3 Time course reduction in nitrate and oxalate in spinach cv
Whitney transferred to RO water for l 92 h (8 days).
Figure 4 Comparison of spinach treated for 192 h (8 d) in RO
water, RO water + tent, or control plants in a nitrate nutrient solution.
Figure 5 Reduction in nitrate and oxalate in spinach cv Alrite
transferred to RO water for 168 h (7 days). Spinach treated in RO water, RO
water + tent, or control plants in a nitrate nutrient solution.
Figure 6 Comparison of spinach cv Whitney treated for 192 h (8 d)
in TAP water, TAP water + tent, or control plants in a nitrate nutrient
solution.
Figure 7 Time course reduction in nitrate and oxalate in spinach cv
Whitney transferred to TAP water for up to 26~ h (1 1 days).
Figure 8 Combined data points from all six oxalate draw-dov=n
experiments show similarity in slope of lines during draw-down regardless of
starting concentration, tap or RO water, and spinach cultivar Whitney or
Alrite.
Figure 9 Line of best fit developed from combining oxalate draw
dwvn data from six experiments.
Figure 10 Combined nitrate concentration data points from four
draw-down experiments show similarity in slope and shape of lines during draw-
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down. Graph combines results from tap water, R(7 water, and spinach eultivars
Whitney and Alrite.
Figure I I Line of best fit developed from combining nitrate draw
down data from three experiments.
Figure 12 During drav~~-down procedure, nitrate is used in vacuoles
and plants can continue to grow (add dry weight).
Figure 13 Decline in three indicator vitamins with increasing
duration of oxalate draw-down period: A) vitamin C, B) Folate, C) Beta-
carotene.
Figure I4 Oxalate concentration in spinach leaves as a function of
nitrate concentration. Data from four nitrate draw-down experiments combined
shows saturation of nitrate influence on oxalate concentration at 200 umoles/g
DW in this set of experiments.
DETAILED DESCRIPTION OF THE INVENTION
The teem C''ontrolled Environment Agriculture (or "CEA"), as is used
heroin, is a combination of horticultural and engineering techniques that
optimize crop production, crop quality, and production efficiency (L.D.
Albright, 1990, Environment Control for Animals and Plants).
The term hydroponic nutrient solution or medium, as is used herein, is a
water-based formulation containing essential nutrients for plant growth. The
solutions contain mineral elements and compounds, containing but not limited
to nitrogen, phosphorous, potassium, calcium, magnesium, oxygen, sulphur,
boron, chlorine, copper, iron, manganese, molybdenum, zinc, in addition to
many minerals present in trace amounfs. The pH of the solution is controlled
to
allow solubility of the mineral elements and compounds in the solution.
(,rones,1997, Hydroponics, A practical Guide for the Soilless Grower). The
nitrogen concentration of the hydroponic nutrient solution or medium may range
from, but is not limited to, slightly under 50 to more than 350 ppm.
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The reduction in concentration of oxalic acid, nitrate, or nutrient quality
indicators, as accomplished by the present invention, may be allowed to
continue
as long as plants maintain a marketable or healthy appearance, or a dark-green
appearance.
The term "nutrient quality indicators" includes vitamins andlor minerals
in a plant that are important for human or animal nutrition, and includes, but
is
not limited to vitamin C, ascorbic acid, folate, folic acid, vitamin B9, (3-
carotene,
a vitamin A precursor, lutein, calcium, iron, vitamin A, vitamin E, and other
vitamins and minerals.
The term "mature plant," as is used in this application, includes plants
that have reached a sufficient stage of growth and contain the desired
characteristics, so that they may be harvested for any useful purpose. As an
example, for spinach, the term "mature plant" may describe a plant that has
reached approximately 2-7 ounces of fresh weight after about 20-35 days from
seeding.
The present invention may be useful in altering the concentrations of
oxalic acid, oxalate, nitrate, or nutritional quality indicators of any plant
species,
including, but not limited to, plants that contain oxalic acid or nitrate,
such as
spinach, beets, nuts, chocolate, cacao, strawberries, wheat bran, tea, leafy
green
vegetables, asparagus, runner beans, beetroot, brussel sprouts, cabbage,
cawots,
cauliflower, celery, chives, duckweed, lettuce, man-ow, mushrooms, onions,
parsley, green peas, potatoes, radishes, rhubarb, spinach, tomatoes, turnips,
apples, apricots, ripe bananas, gooseberries, grapefruits, melons, oranges,
peaches, pears, pineapples, plums, blueberries, raspberries, strawberries,
arugula,
beet greens, collard greens, kale, endive, bole choy, dandelion greens,
escarole,
sole, mache, mustard greens, radicchio, rapini, swiss chard, and watercress.
The present invention may also be useful in altering the concentrations of
o~:alic acid, oxalate, nitrate, or nutritional quality indicators of other
plant
species, including, but not limited to, corn, rapeseed, alfalfa , rice, rye,
millet,
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pearl millet, proso , foxtail millet, finger millet, sunflower, safflower,
wheat,
soybean, tobacco, potato, peanuts, cotton, potato, cassava, coffee, coconut,
pineapple, cocoa , banana, avocado, Gg, guava , mango, olive, papaya , cashew,
macadamia, almond, sugar beets, sugarcane, oats, duckweed, barley, vegetables,
ornamentals, conifers, tomatoes, green beans, lima beans, peas, Cncttf~iis
(including cucumber, cantaloupe, and musk melon), azalea, hydrangea, hibiscus,
roses, tulips, daffodils, petunias, carnation, poinsettia, chrysanthemum,
legumes
(including peas and beans, such as guar, locust bean, fenugreek, soybean,
garden
beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea), peanuts,
crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, lupine,
trifolium, field bean, clover, Lotus, trefoil, lens, lentil, false indigo,
alfalfa,
orchard grass, tall fescue, perennial ryegrass, creeping bent grass, redtop
grass,
aneth, artichoke, blackberry, canola, cilantro, clementines, eucalyptus,
fennel,
grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra,
orange, parsley, persimmon, plantain, pomegranate, poplar, radiata plne,
Southern pine, sweetgum, tangerine, triticale, vine, yams, apple, pear,
quince,
cherry, apricot, melon, hemp, buckwheat, grape, raspberry, chenopodium,
blueberry, nectarine, peach, plum, watermelon, eggplant, pepper, cauliflower,
broccoli, onion, carrot, leek, beet, broad bean, celery, radish, pumpkin,
endive,
gourd, garlic, snapbean, squash, turnip, asparagas, and zuchini.
E~CAMPLES
Pre-sowring seed treatment
Spinach seeds, cv VThitnoy, were treated using a modified-Katzman
protocol in Experiments I, II, IV and V and VI. Experiment III was performed
using the spinach cultivar Alrite. Calcium hypochlorite was removed from the
pre-sowing treatment to avoid seed contact with potential sources of chloride
ions. Seeds wore placed in a ? liter beaker containing glass distilled water
and
wero stirred in the room temperature water bath for ~ hours with a magnetic
stir
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plate. Water was drained from the beaker and replaced with a 0.3% H~U
solution. Stirring continued overnight. After 15 hours, seeds were removed
from packets and placed on blotter paper in germination boxes. Seeds were
spread evenly on the blotter paper and distilled water was added to keep
germinating seeds moistened. Seeds in germination boxes were then placed in a
°C cooler for 7 days while the imbibition and germination continued.
Seed planting and hydroponic pond system seed liolders
EXPERIMENT I.
Floating polystyrene trays were prepared by cutting eleven 28 cm x O.G
cm ( 11 x 0.25 in) strips out of each 0.3 m x O.G m ( 1 x 2 ft) tray. Thirty
felt
strips were cut from a bolt into rectangles 28 cm x 10 cm ( 11 x ~. in). Two
pieces of felt were placed together to form cloth sandwich-strips and were fed
through the polystyrene strip-holders. Felt-strip pairs were held in place by
wedging a small piece of polystyrene at both ends of each felt-strip pair. The
felt-strip seed holders wicked up water and nutrients from the pond solution
and
maintained a moist environment around the seeds and roots. The foam boards
were 2.5 em (1 inch) thick and roots grew downward, between the two felt
strips
into the oxygenated hydroponic solution below. Seeds were placed with root
oriented downward between two pieces of felt and held by friction fit over the
hydroponic nutrient solution. Only seeds that had germinated during the one-
week in the cooler were planted. Three seeds were planted between each felt
strip pair. Seedling radicals were approximately 2 to 5 mm long at time of
sowing.
EXPERIMENTS II - V
Seeds were sown into reverse-osmosis (RU) water-leached rockwool
flats. The moistened seeded rockwool flats were covered and maintained at
18°C
For 3 days after sowing without need for additional watering. At age 3 days,
growth chamber temperature was increased to ?' "C and ebb and flow watering
was initiated at a rate of twice per day. Seedlings were watered automatically
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with a nutrient solution containing no ammonium and no chloride. Cool white
fluorescent lamps in the growth chamber were set to 25% from initiation of
imbibition to age 3 days. At age ~ days, lights were increased to
50°!° maximum
intensity. At age 12 days after planting seeds in rockwool, Whitney seedlings
in
rockwool cubes were transferred from the growth chamber to polystyrene
floaters in an 18 °C hydroponic pond.
Greenhouse environmental parameters
Greenhouse temperature vas controlled at 2~ °C continuous. Light
control program was set to deliver 16 mols of light per day. Supplemental
light
was provided by an array of high-pressure sodium lamps.
Culture phase hydroponic pond conditions
The pond solution nutrient medium (or hydroponic nutrient medium)
recipe was adjusted to eliminate sources of ammonium and chloride, due to
reported toxicity problems in spinach with these ions (Elia et al, 1999). Pond
temperature was maintained at 18 °C. Nutrient solution pH and
electrical
conductivity (EC) were monitored and corrections made as needed. Set point for
pH was 5.8. Nitric acid was used to decrease pH, and potassium hydroxide was
used to increase pH. Electrical conductivity set point was 920 micro S for
this
particular nutrient solution. Daily water samples were taken and frozen for
nitrate analysis. Nitrate concentration in the N03 solution ranged from 0.2 to
2.5
mM. Nitrate concentration in the urea pond ranged from 0 to 1.1 mM. Nitrate in
the RO water pond ranged from 0 to 0.01 mM.
Nitrate draw-down phase hydroponic pond conditions
EXPE12IIVIENT I
At age 21 days after sowing, the seedlings in felt strips were divided into
three groups. Plants from groups 1 and ? were transferred to a pond lined with
RO water (no nutrient solution). Plants from group 3 (control) remained in the
culture nutrient solution. Pond temperature for both ponds was 18 °C.
Plants
were maintained in this condition for 5 days.
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EXPERI1VIENTS II-VI
At plant maturity (approximate age ?8 days) plants were transferred to
one of three RO water-filled tanks for 5 days to draw down nitrate in cell
vacuoles. Plant from experiments V and VI used tap water as RO water line was
not functioning in greenhouse (GH). Tanks were not oxygenated, but were
stirred regularly by hand to aerate. Tanks were located in GH 15 C with
photoperiod from 6 am to 10 pm, continuous HPS lighting. Greenhouse
temperature during nitrate draw-down phase was 20 °C. There were three
tanks
used in the test, with six plants per tank.
Oxalate draw-down phase hydroponic pond conditions
EXPERIIYIENT I
After 5 days in the nitrate-draw down pond or control pond, plants were
divided into G groups. The following list describes the PRE-HARVEST
treatment conditions:
1)RO water ~ RO water open air
~)RO water ~ RO water, inside CO~ draw-down tent
3) RO water ~ Urea solution, open air
4) RO water ~ Urea solution, inside CO~ draw-down tent
5) Nutrient solution ~ Nutrient solution open air
6) Nutrient solution ~ Nutrient solution, inside CO~ draw-down tent
Each C02 draw-down tent was fabricated by placing clear plastic bags
over a wire-mesh cage. A beaker containing 150 ml of 1 N KOH with fluted
germination paper fan was placed inside each CO~ draw-down tent. There was
no humidity control in the tents for Experiment I.
EXPEI2.Ilt'IENTS II-VI
Six plants in each tank were divided in two. Three plants were placed in
a CO~ draw-down tent. Humidity in tent was controlled with a de-humidifier. A
-1 liter open reservoir of 1 N KOH was placed inside the tent to assist in
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down of CO~. The other three plants in each tank remained outside the tents.
Oxalate draw-down time was two days. Tanks were located in GH 15
°C with
photoperiod from 6 am to 10 pm, continuous HPS lighting. Greenhouse
temperature during oxalate drav~~-down phase was 20 °C.
Harvest
After 8 days in pre-harvest treatment, plants were harvested by separating
above-root portion from roots with a razor. Plants were placed in a 70
°C drying
oven inside brown paper bags and dried for two days. Plants in the second tap
water experiment were freeze dried rather than oven dried for additional
nutrient
analyses.
Tissue preparation
Dried plant samples were bulked by treatment and age at harvest and
were ground on a Wiley Mill, using a #20 screen.
Chemical analysis of oxalate and nitrate contents
Soluble oxalate and nitrate were extracted together in the same
supernatant. A sample of ground tissue was weighed into a 15 ml tube. Ten
milliliters of HC1 (0.1 N) was added and mixed on a vortex mixer. Test tubes
were sealed and placed on a shaker for one hour at room temperature. Samples
were centrifuged and 2 ml of supernatant removed and centrifuged again in 2.2
ml microcentrifuge tubes. A 0.5 ml portion was removed and diluted with 4.5
ml of 18 M ohm water. A 500 mieroliter aliquot of the diluted sample was
placed in a Dionex autosampler vial (0.5 ml vial, Dionex Corp., Sunnyvale, CA)
For each spinach cultivar.
Chemical analyses of vitamin C, Folate, and (3-carotene - Experiment VI
only
To determine whether the draw-down technidue was also reducing
vitamin content of the spinach, freeze dried tissue samples from three time
points
during the second tap-water draw-down experiment were sent to ENI
Laboratories (www.eurofins.com, Eurofms Scientific, Dayton, NJ) for accredited
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nutrient analysis. The time points selected for analysis were Day D (starting
point control), Day 5 (day nitrate reserves are depleted), and Day 8 (last day
spinach had marketable appearance). The number of samples and types of
analysis were limited due to cost. Three nutrients were selected as indicators
of
potential loss of nutrients with the pre-harvest nitrate/oxalate reduction
technique. The water soluble vitamin, vitamin C was selected as the first
indicator because spinach contains ample amounts of vitamin C for
measurement, and ascorbic acid decline with post-harvest food preparation is
well documented. Folic acid (total vitamin B9) was selected because spinach is
among the vegetables high in folic acid and this would be an important
nutrient
to preserve. The vitamin A precursor, (3-carotene, was selected to represent
fat-
soluble vitamins.
Statistical anal3~sis
Statistical analysis was performed using Minitab software (V 12, Minitab
Inc., State College, PA), Microsoft EXCEL 97 (Microsoft Corp> USA) and
SigmaPlot software (SigmaPlot 200D, V6.X, SPSS Inc., Chicago, IL).
RESULTS AND DISCUSSION
Soluble nitrate and oialic acid reduction
The practice of pre-harvest transfer of spinach plants into RO water and
tap water reduced nitrate concentration. In Experiment I, plants in CO~ draw-
down-tents with either NO~ nutrient solution or urea nutrient solution had
higher
levels of nitrate than plants in nutrient solutions outside of the tents
(Figure 1).
This increase in nitrate concentration was probably due to lowered light
levels
under the plastic tents.
Transfer of the seedlings into RO water (without tent) was the most
effective treatment for reduction of nitrate and oxalate within 8 days. Plants
in
the nitrate solution (Control) had an average leaf concentration of 1 D63 ~mol
oxalate/g dry weight (DW) ( 1 D ° °). Plants in the RO water
solution had 655
l.tmol o~alate/g DW (6°°). Nitrate levels were ~B.I ~tmul/g DVl'
(~°°) in the
1'_
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control plants, and 34 ymollg DW (:> 1 ° o) in the RO water treated
plants (Figure
I). From a qualitative standpoint, there were no obvious changes in plant
appearance between the treatments. To determine whether this time could be
shortened, Experiments II-VI were designed to examine time-course patterns of
reduction.
In Experiment II, oxalate was reduced from 1120 to G33 moll g DW
(i.e., from 10 °.% to G °'o DW) in five days of RO water
treatment. Nitrate was
reduced from 548 to 15 ymol/g Dug (i.e., 3% to 0% DW) in five days (120
hours) (Figure 2). The experiment was extended to eight days in Experiment
III,
and a humidity-controlled tent treatment was added as an end-point for
comparision with controls. For the time-course portion of Experiment III,
oxalate concentration was reduced from 1203 to 387 ~mollg DW (i.e., from
11°0
to 3% DW) in eight days (Figure 3). Nitrate concentration was reduced from
4-19 to 0 Ermollg DW (i.e., 3% to 0°o Dul) in eight days. The RO water+
tent
treatment plants were not significantly different from the plants given the RO
water treatment alone (Figure 4).
The same procedure was performed for seven days on another cultivar of
spinach, Alrite (Figure 5). Alrite has a faster bolting tendency than Whitney.
Alrite leaf nitrate concentration was reduced from 299 to 0 umol/ g DW (i.e.,
from 2% to 0 ° o DW) and leaf oxalate concentration was reduced from
1181 to
432 ~mollg DW (i.e., from 10° o to 4 °r DW) after 7 days of the
draw-down
treatment in RO water (Figure 5). Soluble leaf nitrate content was lower in
Alrite (299 ~mollg DV') than in Whitney (449 Itmol/g DW). The lower
concentration of nitrate in Alrite than in Whitney is consistant with findings
in
other experiments (e.g., Table 4.4) and may be a genetic difference in leaf
nitrate
concentration. The results of the oxalate drawl-down procedure were the same
for Alrite as for Whitney.
In Experiment V, the draw ~-down method was performed using tap-water
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instead of RO water to determine whether cost could be lowered by using tap
water for the method. Nitrate was 691 E~mol/g DW (4°I° DW) in
the control
treatment, and 83 pmol/g DW (I °,lo DW) in the tap water treament. Tap
water
contains nitrate, so it is not surprising that the tap water treated plants
did not
reach 0 nitrate in the same time that the RO water treated plants reached 0
nitrate
concentration. Oxalate was 945 ~mol/g DW (8% DW) in the control plants and
552 ~mol/g DW (5% DW) in tap-water treated plants (Figure 7).
The final draw-down test, Experiment VI, was a time-course sampling
with plants treated in tap water. Three samples were also analyzed for three
nutrient indicators (vitamin analysis - next section). Oxalate levels were
reduced
from 1317 to 461 pmol/g DW (i.e., from 12% to 4 °'o DW). The plants
were
yellowed on the last hardiest day (264 h, or day 11) and would not have been
marketable. Daily qualitative observations recorded note that day 8 ( 192 h)
was
the last day the plants appeared healthy and marketable. Leaf oxalate
concentration on day 8 (192 h) v'ras 565 ~mol/g DW (~°,o DVT). Leaf
nitrate
concentration was reduced from 284 to 0 pmol/g DW (2 % to 0% DW). Fresh
tap water vas added to the treatment ponds after to 120 h harvest (day 5). Tap
water contains nitrate, and the increase in pond-water nitrate due to adding
fresh
wlater resulted in leaf concentration increasing on day 6 (144 h) and then
falling
again within 2 days. Interestingly, when leaf nitrate concentration rose, leaf
oxalate concentration followed and increased one day after the nitrate peak
(day
7) at 168 hours. It is notable that the increase in oxalate following the
water-
refresh did not shift the draw-down to a higher recovery level. Figure 7 shows
nitrate and oxalate leaf concentrations during the course of the tap-water
draw-
down method performed on spinach, cv Whitney. Also indicated are the three
sampling dates for vitamin Analysis. The last marketable day is indicated, but
oxalate concentration continued to fall until the last plants were removed on
day
11 ('64 h).
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Use of RO water was superior to tap when water-replenishment was
needed due to presence of nitrate in tap water. Oxalic acid was reduced by one-
half to two-thirds through the 7 to 8 day pre-harvest treatment. 'I'he
reduction in
oxalic acid paralleled nitrate reduction in time-course studies.
By combining data from all six draw-down experiments, the reduction in
soluble oxalate is linear, with a slope that was constant in all experiments,
regardless of starting point of soluble oxalic acid concentration in the
leaves
(Figure 8).
From these data points, a combined line of best Ft was calculated by
EXCEL (Figure 9).
From the linear line of best fit for soluble oxalate reduction:
[Oxalate Concentration (umol/g DW)] _ -3.2715* (hrs in treatment) +
1 U87.1
The slope of the line is approximately -3.3 and indicates that in the
method developed here, oxalate is removed at a rate of 3.3 umol per hour per g
DW. 'This is equivalent to a removal rate of approximately U.U2°.%
oxalate/g DV'
per hour. It would be expected then, that 78.5 umol oxalate are removed per
day
per g DW (i.e., U.~9°o DW per day)
Combining data of the nitrate concentration during draw-down does not
show a linear pattern (Figure 1U).
The response to draw-down is not as rapid from the tap water as RO
water, as would be expected because tap water contains nitrate. For this
reason,
only the other three experiment lines are used for the trend-line fitting
(Figure
11)
Plant growth during nitrate draw-down
Plant dry weight data from the final draw-down experiment demonstrates
that the spinach plants continue to grow during the nitrate draw-down period,
as
the plants are using up nitrate stares in the vacuoles. When the nitrate
reaches U,
plant growth stops (Figure 1?).
CA 02425687 2003-04-28
WO 02/34755 PCT/USO1/31254
Vitamin reduction with pre-harvest treatment
Nutrient analysis of tissue from days 0, 5, and 8 of the oxalic acid draw-
down treatment show that all three indicator vitamins declined with increasing
length of oxalate draw-down period (Figure 13). Vitamin C (ascorbic acid)
concentration declined from 39.6 to 19.8 mg/100 g DW in 8 days. Vitamin B9
(Folic acid) concentration in the leaves declined from 1.71 to 1.10 mg/100 g
DW
during the 8 day period. Vitamin A precursor (Beta-carotene) concentration
declined from 76900 to 64300 IU1100 g DW. The linearity of the decline in
nutrients is indicated by the r~ correlation coefficients of the lines.
For vitamin C decline, r~ = 0.9611, and equation:
[vitamin C (mg1100 g DVV)] _ -2.549*(Days of draw-down) + 38.77 2.
For Folate decline, r' = 0.9997, and equation:
[Folate (ug/100 g D~~')] _ -0.0764*(Days of draw-down) + 1.7079
For vitamin A precursor, Beta-carotene, r' = 0.9776, and equation:
[Beta-carotene (IU/100 g DW)] _ -1610.2*(Days of draw-down) +
76478
By combining all data points for the six nitrate and oxalate draw-down
experiments, the graph of oxalate concentration as a function of nitrate
concentration shows a narrow range of response before the influence of
increasing nitrate was saturated (Figure 14).
The pre-harvest technique reduced soluble nitrate levels in the spinach
leaves to undetectable levels by day 5 of the pre-harvest draw r-down
treatment.
Soluble oxalic acid also reduced by one-half to two thirds by the method.
Reduction in oxalic acid occurred and paralleled the reduction in nitrate in
all
time-course studies. 'fhe leaf concentration of the three vitamin indicators
also
declined with increasing time in the oxalate draw-down treatment.
16
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WO 02/34755 PCT/USO1/31254
REFERENCES:
All publications, patents and patent documents are incorporated by
reference herein, as though individually incorporated by reference. The
invention has been described with reference to various specific and preferred
embodiments and techniques. However, it should be understood that many
variations and modifications may be made while remaining within the spirit and
scope of the invention.
In addition, the following references have been cited in the text and their
complete citations are as follows:
Elia, A., P. Santamaria, and F. Serio ( 1998) Nitrogen nutrition, yield and
quality
of spinach, ,Ioicrnal of the Scieyice ofFoc~d and Agricttltztr~e 76:341-346.
Hodgkinson, A. (1977) Oxalic Acid in Biology and Medicine, Academic Press,
London.
Massey, L.K., H. Roman-Smith, and R.A.L. Sutton (1993) Effect of dietary
oxalate and calcium on urinary oxalate and risk of formation of calcium
oxalate kidney stones, Jattt~yaal of the ~lr~aet°icara Dietetic
Association
93:901-906.
Oke, O.L. (1969) Uxalic acid and plants and in nutrition, 1%~ould r~eviei~~ of
Nuty~itivn crud Dietetics 1Q:?62-303.
17
