Increasing Nutrient Density of Food Crops through Soil Fertility
Management and Cultivar Selection
Proposal presented
By
Md J Meagy
Department of Plant, Soil, and Insect Sciences
University of Massachusetts Amherst
Table of Contents
1. Literature Reviews
2. Research
i. Justification
ii. Objectives
iii. Experiments
a. Experiment 1:
b. Experiment 2:
c. Experiment 3:
d. Experiment 4:
3. Experiment 1:
i.
Introduction
ii.
Materials and Methods
a. Materials
b. Methods
iii.
Results
iv.
Discussion
4. Experiment 2:
i.
Introduction
ii.
Materials and Methods
a. Materials
b. Methods
iii.
Results
iv.
Discussion
5. Experiment 3:
i.
Introduction
ii.
Materials and Methods
a. Materials
b. Methods
iii.
Results
iv.
Discussion
6. Experiment 3:
i.
Introduction
ii.
Materials and Methods
a. Materials
b. Methods
iii.
Results
iv.
Discussion
7. Bibliography
Increasing Nutrient Density of Food Crops through Soil Fertility
Management and Cultivar Selection
Proposal presented
By
Md J Meagy
Department of Plant, Soil, and Insect Sciences
University of Massachusetts Amherst
Increasing Nutrient Density of Food Crops through Soil Fertility
Management and Cultivar Selection
A Disertation Proposal
Presented
By
MD J. MEAGY
Approved as to style and content by:
____________________________________________________
Allen V. Barker, Chair
____________________________________________________
Geunhwa Jung, Member
____________________________________________________
Touria El Jaoual Eaton, Member
Department of Plant, Soil, and Insect Sciences
University of Massachusetts Amherst
Increasing Nutrient Density of Food Crops through Soil Fertility
Management and Cultivar Selection
Abstract
Lettuce is the most widely used leafy vegetable around the world. This experiment will
be conducted on lettuce crop, organic and conventional fertility regimes, fertility
management practices, assessing the relationship between fertility regimes and cultivars
selection for increasing nutrient densities, and assessing the molecular similarities and
variations among the cultivars. Experiments will be conducted in greenhouse and field
sites. Organic and conventional fertilizer regimes will be used as treatment for the plant.
Growth parameter such as height, fresh wt and dry wt will be reported for each
experiment. Elemental analysis will be resulted accumulated by plants afterwards.
Nutrients densities in plants might be increases through fertility and management
practices.
1
Increasing Nutrient Density of Food Crops through Soil Fertility
Management and Cultivar Selection
Background
Malnutrition is a primary factor limiting human productivity in modern times, and
deficiencies of certain elements including calcium, magnesium, potassium, phosphorus,
zinc, copper, and others known as mineral nutrients, in diets of humans are a substantial
nutritional problem throughout the World (Darnton-Hill et al., 2005; Kataki and Babu,
2002; Schaetzel and Sankar, 2000). Davis (2009) reports 5% to 40% declines in mineral
contents of vegetables and fruits in the past 50 to 70 years in the United States. Reports
from the United Kingdom indicated that foods were depleted by about 20% during this
time (Anonymous, 2005; Anonymous, 2006; Mayer, 1997). Research on the diets of
subjects in Philadelphia warranted a downward revision in the intakes of iron,
magnesium, and vitamins (Guenther et al., 1994). The result of a study concluded that
many Americans are not meeting the current recommendations for calcium intake
through diet alone or with supplements (Ma et al., 2007).
Davis (2009) suggests that the decline in mineral and protein content in fruits and
vegetables is due partly to a dilution effect of high yields. Side-by-side comparisons of
low- and high-yielding vegetables and grains showed negative correlations between yield
of produce and concentrations of minerals. However, White et al. (2009) reported that
with potato (Solanum spp. L.), a dilution effect of high yields on nutrient concentration
was not observed universally and that soil fertility affected the mineral nutrients more
than the dilution effect.
2
Soil fertility problems associated with nutrient depletion by crop production are
worldwide (Tan et al., 2005). In the United States, potassium and phosphorus contents
are being drawn down in soils at an increasing rate every year, and the depletion
occurring for 40 years for potassium and for nearly 30 years for phosphorus (Stewart,
2004). Similar nutrient mining of essential elements occurs throughout the World
(Ayoub, 1998; Dobermann et al., 1996; Lal and Singh, 1998; Nandwa and Bekunda,
1998). Elemental depletion of soils must be compensated for by fertilization for
sustainable production of nutrient-sufficient foods (Buol, 1995).
Organically grown fruits and vegetables might differ from conventionally grown
produce due to differences in the types of fertilizers used in the two practices. Low
availability of nutrients in organic fertilizers could limit mineral accumulation in plants
relative to fertilization with chemical fertilizers with high nutrient availability, thereby
making the chemical fertilizers a superior nutrient source. On the other hand, high
availability of certain nutrients or failure to provide elements in chemical fertilizers could
lead to nutrient imbalances in foods (Lundegaardh and Maartensson, 2003). A study
noted that compost increased nutrient concentrations in soils but not always in plants
(Roe, 1998). The result of another study reported no consistent differences in nitrogen,
phosphorus, and potassium concentrations in several vegetables in crops fertilized with
composts and crop residues or with synthetic mineral fertilizers (Herencia et al., 2007). A
recently published study (Benbrook et al., 2008) reported that organically grown foods
derived from plants were superior to those grown conventionally with respect to
phosphorus, potassium, nitrates, several antioxidants, and vitamin C. An experiment
result suggested that the high yields achieved through farming systems with high nitrogen
3
fertilizer inputs led to a dilution of nutrient density in vegetables relative to organic
systems with low nitrogen inputs (Benbrook and Scientist, 2009). High nitrate
concentrations in foods are considered as a factor adversely affecting human health
(Maynard et al., 1976). Organically grown vegetables may have less nitrate accumulation
than conventionally fertilized vegetables (Benbrook et al., 2008). The result of a study
reported that farm manure had a more favorable effect on certain plant constituents
(nitrate and oxalic acid) than potassium nitrate (Turan and Sevimli, 2005). However,
Maynard et al. (1976) noted that the amount of nitrogen fertilization regardless of source
was the principal factor leading to nitrate accumulation in vegetables. A study noted that
with fertilization for optimum yield, nitrate concentrations of vegetables were not
different between organically and chemically fertilized vegetables (Barker, 1975).
Regardless of soil conditions, fruits and vegetables, although high in vitamins, are
typically low in mineral nutrients (Elless et al., 2000). Except for potassium, the fruitvegetable food group contributes less than 30% of the total dietary intake of mineral
nutrients (Levander, 1990). To counter this problem, several attempts have been made to
increase the mineral nutrient content of fruits and vegetables. These actions have
included enrichment of foods in processing, but not much attention has been directed
toward improvement of foods through enhanced soil fertility and cultivar selection,
although these two factors appear to be principal agents affecting the nutrient density of
fruits and vegetables.
Methods of increasing the nutrient content of foods must be developed through
improved practices in fertilization and in the development and selection of crop varieties
that accumulate the nutrients in amounts that are adequate for intake in normal diets of
4
humans. This research will involve studies with heritage and modern hybrids of several
types of vegetable crops grown under differing regimes of fertilization. It is likely that
heritage and modern hybrids will differ in susceptibility to diseases. . The high levels of
horizontal or broad resistance to pathogens are often observed in heritage cultivars due to
their adaptation over time to climate and soil conditions where they have grown in when
they are compared to modern cultivars. The modern cultivars drop in horizontal or broad
resistance to pathogens during breeding and under protection of pesticides. Soil fertility
practices also are likely to affect the prevalence of diseases in the soil and plant
susceptibility to diseases. Plant pathogenic diseases can be stimulated by excess levels of
available nitrogen under favorable conditions.
Justification
The need for this project was raised by the food consumers and producers as well as by
the scientific community. Literature on food composition demonstrates that the mineral
nutrient density of vegetables has fallen in the past 50 years. This decline is associated
with declines in soil fertility and with the genetics of plant cultivars that increase yield at
higher rates than mineral nutrients increases. Research is needed to develop systems of
food crop production that will supply adequate mineral nutrition to people directly. Use
of nutrient-dense crops provide an opportunity for vegetable producers to diversify
production and market, and to increase income and profitability as there appear to be a
ready market for these crops.
5
Research Objectives
1. To determine if the mineral nutrient densities of selected vegetable crops can be
increased through cultivar selection.
2. To determine if the nutrient densities of selected vegetable crops can be increased
by elevating nutrient contents in the medium in which these plants grow.
3. To determine if the nutrient densities of selected vegetable crops can be increased
through soil fertility practices (e.g., organic vs conventional fertilizers; different
fertilizer regimes), and
4.
To assess genetic similarities among selected cultivars within species, for genetic
purity and diversity, using molecular markers.
Research
Four experiments will be set up for this project based on objectives. The brief discussions
of the experiment are as follows:
Experiment 1. Assessment of the relationship between nutrient density in crops and
cultivar selection and nutrient availability in the greenhouse.
This experiment will be designed to determine the mineral nutrient contents of lettuce
through cultivar selection using different fertilizer regimes.
The experiment will be conducted in the greenhouse to evaluate regimes of fertilization
and cultivars for evaluation in fields. The same fertilizers suggested for the field
evaluation will be used in this experiment. The greenhouse experiments will help to
evaluate fertility regimes before and after application in the field. The lettuce crop will be
6
grown in peat-based media (Canadian Growing Mix 1-PV, Conrad Fafard Inc, Agawam,
MA) (Boodley et al., 1996). The elemental nutrient composition will be determined in
edible parts (fresh leaves) of lettuce. Because of their adaptability to greenhouse
production, the lettuce crop to be evaluated using different lettuce cultivars. Each crop
will be evaluated for their growth and nutrient contents. The experimental design of the
experiment will be followed by complete random block design.
Experiment 2. Assess the effect between mineral nutrient density in crops and cultivar
selection by elevating nutrient contents in the medium in which the plants grow.
This experiment will be assessed differences in the mineral densities of lettuce cultivar
through increasing the nutrient contents in the medium. The differences of this
experiment from the experiment one will be in elevating nutrient contents in the medium
in which the plant grow. The experiment will be conducted in greenhouse and field to
assess the response of lettuce using the increasing nutrient contents. The same fertilizers
will be used in the field experiment. The crop will be grown in peat-based media
(Canadian Growing Mix 1-PV, Conrad Fafard Inc, Agawam, MA) (Boodley et al., 1996).
The fertilizer regimes will be chemical fertilizer organic fertilizer with increasing nutrient
contents. The increasing nutrient contents in the fertilizer regimes will be calcium based
on nutrient demand for human dietary nutrient requirement. The nutrient contents will be
measured in the edible parts (fresh leaves) of the plant. This crop will be evaluated for
growth and nutrient densities in produce. The experimental design of the experiment will
be followed by complete random block design.
7
Experiment 3. Assess the effect of organic and conventional soil fertility practices on
mineral accumulation in vegetables in field experimentation.
Fertilization will be provided in the experiment using organic and conventional fertilizer
regime. Two regimes of organic and one conventional fertilizer will be selected to
provide nutrients. Organic fertilizer will come from the individual sources includes
soybean meal for nitrogen, colloidal rock phosphate for the phosphorus, and mined
potassium sulfate for the potassium. The compost will be obtained from the University
Office of Waste Management, which produces compost from dining commons food
waste and yard waste from the Amherst campus. Produce will be harvested and evaluated
for yield and nutrient densities at edible plant parts (e.g. fresh leaves). In addition,
whenever pathogen resulted damages or symptoms are observed under natural conditions
(no artificial inoculations), accurate identification of the disease will be carried out in
conjunction with Plant Disease Diagnostician and a vegetable pathologist (Dr. Robert
Wicks, Plant, Soil, and Insect Sciences) at the University of Massachusetts Amherst. The
disease severity per cultivar will be rated. The experimental design of the experiment
will be followed by complete random block design.
Experiment 4. Assessment of the genetic purity and genetic diversity among cultivars of
lettuce using molecular markers in greenhouse experiment.
8
This experiment is planned to determine the genetic purity and genetic diversity among
cultivars of lettuce crops using molecular markers. The experiment will be conducted in
the greenhouse using the same cultivars as used in the previous experiments. Standard
fertilizer will be used to grow the cultivars. Fresh tissue will be sampled in the middle of
the growth period for genomic analysis. Genomic DNA will be extracted from plant
tissues according to the procedures used in (Curley and Jung, 2004; Simko, 2008) and
(Sim et al., 2009). EST- and genomic-SSR markers of respective vegetables, these will be
selected from the most recent papers (Cavagnaro et al., 2009; Kong et al., 2006; Liu et
al., 2007b; Liu et al., 2007c; Richards et al., 2004; Simko, 2008; Yi et al., 2006) will be
screened against selected cultivars within the vegetable species and used for genetic
similarities. PCRs will be conducted in conditions as described in the respective papers.
DNA similarities among cultivars within each of species will be estimated. The
experimental design of the experiment will be followed by complete random block
design.
Statistical analysis
Plant heights, fresh weights, and dry weights data will be processed as analysis of
variance using the SAS (SAS 9.1.3, SAS Institute Inc., SAS Campus Drive, Cary, NC
27513) PROC GLM procedure. Mean separation will be conducted by F test and
Duncan’s New Multiple Range Test (P=0.05).
9
Total N and elemental analysis data will be analyzed using SAS PROC GLM procedure.
Polynomial comparison among cultivars will be performed by orthogonal polynomial test
using PROC GLM and PROC IML procedure.
.
Assessment of the relationship between nutrient density in crops and cultivar
selection and nutrient availability in the greenhouse.
Introduction
Mineral deficiency adversely affect a third of world’s population. Consequently,
micronutrient deficiencies affect global health and limit human productivity in recent
times, and deficiency of certain mineral nutrients like calcium, potassium, magnesium,
phosphorus, zinc, iron, copper, manganese and others are a substantial nutritional
problem in diets of human around the world (Darnton-Hill et al., 2005; Kataki and Babu,
2002). Nutrient content in vegetables and fruits reported declines of 5% to 40% or more
in some soils in the past 50 to 100 years in the United States (Davis, 2009). Reports
indicated the in the United Kingdom foods contents decreased by about 20% during this
time (Anonymous, 2005; Anonymous, 2006; Mayer, 1997). Study on the diets of subjects
in Philadelphia, PA warranted a downward revision in the intakes of iron, magnesium,
and vitamins (Guenther et al., 1994). Another study concluded that many Americans,
particularly men, social disadvantaged groups, and ethnic minorities are not meeting the
current recommendations for calcium intake through diet alone or with supplements (Ma
et al., 2007).
10
Davis (2009) suggests that the decline in mineral and protein content in fruits and
vegetables is due partly to a dilution effect of high yields. Side-by-side comparisons of
low- and high-yielding vegetables and grains showed negative correlations between yield
of produce and concentrations of minerals. However, a dilution effect of high yield on
nutrient concentration was not observed universally on potato (Solanum spp L.) and that
soil fertility affected the mineral nutrients more than the dilution effect (White et al.,
2009).
Micronutrient malnutrition is a growing concern all over the developing world having
consequence a mental retardation, impairments of the immune system, and overall poor
health (Cakmak, 2009).
Soil fertility problems associated with nutrient depletion by crop production are
worldwide, and also nutrient depletion can be caused to insufficient fertilizers use and
unbalanced fertilization (Tan et al., 2005). In United States, potassium and phosphorus
are being drawn in soils on a national basis at an increasing rate every year, and the
depletion has occurred for 40 years for potassium and for nearly 30 years for phosphorus
(Stewart, 2004). A study suggest prediction of nitrogen uptake for sustainable nitrate pool
of lettuce was depended on the water content (Seginer et al., 2004).
The objective of the study will be to determine if the mineral nutrient densities of
selected vegetable crops can be increase through cultivar selection.
Materials and Methods
Materials
11
Eighteen lettuce (Lactuca sativa) cultivars will be used in this experiment. Nine varieties
will be heritage and nine will be modern for this study. Three varieties (Table 1) of each
group (Butterhead, Romaine/Cos, and Loose leaf) will be chosen in heritage and modern
type of lettuce based on widely used and marketability. Then eighteen varieties (nine
heritages and nine moderns) of both groups will be used for the treatment. Three
fertilizer regimes will be used for this experiment.
Methods
Eighteen different cultivars of lettuce will be grown in the Bowditch greenhouse at
University of Massachusetts Amherst. Seeds of those cultivars were collected from
professional seed producers. Heritage type of seeds will be collected from certified
organic seeds producer (Seed of Change Seeds Co., Spicer, MN 56288) and modern type
of seeds will be collected from another successful seeds producer (Johnny Seeds Co.,
Winslow, ME 04901). All seeds of these cultivars will be planted in peat moss medium
on December 18, 2009 in the greenhouse at 2-3 seeds/cube. . For best germination of
seeds media temperature will be maintained of 68oF (20oC). After 32 days, lettuce
seedlings will be transferred to plastic containers (6 inch diameter) in a peat-based
medium (MetroMix 360, SunGro Horticulture, Vancouver, Canada).
Three different combinations (regimes) of fertilizers will be chosen for nutrient sources
of lettuce cultivars. With different cultivars different nutrient regimes will be tested if
nutrient content increases through cultivars selection. Three treatments will be Hoagland
#1 solution (complete), Jack’s Fertilizer (20-10-20) Peat-Lite (add CaSO4 to medium),
and organic Pure blend Pro Grow (3-1.5-4) for this experiment. The purpose of the
application of different fertilizer regimes is to investigate the nutrient content of the
12
lettuce through cultivar selection. The lettuce will be grown in greenhouse for about 7-9
weeks from seedlings to maturation.
Table 1. Varietal selection of each cultivar
Heritage variety (H)
Modern variety (M)
Butterhead (C1):
Butterhead (C1):
1. Buttercrunch (V1)
1. Adriana (V1)
2. Bronze Mignonette (V2)
2. Focea (V2)
3. Tom thumb (V3)
3. Australe (V3)
Romaine/Cos (C2):
1. Coastal Star (V1)
2. Cosmo-Savoy Leaf (V2)
3. Forellenschluss (V3)
Romaine/Cos (C2):
1. Red rosie (V1)
2. Claremont (V2)
3. Winter density (V3)
Loose leaf (C3):
1. Salad Bowl (V1)
2. Simpson black-seeded (V2)
3. Red deer tongue (V3)
Loose leaf (C3):
1. Tropicana (V1)
2. Simpson elite (V2)
3. Two star (V3)
Each regime will carry a fertilizer treatment with different combination. The first
fertilizer treatment contained of 210 ppm N, 31 ppm P, 234 ppm K, 200 ppm Ca, 48 ppm
Mg, 64 ppm S, and micronutrient composite and Fe chelate 1 ppm from a Hoagland’s
solution (Hoagland and Arnon, 1950). The second fertilizer treatment carried of using
3% N, 1.5% P, 4% K with trace minerals 1.0% Ca and 0.5% Mg from a 3-1.5-4 formula
organic fertilizer (Pure Blend Pro Grow, Botanicare, Tempe, AZ) and with this fertilizer
4 ml 0.5 M MgSO4/liter will be added for Mg and S. The third fertilizer treatment
consisted of 20 % N, 10 % P, 20 % K, 1.5 % Mg, 0.02 % B, 0.01 % Cu, 0.1 Fe, 0.05 %
13
Mn, 0.01 % Mo, and 0.05 % Zn from a Jack’s 20-10-10 Peat-Lite commercial fertilizers
(Jack’s Professional Fertilizers, J.R. Peters, Inc., Allentown, PA 18106) and with this
fertilizer I g CaSO4/pot will be mixed in media for Ca, and additionally 4 ml 0.5 M
MgSO4/liter will be added for Mg and S. The treatment of fertilizer regime will be
applied one time in one day interval at the amount 100 ml/plant for 1st 10 days after
transplanted of lettuce seedlings. Then amount of treatment of each regime then will be
increased at the rate 200 ml/plant for one day interval and continued at maturity. The
fertilizer regimes will be applied to a plant in every two days.
Media pH values will be measured in a water extract after shaking for 15 m (Hesse,
1971) using a pH meter (CRISON micro pH 2002). Media EC values will be determined
in a media/water extract after shaking for 15 m (Hesse, 1971) using a conductivity meter
(CRISON micro pH 2002).
At harvest, growth parameters including plant height, plant size and color, leaf size or
whole heads, leaf color, number of leaf, and fresh weights of leaf will be recorded.
Oven-dry weights of plant leaf and stem will be recorded afterwards.
Portions of dry plant sample of leaves will be ground to pass a 30-mesh screen and sub
sampled and stored in coin envelops for elemental analysis of N, P, K, Ca, Mg, Cu, Zn,
Fe, Mn and Cr accumulated by lettuce leaves. Kjeldhal N will be determined by aciddigestion and posterior determination by auto analyzer (BRAN+LUEBBE, method G188-97, BRAN+LUEBBE, Norderstedt, Germany). Pulverized samples (0.5 g) will be
ashed at 500oC for 12 h in a muffle furnace and the ash will be dissolved in 10% HCl
14
solution prepared in deionized H2O. For P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr, the ash
extracts will be analyzed by inductive coupled plasma (ICP) spectrophotometric analysis
(Jones Jr et al., 1991; Kalra, 1998) at UMass Soil and Plant Tissue Laboratory. Lettuce
growth parameters will be used for evaluating the consequences of different fertilizer
regimes and their combination on lettuce growth and nutrient management through
cultivars selection.
Pest will be managed by using certified organic or conventional herbicides, fungicides or
insecticides as required to bring about control. During the growing season, appearance of
diseases symptoms and occurrences will be carefully monitored and diseases severity will
be evaluated for each vegetables at a scale of 0-9 scale indicating 0 being no visible
symptoms and 9 being severe symptoms.
Summary
Assess the effect between mineral nutrient density in crops and cultivar selection by
elevating nutrient contents in the medium in which the plants grow.
Introduction
15
Mineral elements are required for human body to maintain good health. Plants are the
main sources for these elements. Fresh fruits and vegetables are high in vitamins but are
aften low in essential minerals (Ashmead, 1982). Plants like Brassica juncea cultivated in
hydrophonics condition contains high levels of nutritionally important minerals such as
Cr, Fe, Mn, Se, and Zn (Elless et al., 2000).
Critical functions in human body are mainly conducted by minerals. Calcium,
phosphorus, and magnesium are required for constructing and maintaining bones
(Anonymous, 1989). Diet with enough calcium helps in maintaining healthy bones and
reduces risk of osteoporosis (Anonymous, 2000). In addition, calcium is required in
blood clotting (Karll, 2000; Krause and Mahan, 1984), and phosphorus and magnesium
are essential in energy metabolism. Iron is a crucial part of hemoglobin and myoglobin
(Anonymous, 1989; Krause and Mahan, 1984), and is required for carrying oxygen in the
blood and muscles (Krause and Mahan, 1984). Anemia is caused by iron deficiency
(Krause and Mahan, 1984).
Deficiency of trace elements like zinc, copper, and selenium has a positive correlation
with risk of cancer (Abdulla and Gruber, 2000; Chan et al., 1998). A number of chronic
cardiovascular diseases are shown to be correlated with magnesium and selenium
(Brown and Arthur, 2007; Fox et al., 2001), and diabetes with zinc (Thompson and
Godin, 1995).
The results of the study suggest that it is possible to improve the dietary intake of local
dwelling elders by increasing consumption of fruits, vegetables and calcium-rich foods
(Bernstein et al., 2002). A study in vitro revealed that vegetables such as kale, celery,
collard, Chinese cabbage, and soybean sprout contained high levels of dialyzable calcium
16
(20-39%) compared to milk powder, which contains 25 mg Ca/100 g (Kamchan et al.). A
study on individuals age 65 and older reported that calcium is required for skeletal
maintenance and that supplemental intakes to a total in the range of 1300-1700 mg
Ca/day stop age-related bone loss and reduce fracture risk (Heaney, 2001).
Magnesium is the fourth most abundant cation in the body. It is believed that more than
300 enzymatic reactions are involved with magnesium. The recommended dietary intakes
(RDI) for magnesium are about 30 mg/day for adults. Though magnesium deficiency is
rare in healthy people, but diabetes, kidney diseases, hypertension, and coronary artery
diseases may induce magnesium depletion (Wahlqvist and Darmadi-Blackberry, 2002).
Green vegetables, some legumes (beans and peas), nuts and seeds, and whole and
unrefined grains are good sources of magnesium (USD-ARS, 2003). Eating lot of green
vegetables, whole grains and legumes will help meet your dietary need for
magnesium(USD-ARS, 2003).
Dark green leafy vegetables are good sources of iron, especially sixty percent of nonheme irons are found in fruits, vegetables, grain, and nut. Recommended dietary intakes
of iron are 10 mg/day for a child, 12 mg/day for a man, and 15 mg/day for a woman
(Baynes and Bothwell, 1990).
An experiment conducted on butter head lettuce reported that plants grown with high Fe
levels (10 mg/L) had notable tip burn incidence but no bolting was observed (Chow et al.,
1999). The results also indicated that high Fe could be beneficial in producing desired
head size. In accordance with another experiment revealed tip burn increased with
nitrogen supply and was related to an increase in head size (Brumm and Schenk, 1992).
17
Fresh leafy vegetables are major sources of minerals for recommended dietary intakes in
the recent times. Lettuce has been an important vegetable crop with high market value,
and its nutritional characteristics have been studied throughout the world (Ashkar and
Ries, 1970; Chow et al., 1999).
The objective of the experiment will be to determine if the nutrient densities of selected
vegetable crops can be increased through increasing the nutrient contents in the medium
in which the plants grow.
Materials and Methods
Materials
Six lettuce cultivars will be used for this experiment. The six cultivars will be taken from
the eighteen cultivars based on nutritional requirement and growth observes in the
experiment above. Three cultivars of each group will be selected as modern and heritage.
Two fertilizer regimes with elevating concentration will be selected for this study. One
fertilizer regime will be chemical fertilizer, and another regime will be organic fertilizer
with varying nutrient concentration in solution.
Methods
The seedlings of lettuce cultivars will be grown in the Bowditch greenhouse at UMass
Amherst. Seeds of those cultivars will be collected from the professional seeds growers.
Heritage type of seeds will be collected from certified organic seeds producer (Seed of
Change Seeds Co., Spicer, MN 56288) and modern type of seeds were collected from
another successful seeds producer (Johnny Seeds Co., Winslow, ME 04901). All seeds
18
will be planted in peat moss medium for raising seedlings. For best germination rate, the
optimum temperature will be maintained of 68oF (20oC). After four weeks from seeding,
same sized seedlings will be transplanted to the 6̋˝ round pot filled with peat moss media
(MetroMix 360, SunGro Horticulture, Vancouver, Canada).
Two different fertilizer regimes will be selected for nutrient sources of lettuce cultivars.
The elevating fertilizer regimes will be tested if the nutrient densities of the lettuce crop
increases through cultivar selection and nutrient management. Two fertilizer regime will
be Jack’s 20-10-10 Peat-Lite commercial fertilizers (Jack’s Professional Fertilizers, J.R.
Peters, Inc., Allentown, PA) and organic fertilizer (Pure Blend Pro Grow 3-1.5-4,
Botanicare, Tempe, AZ) with varying concentration of Ca for this experiment. The goal
of the application of different fertilizer regimes with varying concentration is to
determine the nutrient densities of the lettuce through cultivar selection and nutrient
management. The lettuces will be grown in the greenhouse for about 4-5 weeks from
transplanting to marketable size.
Each regime of fertilizer will be divided into three treatment groups based on varying
internal nutrient concentration. The first fertilizer regime will be contained N, P, K,
and micronutrient composite from a Jack’s 20-10-10 Peat-Lite commercial fertilizers
with varying Ca (200, 300, 400 ppm) as CaCl2. Another fertilizer regime will be obtained
from organic fertilizer with varying amount of Ca (200, 300, 400 ppm) as CaCl2. Three
treatments of each group of fertilizer regime will be tested for this study. Therefore six
treatments will be selected in two groups of fertilizer regimes. The treatment will be
applied in one day interval at 100 ml/plant for first week then at 200 ml/plant until
harvest.
19
At harvest, growth parameters including plant height, plant size and color, and fresh
weights of plant will be recorded. Oven-dry weights of plant will be recorded afterwards.
Portions of dry plant sample of leaves will be ground to pass a 30-mesh screen and sub
sampled and stored in plastic containers for elemental analysis of N, P, K, Ca, Mg, Cu,
Zn, Fe, Mn and Cr accumulated by lettuce leaves. Kjeldhal N will be determined by
acid-digestion and posterior determination by auto analyzer (BRAN+LUEBBE, method
G-188-97, BRAN+LUEBBE, Norderstedt, Germany). Pulverized samples (0.5 g) will be
ashed at 500oC for 12 h in a muffle furnace and the ash will be dissolved in 10% HCl
solution prepared in deionized H2O. For P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr, the ash
extracts will be analyzed by inductive coupled plasma (ICP) spectrophotometric analysis
(Jones Jr et al., 1991; Kalra, 1998) at UMass Soil and Plant Tissue Laboratory. Lettuce
growth parameters will be used for evaluating the consequences of different fertilizer
regimes and their combination on lettuce growth and nutrient management through
cultivars selection.
Pest will be managed by using certified organic or conventional herbicides, fungicides or
insecticides as required to bring about control. During the growing season, appearance of
diseases symptoms and occurrences will be carefully monitored and diseases severity will
be evaluated for each vegetables at a scale of 0-9 scale indicating 0 being no visible
symptoms and 9 being severe symptoms.
20
Assess the effect of organic and conventional soil fertility practices on mineral
accumulation in vegetables in field experiment.
Introduction
Organic farming differs from conventional farming mainly tillage methods, crop rotation,
fertilizer applications, and pest control methods. Organically grown vegetables have
greater market values than conventional farms. Synthetic chemical fertilizers and
pesticides uses in intensive agriculture may cause serious impact on public health and the
environment (Pimentel et al., 2005). Integrated pest and nutrient management systems
21
and certified organic agriculture may reduce reliance on agrochemical inputs as well as
make agriculture environmentally and economically sound (Pimentel et al., 2005).
Applications of compost in vegetable crop production systems have been shown better
yield and fruit quality responses a compared with those obtained from standard vegetable
crop commercial practices (Stoffella et al., 2001). The impact of organic production
practices on soil quality revealed that there was 22% more organic carbon (1257 kg/ha)
and 20% more total N (970 kg/ha) on organic farm than conventional farm (Liebig et al.,
1999). The findings of the study also reported that soils of organic farms had soil pH
closer to neutral, lower bulk density, and higher available water-holding capacity,
microbial biomass C and N, and soil respiration compared with conventional farms
(Liebig et al., 1999). Ultimately, organic farming resulted better in maintaining soil
productivity and reducing soil erosion than conventional farming (Reganold et al., 1987).
It has been believed that by applying good organic composts supplemented with lower
amount of chemical fertilizers, high crop production and maintenance, or even increasing
soil fertility can be achieved.
The objective of the study will be to determine if the nutrient densities of selected
vegetable crops can be increased through soil fertility practices (e.g., organic vs
conventional fertilizers; different fertilizer used).
Materials and Methods
Materials
Eighteen different lettuce cultivars will be used in this study. Nine heritage and nine
modern cultivars will be used for this experiment. Two regimes of organic fertilizer and
22
one conventional fertilizer regime will be used for this study. One kind of organic
fertilizer regime will be obtained from individual nutrients sources providing nitrogen,
phosphorus, and potassium requirements of crops; and other kind of organic fertilizer
regime will be obtained from compost source providing those nutrients. Fertilizer for
individual nutrients will be included soybean meal for nitrogen, colloidal rock phosphate
for the phosphorus, mined potassium sulfate for the potassium. These materials are used
commonly by organic growers in the region. The compost will be collected from the
University of Massachusetts Amherst office of waste management, which produce
compost from dinning commons food waste and yard waste from the Amherst campus.
Conventional fertilizer will be obtained from a commercial fertilizers (10-10-10 all
purpose) providing nutrient requirements of crops.
Methods
The experiment will be conducted at UMass field experiment station. Eighteen different
cultivars of lettuce will be grown in the field at UMass Amherst field experiment station.
Seeds of those cultivars will be collected from the professional seed producers. Heritage
type of seeds will be collected from certified organic seeds producer (Seed of Change
Seeds Co., Spicer, MN) and modern type of seeds will be collected from another
successful seeds producer (Johnny Seeds Co., Winslow, ME). Both types of seed will be
planted in mixed soil (soil with peat moss 1:1) at Bowditch greenhouse. For best
23
germination of seeds soil temperature will be maintained of 68oF (20oC). At 4 weeks of
age tomato seedlings will be transplanted in UMass field experiment station.
Therefore, three different regimes of fertilizer will be chosen for this experiment. Two
will be organic fertilizer regimes and one convention fertilizer regime. One group of
organic regime will be obtained from some individual sources mentioned above, another
group of organic regime will be obtained from compost sources collected from UMass
Amherst office of waste management, and a conventional fertilizer regime will be
obtained from a 10-10-10 all purpose commercial fertilizer. The compost will be
analyzed for its nutrients contents before application. The reason for the use of different
fertility regimes is to find out the higher nutrient densities in plants, growth and soil
fertility management practices. The fertilizer will be applied based on the nutrient
requirement of the crops according to the management guide (Rosen and Eliason, 2005).
The organic fertilizer regime will be applied at the rate of 20 ton/acre during soil
preparation in one time as basal dose. The conventional fertilizer regime will be applied
at the rate of 100 Ib/acre before transplant in one time as basal dose. In addition, 100 Ib
CaSO4 /acre will be added during land preparation for calcium.
Soil samples will be taken after plant harvest for N, P, K, Mg, and Ca measurement to
determine the effect of the treatment on soil nutrient availability. A soil testing method
proposed by Morgan will be used to extract the soil available nutrients (Morgan, 1941).
Nitrogen will be assessed distillation and titration of NH3 in solution (Bradstreet, 1965);
P will be determined by colorimetry (blue molybdophosphoric acid method) (Olsen and
24
Dean, 1965); Ca and Mg will be determined by atomic absorption spectrometry; and K
by atomic emission spectrometry (Lierop, 1976; Thomas et al., 1967).
At harvest, growth indices including plant height, plant size, leaf color, leaf sizes, and
fresh weights of whole plant will be recorded. Oven dry weights of whole plants will be
recorded afterwards.
Dry leaves sample will be grounded to pass a 30-mesh screen for tissue nutrient analysis
of N, P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr. Kjeldhal N will be determined by aciddigestion and posterior determination by auto analyzer (Bran+luebbe method G-188-97,
Bran+luebbe, Norderstedt, Germany). Pulverized samples (0.5 g) will be ashed at 500oC
for 12 h in a muffle furnace and the ash will be dissolved in 10% HCl solution prepared
in deionized H2O. For P, K, Ca, Mg, Cu, Zn, Fe, Mn and Cr, the ash extracts will be
analyzed by inductive coupled plasma (ICP) spectrophotometric analysis (Jones Jr et al.,
1991; Kalra, 1998) at UMass Soil and Plant Tissue Laboratory. Lettuce growth
parameters will be used for evaluating the consequences of different fertilizer regimes
and their treatment on nutrient management through cultivars selection and soil fertility.
Pest and disease symptoms will be managed by using certified organic herbicides and
fungicides or insecticides to bring about control. The severity of diseases will be
evaluated using a scale of 0-9 indicating 0 being no visible symptoms and 9 being severe
symptoms.
Assessment of the genetic purity and genetic diversity among cultivars of lettuce
using molecular markers in the greenhouse experimentation.
Introduction
25
Molecular markers are having uses as an effective tool for efficient selection of desired
agronomic traits. Molecular markers can facilitate tomato breeding by means of marker
assisted selection (MAS) to improve agronomical important characteristics such as yield,
fruit quality, and disease resistance. Molecular markers such as RFLP (Restriction
fragment length polymorphism) (Ooijen et al., 1994; Sandbrink et al., 1995), RAPD
(Random amplification of polymorphic DNA) (Qian et al., 2001; Stevens et al., 1995),
ISSR (Inter simple sequence repeat) (Joshi et al., 2000; Zietkiewicz et al., 1994), AFLP
( Amplified fragment length polymorphism) (Vos et al., 1995), and Microsatellite
polymorphism (Panaud et al., 1996) have been developed in tomato and other crops since
last decade. However, RFLP uses breeding purposes is limited because it requires the use
of radioactivity and is laborious. RAPD, ISSR and AFLP markers either identify only
dominant alleles or are sensitive to PCR amplification conditions.
Microsatellites or Simple Sequence Repeats (SSRs) are short (mostly 2-4 bp) tandem
repeats of DNA sequences. The variation or polymorphism of SSRs are a result of
polymerase slippage during DNA replication (Levinson and Gutman, 1987). SSR
markers are becoming the preferred molecular markers in crop breeding because of their
properties of genetic co-dominance, high reproducibility and multi allelic variation. They
are the most practical markers for genomic mapping, variety identification and markerassisted selection. In lettuce, some microsatellite markers have been used
(Areshchenkova and Ganal, 1999; Smulders et al., 1997), but the number of SSR markers
are available for molecular breeding is still few and only a limited number of SSR
26
markers have been mapped to the tomato genome (Areshchenkova and Ganal, 1999;
Broun and Tanksley, 1996).
The objectives of the present study will be to assess the genetic similarities, variation, and
purity among Lactuca sativa accessions in the USDA collection; and to develop and
characterize the more SSRs markers for lettuce.
Materials and Methods
Materials
Plant material
Eighteen lettuce cultivars will be used for this experiment. Nine will be modern and nine
will be heritage lettuce cultivars. The reason for choosing these plant cultivars for
assessing the similarities and variation among cultivars are using in the previous
experiment. All cultivars and its parental lines will be collected from the U.S. National
Plant Germplasm System (NPGS) at North Central Region Plant Introduction Station in
Ames, IA, USA; and heritage cultivars line will be collected from a commercial source
(Seed of Change Seeds Co. Inc, Spicer, MN). All purposes commercial fertilizer of
peters professional 20-20-20 TE formula will be used to grow lettuce. The rate of the
fertilizer will be used at1 g/L, and additional CaSO4 will be added in the media at 1g/pot
for Ca.
Methods
Genomic DNA Isolation
27
The heritage and modern seeds of the cultivars will be grown in the greenhouse with a
day temperature of 24 ± 3oC and a night temperature of 18 ± 3oC in the Bowditch
greenhouse at University of Massachusetts Amherst. One-month-old seedlings will be
transplanted to the pots where individual of each cultivar will randomly be selected and
numbered. Regular irrigation, fertilization, staking and crop protection measures will be
adopted and purity visual evaluation will be conducted based on the main important
morphological characters throughout the growth period. Total genomic DNA of the 18
lines will be isolated from young leaves following the method described by (Liu et al.,
2003; Yu and Pauls, 1994) with some modifications. Leaves will be collected and
immediately frozen in liquid nitrogen and stored at -70°C until use. For each sample, four
fresh leaf obtained by punching leaves with the cap of a 1.5-ml Eppendorf tube, will put
into 400 μl of DNA extraction buffer (200 mM Tris–HCl, pH 7.4, 250 mM of NaCl, 25
mM of EDTA, pH 8.0, 0.5% SDS) and homogenized with a plastic pestle (Mandel
Scientific Company Ltd.). Then 400 μl of 24:1 chloroform/isopropyl alcohol will be
added to the homogenized solution, vortexed and left at room temperature for 30 min.
The homogenate will spun in a micro centrifuge at a speed of 10,500 rpm for 2 min and
350 μl of the supernatant were transferred into a new Eppendorf tube. For DNA
precipitation, an equal volume (350 μl) of isopropanol will be added to the tube that will
left at room temperature for 5 min and then spun at 11,000 rpm for 5 min. Then, the DNA
pellet will be air-dried at room temperature for 30 to 60 min before it will be dissolved in
200 μl of water at 4 °C overnight. The supernatant will be collected after micro
centrifugation at 1,300 rpm for 2 min, yielding about 25 ng/μl of DNA.
28
Search of DNA sequences and primer design
A list of about 1,000 asteraceae microsatellites (the majority were L. sativa) showing the
GenBank database accession numbers with their motifs, and the number of repeats will
kindly provided by Andreas Matern, Cornell University, Ithaca, New York. The entire
DNA sequence for each accession is searched, retrieved from the GenBank database and
verified for the presence of SSRs. If the SSR is not at, or very close to, either the 5′ or 3′
end, the sequence is collected. Prior to primer design, all the saved DNA sequences will
be analyzed using the program DNASIS (Hitachi America Ltd., San Bruno, Cal.) for
homologous sequences. Each sequence will be compared with the rest of the DNA
sequences. If homologous sequences are found, only one unique sequence will kept for
primer design while the rest of the homologous DNA sequences will be eliminated
because of their redundancy. PCR primers (forward and reverse) flanking the repeat
sequence will be designed using the computer program GENE RUNNER (Hastings
Software, Inc., N.Y.). The core parameters used in the primer design include the
following: (1) the primer length is between 18 bp and 25 bp, (2) the percentage of GC is
between 35% and 60%, (3) the Tm of the primers is over 40 °C which will be calculated
using Tm = 59.9 + 0.41 (%G+C) – (675/primer length) based on the standard PCR
conditions at a salt concentration of 50 mM (Sharrocks, 1994), and (4) the predicted PCR
products range from 100 to 350 bp in length with a preference of between 100 bp and 250
bp. In addition, the primer internal structures, such as hairpin loops, possible primer
dimers, length of single base pair run at the 3′ end and the number of short repeats (such
as CT, GA etc.) will also be taken into consideration. When two or more SSRs are
located in the same DNA sequence but are at different sites, two flanking primers are
29
designed separately for each of the SSRs. All designed oligonucleotides will be
synthesized commercially by Sigmagenosys, Incorporated.
SSR primers will be obtained from Operon Technologies Inc. (Alameda, CA) and tested.
Single DNA molecular marker system, SSR, will be used to test seed genetic purity. SSR
primers will be obtained from OperonTechnologies Inc., SSR-PCR will be performed in
PTC-100 thermocycler (MJ Research Inc., USA) according to the protocol of (Williams
et al., 1990) and (Zietkiewicz et al., 1994) respectively. The SSR amplification products
will be separated and detected according to the reported methods (Liu et al., 2007a).
The oligonucleotide primers for SSR analysis will be synthesized by Invitrogen Biotech
(Shanghai) Co., China, according to the reported sequences of microsatellite markers (He
et al., 2003). The SSR-PCR reactions will be performed and products will be detected
according to the reported protocol (Liu et al., 2007a) .
PCR amplification and product electrophoresis
PCR reactions will be performed in 96-well plates using either the Perkin Elmer
GeneAmp PCR system 9600 (PE Biosystems) or the TECHNE Genius themal cycler
(Techne Ltd., U.K.) with the same amplification program. Each 10-μl reaction mixture
contained about 25 ng of lettuce genomic DNA, 0.3 μM of forward and reverse primers,
300 μM of each dNTP, 1 μl of 10 × PCR buffer containing 100 mM of Tris–HCl, pH 8.3,
500 mM of KCl, and 1 unit of Taq DNA polymerase. The PCR amplification conditions
will be programmed as one cycle of denaturation at 94 °C for 2 min, followedby 35-
30
cycles amplification with a 25 s denaturing at 94 °C, a 25 s annealing at the Tm (Tm
varies for the individual primers) and a 25 s extension at 68 °C. After PCR amplication,
the products will be mixed with 3 μl of stop buffer (97% deionized formamide, 0.3%
each bromophenol blue and xylene cyanol FF and 10 mM of EDTA, pH 8.0) and then
denatured at 94 °C for 5 min in a PCR machine. Four micro litres of each denatured PCR
product will be used for fragment separation on a DNA sequencing gel
(6% polyacrylamide, 8 M urea and 1 × TBE buffer) running at a constant power of 55 W
for 2–2.5 h, using an S2 sequencing-gel apparatus (GIBCO BRL). A 1-kb-plus DNA size
marker will also be loaded along with the samples for each run to estimate the fragment
sizes of the separated DNA fragments. After each run, the gel was placed in 10% glacial
acetic-acid fixation solution for 20 min with gentle shaking, silver-stained for 30 min and
then immediately developed in a 3% sodium carbonate solution according to the DNA
silver-staining kit (Promega).
Nomenclature of SSR markers
The nomenclature of the SSR markers will be based on the method described by (Yu et
al., 2000). The SSR name will be prefixed with LS standing for L. sativa, followed by the
repeat motif in lowercase and a number starting from 001 for each distinct repeat motif.
For example, LSaat001 and LSaat002 re-present, respectively, the SSR markers at two
different loci with the same repeat motif “aat”. For the imperfect or compound repeats,
such as (AAG)3T(TGA)7, only the motif with the highest repeat number, in this case
TGA, is used. When two or more different repeats such as the SSR locus
31
(CT)12(GATA)12(AT)2(AC)10 have the same number of repeats, the repeat motif at the
5′ end is used. Thus, the SSR name for (CT)12(GATA)12(AT)2(AC)10 is designated as
LSct rather than LSgata. This SSR nomenclature system can be applied to any newly
developed microsatellites and provides a simple way to track SSR loci for use in a
breeding program.
Genetic analysis
All 18 genotypes from different geographic origins will be used to screen the SSR
primers for PCR amplification and product-length polymorphism. For primers that
produced the expected fragments after PCR reactions, the number of alleles will be
recorded and the polymorphism information content (PIC) of an SSR locus will be
calculated as described by (Saal and Wricke, 1999):
𝐾
PIC= 1-∑𝑖=1 𝑝𝑖 2
where pi is the frequency of the ith allele out of the total number of alleles at an SSR
locus, and k is the total number of different alleles for that locus. For phylogenetic
analysis, only the data for the polymorphic SSR loci are entered for all DNA samples,
and a “1” or “0” is used if an allele is present or absent for a genotype, respectively. The
data will be analyzed using the computer program TREECON (Van de Peer and De
Wachter 1994). The genetic-distance estimation will be based on the method described
by (Nei and Li, 1979). All 18 different lettuce genotypes will be clustered based on the
estimated genetic distance, and the phylogenetic tree topology was inferred with the
clustering method of the Unweighted Pair Group Method Using Arithmetic Average
(UPGMA).
32
Summary
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