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Physiological Studies of the Halophyte Salicorniabigelovii: A Potential Food and Biofuel Crop forIntegrated Aquaculture-Agriculture Systems
Item Type text; Electronic Dissertation
Authors Martinez Garcia, Rafael
Publisher The University of Arizona.
Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.
Download date 23/05/2018 22:57:27
Link to Item http://hdl.handle.net/10150/193968
PHYSIOLOGICAL STUDIES OF THE HALOPHYTE Salicornia bigelovii: A
POTENTIAL FOOD AND BIOFUEL CROP FOR INTEGRATED AQUACULTURE-
AGRICULTURE SYSTEMS
by
Rafael Martinez-Garcia
_____________________
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF SOIL WATER AND ENVIRONMENTAL SCIENCE
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2010
2
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Rafael Martinez-Garcia
entitled PHYSIOLOGICAL STUDIES OF THE HALOPHYTE Salicornia bigelovii: A
POTENTIAL FOOD AND BIOFUEL CROP FOR INTEGRATED AQUACULTURE-
AGRICULTURE SYSTEMS
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy
_____________________________________________________ Date: 9/17/2010
Dr. Kevin Fitzsimmons
_____________________________________________________ Date: 9/17/2010
Dr. Edward Glenn
_____________________________________________________ Date: 9/17/2010
Dr. Stephen Nelson
Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement.
________________________________________________ Date: 9/17/2010
Dissertation Director: Dr. Kevin Fitzsimmons
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an
advanced degree at the University of Arizona and is deposited in the University Library
to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided
that accurate acknowledgment of source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by
the head of the major department or the Dean of the Graduate College when in his or her
judgment the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.
SIGNED: Rafael Martinez-Garcia
4
ACKNOWLEDGEMENTS
I would like to thank Dr. Kevin Fitzsimmons for giving me the opportunity to become his
student and be part of his working team as well as his guidance throughout my studies. I
also like to thank Dr. Stephen Nelson key to the achievement of this dissertation, for
giving me the opportunity to work with him in his research projects and also for all his
mentorship and valuable comments for the development of this dissertation. Also, I want
to thank Dr. Edward Glenn for his valuable comments and guidance. Thanks to the
CONACYT (Science and Technology National Council of Mexico) for all the valuable
support throughout the achievement of my degree as well as the USAID/ AQUAFISH
CRSP, NASA, and OASE Foundation for support to complete my research projects.
Thanks to my laboratory partner and friends Dr. Pablo Alanis, Dr. Mario Hernandez,
Brendan, David Moore, Naim, Cesar and Tekie for their friendship and help. Also I
would like to thank the staff at the ERL and SWES department. They were very nice and
helpful throughout my studies. And finally many thanks to my parents, siblings and my
love Ana, they have been the source of my energy to get to this point.
The AquaFish CRSP grant at the University of Arizona is funded in part by United States
Agency for International Development (USAID) Cooperative Agreement No. EPP-A-00-
06-00012-00 and by US and Host Country partners.
5
TABLE OF CONTENTS
LIST OF FIGURES .………………………………………………………………… 7
LIST OF TABLES .…………………………………………………………………... 8
ABSTRACT ……..………………………………………………………………..….. 9
INTRODUCTION ………………………………………………………………..…. 11
Seawater Farming …………………………………………………………....11
Halophytes ……………………………………………………………….…. 13
Salicornia bigelovii …………………………………………………….…… 16
Salt and drought tolerance of Salicornia bigelovii ……………………….…. 17
Bioenergy crops ……………………………………………………………… 20
CHAPTER ONE
SALINITY TOLERANCE AND CATION UPTAKE OF Salicornia bigelovii
(CHENOPODIACEAE) IN DRYING SOILS ….…………………….……………. 23
Abstract ……………………………………………………………..……… 24
Introduction ………………………………………………………..……….. 25
Materials and methods …………………………………………..…………. 27
Germplasm ………………………………………………..……….. 27
Greenhouse growth experiment …………………………..……….. 28
Results ………………………………………………………...……………. 31
Survival and growth …………………………………………………31
Water consumption ………………………………………………….33
Cation contents …………………………………………………….. 36
Osmotic adjustment ………………………………………………... 37
Discussion …………….……………………………………………………. 38
Salicornia bigelovii requires Na+ for optimal performance
on drying soils …………………………………….……………..… 38
Use of Na+
for osmotic adjustment ……………….……………….. 39
Drought and salt tolerance ………………………….………….….. 39
6
CHAPTER TWO
BIOMASS PRODUCTION, SEED YIELD, AND TISSUE OSMOLARITY OF
Salicornia bigelovii Torr. (CHENOPODIACAE) IN RELATION TO IRRIGATION
SALINITY …………………………………………………………………………. 41
Abstract ………………………………………..…………………………… 42
Introduction ………………………………….…………………………...… 43
Materials and methods …………………………………………………….. . 46
Germplasm selection ………………………………………………. 46
Greenhouse growth experiments ………………………………….. 47
Effect of irrigation salinity ………………………………………….. 48
Statistics …………………………………………………………….. 52
Results ……………………………………………………………………… 52
Biomass production ………………………………………………… 52
Seed analysis and yield ……………………………………………... 53
Shoot tissue osmolarity …………………………………………….. 56
Discussion ……………………………………..…………………………… 57
Biomass production in relation to irrigation salinity ………………. 57
Osmoregulation ……………………………………………………. 59
Seed production …………………………………………………......60
CONCLUSIONS AND RECOMMENDATIONS ………………………………… 61
REFERENCES ………………………………………………………………….……63
7
LIST OF FIGURES
Figure 1.1 Water use and growth parameters of Salicornia bigelovii plants grown along
a salinity gradient in drying soils, showing water use per pot………………………… 32
Figure 1.2. Cation content of Salicornia shoots grown along a salinity gradient in drying
soils. Results are single analyses of shoot tissues pooled across replicates ………… 35
Figure 2.1. Picture of the growth of Salicornia bigelovii irrigated with three different
salinities (5, 15 and 30 ppt of NaCl) ………………………………………………… 47
Figure 2.2. Salicornia bigelovii irrigated with three salinities (5, 15 and 30ppt of NaCl) in
the green house of the Environmental Research Laboratory ……………… 50
Figure 2.3. Procedures to obtain dry weight and seed cleaning of Salicornia bigelovii
growth experiment at the Environmental Research laboratory………………………. 51
Figure 2.4. Final dry mass (g) of Salicornia bigelovii plants grown in 2-L- pots with
irrigation salinites of either 5, 15, or 30ppt …………………………………………. 53
Figure 2.5. Mean dry mass (g) of individual seeds from two varieties (one from Texas
and one from Florida) of Salicornia bigelovii plants grown in pots irrigated with saline
water at either 5, 15, or 30ppt ………………………………………………………. 54
Figure 2.6. Seed yield (grams) per plant in greenhouse trials with two varieties (one from
Florida and one from Texas) of Salicornia bigelovii grown along a salinity gradient .. 55
Figure 2.7. The color and size of Salicornia bigelovii seeds from two lines (Texas and
Florida) irrigated with three different salinities ( 5, 15 and 30ppt) …………………. 56
Figure 2.8. Shoots tissue osmolarity (mM kg-1
) of Salicornia bigelovii plants grown at
three different salinity irrigation (5, 15 and 30ppt) …………………………………. 57
8
LIST OF TABLES
Table 1.1 Characteristics of halophyte plants …………………………………………. 14
Table 1.2. Analyses of variance (ANOVA) of Salicornia bigelovii growth parameters in
drying, salinized soils. The data were analyzed as one-way ANOVAs with salinity as the
categorical variable ……………………………………………………………………. 34
Table 1.3. Analyses of variance (ANOVA) of Salicornia shoot tissues for plants grown
in drying salinized soils. Samples were pooled across replicates and results were
analyzed as one-way unreplicated ANOVAs with salinity as independent variables . ..36
Table 1.4. Percent contribution of individual cations to the calculated osmolarity of
shoots of Salicornia bigelovii grown at different salinities in drying soil ……………. 37
9
ABSTRACT
Numerous studies over the past thirty years have demonstrated the technical
feasibility of using seawater and other saline water supplies for irrigation. Through the
use of saline water for irrigation, highly salt-tolerant crops derived from wild halophytes
could greatly increase global water and land resources available for agriculture. Large
supplies of brackish water and seawater from different sources are available in areas
suitable for production of salt-tolerant crops to grow. Dwarf glasswort Salicornia
bigelovii Torr. (Chenopodiaceae), is a leafless, succulent, small-seeded, annual saltmarsh
plant, with demonstrated potential as a saline water crop for coastal deserts. Currently, S.
bigelovii is commercially cultivated as a minor specialty vegetable for U.S. and European
fresh produce markets. It is also a potential oilseed, forage, biomass crop, and a
promising carbon sequestration plant. In the first chapter of this document we describe a
study where we grew Salicornia bigelovii from seedlings, of plants originating along the
Texas coast, in saline, drying soils in a greenhouse experiment. The effects of drought
and salinity stress were additive. Optimal growth and water use efficiency coincided at
0.35-0.53 M NaCl. The plants were tolerant of high salinity but exhibited little drought
tolerance. Salicornia bigelovii plants varied little in their uptake of Na+ for osmotic
adjustment, with final Na+ contents of 18% on a dry mass basis. Both growth and water
use efficiency of Salicornia bigelovii were affected by salinity. Also, Na+,
the primary
cation involved in osmotic adjustment of this species, apparently stimulates growth by
mechanisms apart from its role as an osmoticum. In the second chapter of this
dissertation we developed a research study where we evaluated the production and
10
osmotic adjustment of two S. bigelovii Torr. (Chenopodiaceae) lines (Texas and Florida),
plants were grown in pot in a green house at the Environmental Research Laboratory,
Tucson, AZ and irrigated with water treated with three different levels of NaCl (5 ppt, 15
ppt and 30 ppt) combined with inorganic fertilizer. Salicornia bigelovii plants from Texas
and Florida lines were started from seeds from the Environmental Research Laboratory
seed collection. At the end of the experiment sixty plants from each line were measured
for height, biomass, seed yield, seed size, dry matter yield, and tissue osmolarity. The
experiment lasted 16 weeks. There was no significant difference among groups in plant
height, or final biomass either in salinity irrigation treatments, or S. bigelovii lines. Tissue
osmolarity differed among salinity treatments but not among S. bigelovii lines. The
highest tissue osmolarity value was 1192 mM kg-1
found at the treatment 30ppt in the
Florida line. Total biomass production was 12 000 kg/Ha.
11
INTRODUCTION
Seawater farming
Declining land and water resources leave half the world’s population threatened
by a shortage of food and fuel; and this has led researchers to turn to coastal deserts,
hitherto seen as barren wastelands, to provide food and biofuel via seawater agriculture.
Seawater agriculture is defined as growing salt-tolerant crops on land using water
pumped from the ocean for irrigation. Unlike freshwater there is no shortage of seawater:
97 percent of the water on earth is in the oceans. Desert land is also plentiful: 43 percent
of the earth’s total land surface is arid or semiarid, although only a small fraction is close
enough to the sea to make seawater farming feasible. It is estimated that 15 percent of
undeveloped land in the world’s coastal and inland salt deserts could be suitable for
growing crops using saltwater agriculture. This potentially amounts to 130 million
hectares of new cropland that could be brought into agricultural production -without
cutting down forests or diverting scarce freshwater resources for development. Seawater
farming involves taking water and nutrients from the sea and to build what is possibly the
most important thing we have destroyed-the productive soils of our lands. In the process
of doing this, we can create carbon fixation within the soil that will allow us to balance
the continuous flux of carbon into the atmosphere. (Boyko, 1969; Hodges et al., 1993;
Glenn et al., 1998).
12
Seawater agriculture must fulfill two requirements to be cost-effective. First, it
must produce crops at yields high enough to justify the expense of pumping irrigation
water from the sea. Second, researchers must develop agronomic techniques for growing
seawater-irrigated, salt-tolerant crops in a sustainable manner (Glenn et al., 1998).
Approaches to the development of seawater agriculture have taken two directions. The
first, some investigators have attempted to breed salt tolerance into conventional crops,
such as barley and wheat. The other approach has been to domesticate wild, salt-tolerant
plants, called halophytes, for use as food, forage and oilseed crops (Glenn et al., 1998).
Numerous studies over the past thirty years have demonstrated the technical
feasibility of using seawater and other saline water supplies for irrigation. Guidelines for
managing salinity have been developed for conventional crops ( Ayars et al., 2006), and
these have been extended to halophytes irrigated with higher salinity water (Watson and
O'Leary, 1993; Glenn et al., 1997, 1998a, 1999; Brown and Glenn, 1999; Noaman and
El-Haddad, 2000; El-Haddad and Noaman, 2001). It has been shown that halophytes can
maintain productivity at salinities up to 60-80 g/L in their root zone (approximately twice
seawater salinity) (Glenn et al., 1997; 1999). Yields of 10-20 ton/ha of dry biomass and 2
ton/ha of seed have been produced by halophytes irrigated with hypersaline seawater (40
g/L) in small-plot trials in a coastal desert environment (Glenn and O'Leary, 1985; Glenn
et al., 1991a).
13
Halophytes
Saline habitats occur along bodies of saltwater, e.g., coastal salt marshes, and also
inland within high-evaporation basins, inland saline lakes, and lowlands of dryland and
desert topography. The electrolytes sodium (a cation) and chloride (an anion) are
extremely toxic to most plants at relatively low soil water concentrations, due to
deleterious effects on cellular metabolism and ultrastructure.
Halophytes belong to two primary families: Chenopodiaceae and Poaceae. They
are plant species that naturally grow in areas of increased salinity in the root area, such as
in saline semi-deserts, mangrove swamps, marshes and sloughs, and seashores (Table 1).
Relatively few plants are halophytes - perhaps only 2% of all plant species. The large
majority of plants are "glycophytes", and are damaged fairly easily by salinity (Watson
and O'Leary, 1993; Glenn et al., 1999).
14
Table 1.1 Characteristics of Halophytes plants
True halophytes are plants that thrive when given water having greater than 0.5% NaCl
and other salts. A small number of plant lineages have evolved structural, phenological,
physiological, and biochemical mechanisms for salt resistance; and true halophytes have
evolved convergently in to two primary families.
Xerohalophytes are the desert species of halophytes. Desert and coastal halophytes
possess the same mechanisms for dealing with salt toxicity and salt stress. Species living
in both saline habitats commonly belong to the same phylogenetic lineages.
There are marine phanerogams (seed-bearing plants) that live completely submerged in
seawater.
Most conventional crops are glycophytes and suffer growth reductions when the
salinity of the irrigation water exceeds more than a few percent of the salt content of
seawater. There are few irrigation districts in the world that can utilize water of greater
than 1,000 ppm total salts (3% of seawater) without damaging crops. This need for high
quality water is a major constraint to irrigated agriculture (Glenn et al., 1985; 1998).
Through the use of saline water for irrigation, highly salt-tolerant crops derived
from wild halophytes could greatly increase global water and land resources available for
15
agriculture (Glenn et al., 1998a; Rogers et al., 2005; Masters et al., 2007). Large supplies
of brackish water and seawater from different sources are available in areas suitable for
production of salt-tolerant crops to grow (Glenn et al., 1994a; 1994b; 1998a; 1999; Pielke
et al., 1993; Hodges et al., 1993). The availability of commercial crops capable of
growing in brackish and seawater could increase agriculture production worldwide (Riley
et al., 1997; Qadir and Oster, 2004; Rogers et al., 2005; Ayars et al., 2006; Masters et al.,
2007). In addition, halophytes could be produced on the problematic, salinized lands
around the globe. It has been proposed that if commercial crops capable of growing on
brackish water and seawater were available, the world's irrigated acreage could
potentially be doubled (Glenn et al., 1991a; 1992a).
There are many different types of halophytes, and they exhibit a broad range of
salt tolerance (Glenn and O'Leary, 1984); but the most tolerant species have proven to be
as productive of biomass and seeds on seawater as conventional crops on fresh water,
even in extreme coastal desert environments, in multi-year field trials (Glenn and
O'Leary, 1984; Glenn et al., 1991a). Many of the productive species are in the family
Chenopodiaceae - salt bushes, succulent salt marsh plants, and other highly tolerant
plants native to the salt marshes and tolerant plants native to the salt marshes and salt
deserts of the world. Biomass yields under agronomic conditions are in the range of 10 -
20 Ton/ Ha.
16
Salicornia bigelovii
Dwarf glasswort Salicornia bigelovii Torr. (Chenopodiaceae) is one particular
halophyte that has the desirable characteristics for seawater agriculture and has been
identified as the most promising crop that can be produced in arid regions through
irrigation with saline water. It is an especially promising crop for coastal deserts (Glenn
et al., 1998c; Zerai et al., 2010). It is a leafless, succulent, small-seeded, annual saltmarsh
plant that colonizes new areas of mud flat through prolific seed production, with
demonstrated potential as a saline water crop for coastal deserts (Glenn et al., 1998a).
Currently, S. bigelovii is commercially cultivated as a minor specialty vegetable for U.S.
and European fresh produce markets; it is perhaps the world's first terrestrial crop
produced exclusively on seawater. It is also a potential oilseed, forage, biomass crop, and
a promising carbon sequestrations plant. The biomass has been evaluated as a source of
pressboard. It has been produced under seawater irrigation in several large projects over
the past two decades (Glenn et al., 1992b; 1992c). Feeding trials have demonstrated the
value of the oil, seed meal, and forage as animal feed ingredients; these feeding trials
have shown that Salicornia bigelovii seed meal can replace conventional seed meals at
the levels normally used as a protein supplement in livestock diets. Hence, every part of
the plant is useful (Swingle et al., 1996; Glenn et al., 1998). Furthermore, Salicornia
bigelovii yields of seed and biomass equate to, or exceed, freshwater oilseed crops such
as soybean and sunflower; and its oil content is similar to safflower oil in fatty acid
composition (>70% linoleic acid, a desirable polyunsaturated oil for human consumption)
(Glenn et al., 1991a). Salicornia bigelovii seed contain 26 to 33 percent oil, and 35
17
percent protein with a salt content less than 3 percent. The oil can be extracted from the
seed and refined with conventional oilseed equipment; it is also edible, with a pleasant,
nutlike taste and a texture similar to olive oil. A small drawback is that the seed contains
saponins, bitter compounds that make the raw seeds inedible. These do not contaminate
the oil, but they can remain in the meal after oil extraction (Glenn et al., 1991a; 1998c).
Also S. bigelovii has been evaluated for breeding improvement through selective
breeding (Zerai et al., 2010)
Salt and drought tolerance of Salicornia bigelovii
Halophytes use the controlled uptake of Na+
(balanced by Cl- and other anions)
into cell vacuoles to drive water into the plant against a low external water potential.
Dicotyledonous halophytes generally accumulate more NaCl in shoot tissues than
monocotyledonous halophytes (especially grasses), which led early researchers to
characterize the former as “includers” and the latter as “excluders”. The enhancement of
exclusion mechanisms still appears to be the principal strategy of researchers trying to
improve the salt tolerance of grains (Glenn et al., 1999).
Despite their polyphyletic origins, halophytes appear to have evolved the same
basic method of osmotic adjustment: accumulation of inorganic salts, mainly NaCl, in the
vacuole and accumulation of organic solutes in the cytoplasm. Differences between
halophyte and glycophyte ion transport systems are becoming apparent. Highly salt
18
tolerant halophytes maintain an osmotic pressure two to three times higher than the
osmotic pressure of the external medium in tissue (Glenn et al., 1999).
Salicornia bigelovii is a member of the family Chenopodiaceae, a group
containing a large number of salt-tolerant species, including xerohalophytes, plants that
are specialized for deserts and capable of tolerating both salt and drought stress (Reimann
and Breckle, 1993; Glenn et al., 1999; Flowers and Colmer, 2008). Some of the
physiological mechanisms of drought and salt tolerance are similar (Chaves et al., 2009),
and salinity has been described as a form of physiological drought (Munn, 2002). The
capacity for Na+ accumulation is positively correlated with salt tolerance and adaptation
to saline environments (Glenn and O’Leary, 1985; Glenn et al., 1999); but the
physiological responses of halophytes to simultaneous stress of salinity and drying have
not been evaluated.
In Chapter One of this dissertation we evaluated the salt and drought tolerance of
Salicornia bigelovii to achieve a better understanding of the physiological responses to
salt and drought as environmental stressors. The achievement of this better understanding
will aid in the development of this species as a commercial crop in areas where
traditional crops cannot be grown. To this end, we conducted greenhouse trials with
Salicornia bigelovii grown in sealed pots in order to evaluate, survival, growth, osmotic
adjustment, and water-use efficiency of the plants in response to the stress of increasing
salinity and decreasing soil moisture.
19
Previous studies explored the functionality of within-species differences in Na+
and K+ uptake in Atriplex canescens varieties in drying, salinized soils (Glenn et al.,
1998a). Our hypothesis was that drought and NaCl salinity would be additive stress
factors, since both factors lower the external water potential, restricting water entry into
the plant (Munns, 2002; Chaves et al., 2009). However, in A. canescens (Glenn et al.,
1998b), as well as in studies with other halophytes (Ueada et al., 2003; Ben Hassine et
al., 2008; Slama et al., 2008a,b), NaCl had a positive effect on plants growing in drying
soil, enhancing their growth and capacity for water uptake as the soil solution became
depleted. A possible simple explanation for this effect is their utilization of Na+ for
osmotic adjustment to both salinity and drought stress (Glenn et al., 1998a).
Contrary to expectations, Na+ had a positive effect on growth and water use
efficiency. Plants growing in drying soils exhibited much greater salt tolerance than when
grown under non-water-limited conditions. The results are interpreted with reference to
the need to develop model systems for mechanisms of drought and salt tolerance in salt-
tolerant plants.
20
Bioenergy crops
With a growing gap between petroleum production and demand, and with
mounting environmental regulations, industry is investigating candidates for alternative
(nonpetroleum-based) fuels and improved fuel efficiency. Bioderived fuels are being
considered to replace or supplement conventional distilled petroleum fuels (Hendrick and
Bushnell, 2008).
Bioenergy crops are crops capable of producing renewable energy from materials
derived from biological sources. Many different perennial and annual crops can be
included under this heading, including oil producing crops and crops as sources of
lignocellulose. Since 1978, the technical feasibility of producing energy crops has
progressed significantly and several such bioenergy projects have been started (Wright,
2006). However, a drawback to conventional biofuel crops is that they require the
diversion of farmland, pastures, and rangelands from food to fuel production. Thus, a
major issue in producing bioenergy crops; is the interaction between food producing or
energy production because there is a limited amount of arable land. That is why saline
agriculture, an undeveloped source of both food and fuel, is so interesting (Hendrick and
Bushnell, 2008).
In the developing field of saline agriculture there is a growing interest in oilseed
halophytes as bioenergy crops for the production of liquid biofuels. The interest is driven
21
by the potential environmental benefits and the fact that energy crops are a renewable
source of energy. Liquid biofuels can theoretically replace or supplement petroleum fuel
supplies, reducing the U.S. dependence on foreign oil, and reducing net carbon dioxide
emissions by replacing fossil fuels with renewable sources. A promising avenue is the
production of biofuels from halophyte crops (Glenn et al., 1999; Hendricks and Bushnell,
2008; Rozema and Flowers, 2009), as they can be produced on land that is not suitable
for conventional agriculture. Recent efforts have resulted in the development of many
processes to convert vegetable oil into a form suitable for use as fuels (Ranganathan et
al., 2008). In addition, oil yielding crop plants are an important component of economic
growth in the agricultural sector. The oilseeds containing unusual fatty acids are
industrially important, due to its potential use as biofuels (Eganathan et al., 2006).
The dwarf glasswort Salicornia bigelovii, which can be produced through
seawater agriculture systems, is a very promising crop for bioenergy, due to its high oil
content comparable with commercial crops. It may also be a promising biomass source
for ethanol production. Zerai et al. (2010) evaluated the potential for improvement of
several varieties of Salicornia bigelovii through selective breeding.
Among the many varieties evaluated by Zerai et al. (2010) we selected two that
were the most promising, lines from Texas and Florida. In Chapter Two of this
dissertation we conducted green house trials in order to evaluate the biomass production,
22
seed yield, seed composition and osmotic adjustment of these two Salicornia bigelovii
Torr. (Chenopodiaceae) lines (Texas and Florida) under three different irrigation
salinities.
23
CHAPTER ONE
SALINITY TOLERANCE AND CATION UPTAKE OF Salicornia bigelovii
(CHENOPODIACEAE) IN DRYING SOILS
Rafael Martinez-Garcia, Edward P. Glenn, Stephen G. Nelson and Brendan Ambrose
Environmental Research Laboratory, University of Arizona
24
Abstract
The dwarf saltwort Salicornia bigelovii (Chenopodiaceae) is an annual, saltmarsh
plant that can produce oil seed and protein-rich meal, as well as vegetable and forage
products, on seawater irrigation. We grew Salicornia bigelovii from seedlings, of plants
originating along the Texas coast, in saline, drying soils in a greenhouse experiment. The
effects of drought and salinity stress were additive. Optimal growth and water use
efficiency coincided at 0.35-0.53 M NaCl. The plants were tolerant of high salinity but
exhibited little drought tolerance. Salicornia bigelovii plants varied little in their uptake
of Na+ for osmotic adjustment, with final Na
+ contents of 18% on a dry mass basis. Both
growth and water use efficiency of Salicornia bigelovii were affected by salinity. Also,
Na+, the primary cation involved in osmotic adjustment of this species, apparently
stimulates growth by mechanisms apart from its role as an osmoticum.
25
Introduction
Dwarf glasswort Salicornia bigelovii Torr. (Chenopodiaceae) has been identified
as a crop that can be produced in arid regions through irrigation with saline water. It is
an especially promising crop for coastal deserts (Glenn et al., 1998c; Zerai et al., 2010).
Currently, Salicornia bigelovii is commercially cultivated as a minor specialty vegetable
for U.S. and European fresh produce markets; it is perhaps the world's first terrestrial
crop produced exclusively on seawater. It is also a potential oilseed, forage, biomass
crop, and it has been produced under seawater irrigation in several large projects over the
past two decades. Feeding trials have demonstrated the value of the oil, seed meal, and
forage as animal feed ingredients (Swingle et al., 1996). Furthermore, Salicornia
bigelovii oil is similar to safflower oil in fatty acid composition (>70% linoleic acid, a
desirable polyunsaturated oil for human consumption) (Glenn et al., 1991a). The biomass
has been evaluated as a source of pressboard and as a carbon-sequestration crop.
This species is a member of the family Chenopodiaceae, a group containing a
large number of salt-tolerant species, including xerohalophytes, plants that are specialized
for deserts and capable of tolerating both salt and drought stress (Reimann and Breckle,
1993; Glenn et al., 1999; Flowers and Colmer, 2008). Some of the physiological
mechanisms of drought and salt tolerance are similar (Chaves et al., 2009), and salinity
has been described as a form of physiological drought (Munn, 2002). The capacity for
26
Na+ accumulation is positively correlated with salt tolerance and adaptation to saline
environments (Glenn and O’Leary, 1985; Glenn et al., 1999) but the physiological
responses of halophytes to simultaneous stress of salinity and drying have not been
evaluated.
A better understanding of the responses of S. biglovii to these environmental
stressors will aid in the development of this species as a commercial crop in areas where
traditional crops cannot be grown. To this end, we conducted greenhouse trials with S.
biglovii grown in sealed pots in order to evaluate survival, growth, osmotic adjustment,
and water use efficiency of the plants in response to the stress of increasing salinity and
decreasing soil moisture.
In previous studies we explored the functionality of within-species differences in
Na+ and K
+ uptake in Atriplex canescens varieties in drying, salinized soils (Glenn et al.,
1998a). Our hypothesis was that drought and NaCl salinity would be additive stress
factors, since both factors lower the external water potential, restricting water entry into
the plant (Munns, 2002; Chaves et al., 2009). However, in A. canescens (Glenn et al.,
1998b), as well as in studies with other halophytes (Ueada et al., 2003; Ben Hassine et
al., 2008; Slama et al., 2008a,b), NaCl had a positive effect on plants growing in drying
soil, enhancing their growth and capacity for water uptake as the soil solution became
depleted. A possible simple explanation for this effect is their
27
utilization of Na+ for osmotic adjustment to both salinity and drought stress (Glenn et al.,
1998a).
Contrary to expectations, Na+ had a positive effect on growth and water use
efficiency. Plants growing in drying soils exhibited much greater salt tolerance than when
grown under non-water-limited conditions. The results are interpreted with reference to
the need to develop model systems of mechanisms for drought and salt tolerance in well
adapted plants.
Materials and Methods
Germplasm
We selected a variety of dwarf glasswort Salicornia bigelovii, originally collected
from the Texas coast, for use in the experiments. This species is a North American C3
annual, leafless, stem-succulent salt marsh plant. It accumulates very high levels of
sodium and grows poorly in low-sodium cultures (Ayala and O’Leary, 1995). It can
complete its entire life cycle on undiluted seawater (Glenn and J. O’leary, 1985), and is
capable of high biomass and seed yields under agronomic conditions (Glenn et al., 1991).
Seeds were collected from a salt marsh near Galveston, Texas.
28
Greenhouse growth experiment
An experiment was conducted in a greenhouse at the Environmental Research
Laboratory in Tucson, Arizona during the spring and summer of 2007. The greenhouse
has approximately 80% light transmission and is evaporatively cooled, with diurnal
temperatures typically ranging from 25-35oC during the course of the experiment.
The trial was conducted from June 6 to August 6, 2007. Seeds were germinated in
trays on fresh water then transplanted to 2 L pots. Each pot was lined with plastic
sheeting to prevent drainage and filled with 1600 grams of soil. The soil was a mixture of
medium-textured, river-washed sand and a peat-based potting mix (Sunshine Mix No. 1,
Sun Gro Horticulture, Bellevue, Washington). Each pot received an initial 800 ml of
treatment solution and received no further irrigation. Treatment solutions were made up
in distilled water, and contained 390 mg soluble fertilizer (20% N -20% P - 20% K, plus
trace metals) (Scotts Miracle-Gro, Inc., Marysville, Ohio) plus NaCl to produce salinities
of 0 M, 0.09 M, 0.18 M, 0.35 M and 0.53 M NaCl. The plastic enclosing the soil was
sealed across the top of the pot, except for a small opening to receive the transplant. After
the seedlings were transferred to the pots, the plastic cover was drawn around the plant
stem. The surface of the pot was then covered with a layer of gravel as an additional
vapor barrier, and the pot was wrapped in aluminum foil as a heat shield. Control pots
were prepared the same as treatment pots but did not receive transplants. Instead they
received a dried, non-living, stem where the transplant would have been located.
Although there was no Na+ in the 0 M NaCl treatment solution, some was present in the
29
soil mix; so these pots were not Na+-free. Analyses showed control pots contained
approximately 730 mg of total dissolved solids, containing 125 mg Na+ (2.7 mM), 90 mg
K+ (2.3 mM), 50 mg Ca+2
(1.3 mM), and 23 mg Mg+2
(1.0 mM) per pot at the start of the
experiment.
Pots were assigned to treatment, based on a random numbers table. There were
five salinity treatments with three plants of Salicornia bigelovii per treatment, 15 pots all.
The initial height of each plant was approximately 1 cm at the start of the experiment.
Plant heights and pot weights were determined approximately weekly. Plants were
harvested when they showed signs of wilting, and when pot weights no longer decreased
from week to week, indicating they had ceased to remove water from the pot.
On harvest, each plant was separated into shoots and roots, were oven dried (65
oC) and both wet and dry weights of each were determined. Plant roots were washed in
tap water to remove adhering soil particles prior to weighing, and this resulted in a loss of
some of the fine roots. The soil in each pot was removed, mixed thoroughly, and sampled
to determine residual moisture content and salt content. Residual moisture content was
determined by oven drying (at 65 oC) a 10-g soil sample. The dried sample was then
extracted in 50 ml of distilled water and electrical conductivity (EC) was determined with
a Markson EC meter (Markson Scientific, Inc., Henderson, North Carolina) calibrated
with standards of NaCl covering the range of salinities encountered in the extracts. These
30
readings were used to determine grams of NaCl per gram of soil and per gram of residual
soil moisture in each pot at harvest.
Shoot tissues were ground in a Willey Mill (Scientific Engineering Corporation,
Delhi, India) and pooled by treatment, then analyzed for Na+, K
+, Mg
2+ and Ca
2+ by mass
spectrometry (ICP) (IAS Laboratories, Tempe, Arizona). Shoot osmolarity was calculated
by summing the molar values of cations plus one balancing anion for K+ and Na
+ and two
balancing anions for Ca2+
and Mg2+
. This simplified calculation assumes that cations and
anions are completely dissociated and uniformly mixed in the shoot tissue water, with an
activity coefficient of 1.0. While these assumptions are unlikely to be completely valid
(Glenn and J. O’leary 1984), they allow a comparison of the relative contribution of
individual cations. Molarity of the soil solution at the end of the experiment was
calculated based on residual water content and NaCl content as determined by electro
conductivity (EC) measurements. Dry weight gain of plants was determined by
subtracting initial dry weights of plants from final dry weights minus their mineral
contents, calculated by the sum of cations and assuming chloride as a balancing anion.
For each plant, water use efficiency (WUE) was calculated by dividing total dry biomass
per plant (minus mineral content) by the amount of water consumed over the experiment.
Results were expressed as g dry mass per kg of water (gdw kg-1
).
31
Growth and water use characteristics of Salicornia bigelovii were analyzed with a
one-way ANOVA (Sokal and Rohlf, 1995) with salinity as the independent variable.
Treatment effects were considered significant if P < 0.05.
Results
Survival and growth
Survival along the salinity gradient and days to harvest were 61 days of growth.
Of the three Salicornia bigelovii on 0 M NaCl, one died; but all of the plants survived on
higher salinities. Plants reached the wilt point nearly simultaneously and were harvested
between 60 and 61 days of growth. Treatments at 0.18 and 0.35 M NaCl were harvested
at 60 days, while treatments at 0.00, 0.09 and 0.53 M NaCl were harvested at 61 days.
Growth parameters are in Figure 1.1C. The final dry mass of the shoots and the
total plant dry mass were each significantly affected by salinity, but root growth was not
significantly different among salinity treatments (Table 1.2). Growth increased with
salinity up to the 0.35 M NaCl treatment then declined with further salinity increases.
32
Figure 1.1.Water use and growth parameters of Salicornia bigelovii plants grown along a
salinity gradient in drying soils.
Water Use Per Pot
Salinity (M NaCl)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Wa
ter
Us
e (
ml)
0
100
200
300
400
500
600
Roots
Shoots
Soil Moisture Per Pot
Salinity (M NaCl)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
So
il M
ois
ture
(g
g-1
)
0.0
0.1
0.2
0.3
0.4
0.5
Root and Shoot Dry Weights
Salinity (M NaCl)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Dry
We
igh
t (g
)
0.0
0.5
1.0
1.5
2.0
2.5
Water Use Efficiency
Salinity (M NaCl)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
WU
E (
gd
w k
g-1
H2O
)
0.0
0.5
1.0
1.5
2.0
2.5
Wa
ter
Co
nte
nt
(g g
dw
-1)
0
1
2
3
4
5
6
7
A B
C D
33
Showing water use per pot (A); soil moisture at harvest (B); shoot (open circles) and root
(closed circles) dry weights (C); and water use efficiency (closed circles) and shoot water
content (open circles) (D). Error bars are standard errors of means.
Water consumption
Water use parameters are in Figure 1.1A, B, and D. Control pots without plants
lost very little water over the experiments (Figure 1.1A). Water use per plant differed
significantly by salinity with maximum water uptake occurring on 0.35 M NaCl (Figure
1.1A). WUE increased over the salinity range and was highest at 0.53 M NaCl (Figure
1.2D). Soil moisture contents of pots at the end of the experiment were significantly
different among salinity treatments (Table 1.2). The water used was less at the three
lower salinities, and did not lower soil moisture below 0.15 g g-1
on any salinity
treatment.
Salinity did not have a marked effect on the growth and water efficiency of
Salicornia bigelovii shoot water contents were not significantly different by salinity
treatment (Table 1.2). The NaCl molarity of the soil solution remaining in the pots at
harvest differed significantly among salinity treatments (Table 1.2). Lowest NaCl
molarities were on the 0 M NaCl treatment, as expected (Figure 1.2).
34
Table 1.2. Analyses of variance (ANOVA) of Salicornia bigelovii growth parameters in
drying, salinized soils. The data were analyzed as one-way ANOVAs with salinity as the
categorical variable.
Dependent Variable Statistical Summary
DF F P
Days to Harvest 4,11 0.643 0.645
Dry Mass (gdw) Tops 4,11 2.54 0.03
Dry Mass (gdw) Roots 4,11 1.18 0.26
Dry Mass (gdw) Total 4,11 2.39 0.03
Water Use Per Plant 4,11 1.68 0.12
Water Use Efficiency 4,11 5.89 0.000
Shoot Water Content 4,11 1.01 0.33
Molarity of Soil Solution 4,11 65.9 0.001
35
Figure 1.2. Cation content of Salicornia shoots grown along a salinity gradient in drying
soils. Results are single analyses of shoot tissues pooled across replicates.
Salicornia Cation Content
Salinity (M NaCl)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Ca
tio
n C
on
ten
t (%
dry
weig
ht)
0
5
10
15
20
Sodium
Potassium
Calcium
Magnesium
36
Cation contents
The cation content of the plant shoots treated with four different salinities are
shown in Table 1.3. Of the four cations analyzed (Na+ K
+ Ca
2+ Mg
2+), only Na
+ showed
a statistical difference among salinities. Na+ in shoots differed significantly among
treatments (Table 1.3) increasing with increased salinity (Figure 1.2). On 0.35 M NaCl,
Na+ was under 18% of dry weight. The shoot tissue contents of K
+, Ca
2+, and Mg
2+ were
not affected by salinity (Table 1.3; Figure 1.2). Tissue K+ content of the plants, 2% of dry
mass, was much lower than that of Na+; and a negative linear relationship was exhibited
between the Na+ and K
+ contents in shoot tissues (F 4,11= 11.5, P = 0.044).
Table 1.3. Analyses of variance (ANOVA) of Salicornia shoot tissues for plants grown
in drying salinized soils.
Salinity
DF F P
Shoot Osmolality 4,11 4.36 0.03
Shoot Na+ 4,11 14.2 0.003
Shoot K+ 4,11 0.001 0.982
Shoot Ca2+
4,11 4.123 0.067
Shoot Mg2+
4,11 0.492 0.498
Samples were pooled across replicates and results were analyzed as a one-way
unreplicated ANOVAs with salinity as independent variables.
37
Osmotic adjustment
The percentages of cations contributing to shoot tissue osmolarity are shown in
Table 1.4. The ratio of Na+ to K
+ increased rapidly in proportion to the starting salinity
level. Shoot osmolarity calculated from total cations plus balancing anions differed
significantly by salinity treatment (Table 1.3). K+ contributed less to the shoot osmotic
adjustment than Na+, which exhibited a marked response in the osmotic adjustment to
salinity (Table 1.4). Although the divalent cations did not increase in response to salinity,
they contributed from 15-50% to calculated shoot osmolality of the plants across the
salinity range, and Ca2+
contributed more than Mg2+
(Table 1.4). Based on mineral
content, calculated osmolarities exceeded the external salinities in the soil solutions at
harvest on all treatments.
Table 1.4. Percent contribution of individual cations to the calculated osmolarity of
shoots of Salicornia bigelovii grown at different salinities in drying soil.
NaCl Concentration (M) Na+ K
+ Ca
2+ Mg
2+
0
0.09
0.18
0.35
0.53
31.9
59.8
63.6
68.5
71.4
19.8
7.6
6.2
5.1
4.3
45.9
30.4
24.7
19.4
19.7
2.4
2.3
5.6
7.1
4.6
38
Discussion
Salicornia bigelovii requires Na+ for optimal performance on drying soils
Plants in this experiment exhibited suboptimal growth, water extraction, and
WUE on 0 M NaCl and were markedly stimulated by 0.09 M NaCl relative to fresh water
controls. Plant growth increased over the salinity range up to 0.35 M NaCl. This is in
contrast to studies with glycophytes, which generally emphasize the detrimental effects of
Na+ on plant growth (Munns et al., 2002; Ghars et al., 2008; Kronzuker et al., 2008;
Chaves et al., 2009). It is clear from this study and others that Na+ can stimulate growth
in halophytes (Glenn et al., 1999; Subbaroa et al., 2003; Tester and Davenport, 2003;
Flowers and Colmer, 2008) up to a point, followed by declines in growth with further
increases in salinity. The mechanisms by which Na+
first stimulates then represses the
growth of halophytes over their tolerance range remain unclear (Tester and Davenport,
2003; Flowers and Colmer, 2008).
Although Salicornia bigelovii is considered to be highly salt tolerant (Ayala and
O’Leary, 1995; Ayala et al., 1996), in our trials, the salinity tolerance of Salicornia
bigelovii was reduced in drying soils. Also, the plants were not very tolerant of reduced
soil-water content as approximately half the water remained in the pots at the wilt point.
Water use efficiency (WUE) of Salicornia bigelovii plants increased steadily with
increases in salinity. Similar to another succulent halophyte (Redondo-Gomez et al.,
39
2006), maximum WUE of Salicornia bigelovii occurred near the optimal salinity for
growth, which presumably indicates increased efficiency of carbon utilization rather than
restricted stomatal conductance.
Use of Na+
for osmotic adjustment
The Salicornia bigelovii plants accumulated much higher levels of Na+ in their
shoot tissues than we found in other Chenopods such Atriplex canescen, A. lentiformis,
and A. hortensis (unpublished data) and growth was stimulated up to 0.35 M NaCl.
Osmolality of Salicornia bigelovii tissue water was about the same as the external
solution on 0 M NaCl. Growth reduction of Salicornia bigelovii at 0 M NaCl was likely
due to insufficient concentration of Na+ in the soil for optimal osmotic adjustment (Glenn
and O’Leary, 1985; Ayala and O’Leary, 1995) as saltmarsh species tend to accumulate
sodium, which is abundant in saline and sodic soils of their natural habitats.
Drought and salt tolerance
Flowers and Colmer (2008) and Zhu (2001) emphasized the need for intensive
study of a restricted number of halophytes as model systems to understand salt tolerance
mechanisms. Dwarf glasswort was considered a candidate for a model species. However,
Salicornia bigelovii flourish in coastal areas, so drought resistance may not have been
selected for these environments. The lack of drought tolerance mechanisms in Salicornia
bigelovii plants could be related in part to different patterns of cation uptake relative to
those of other halophytes. Our results suggest that Salicornia bigelovii crops would be
40
most suitable for production in areas, such as coastal deserts, where there are abundant
supplies of saline water for irrigation.
41
CHAPTER TWO
BIOMASS PRODUCTION, SEED YIELD, AND TISSUE OSMOLARITY OF
Salicornia bigelovii Torr. (CHENOPODIACAE) IN RELATION TO IRRIGATION
SALINITY
Rafael Martinez-Garcia, Stephen Nelson, Edward Glenn and Brendan Ambrose
Environmental Research Laboratory, University of Arizona. Tucson, Arizona
42
Abstract
Salicornia bigelovii Torr. is a small-seeded, annual saltmarsh plant that produces seed oil,
protein meal and forage products on seawater irrigation. This research evaluated the
production and osmotic adjustment of two S. bigelovii Torr. (Chenopodiaceae) lines
(Texas and Florida). Plants were grown in pots in a green house at the Environmental
Research Laboratory, Tucson, AZ and irrigated with water amended with three different
levels of NaCl (5 ppt, 15 ppt and 30 ppt) combined with inorganic fertilizer. Salicornia
bigelovii plants from Texas and Florida lines were started from seeds from the
Environmental Research Laboratory seed collection. At the end of the experiment sixty
plants from each line were measured for height, biomass, seed yield, seed size, dry matter
yield, and tissue osmolarity. The experiment lasted 16 weeks. There was no significant
difference among groups in plant height, or final biomass either in salinity irrigation
treatments, or S. bigelovii lines. Tissue osmolarity differed among salinity treatments but
not among S. bigelovii lines. The highest tissue osmolarity value was 1192 mM kg-1
found at the 30ppt treatment in the Florida line. Total biomass production was a rate of
12 000 kg/Ha.
43
Introduction
Highly salt-tolerant crops derived from wild halophytes could greatly increase
global water and land resources available for agriculture (Glenn et al., 1998a; Rogers et
al., 2005; Masters et al., 2007). Large supplies of brackish water and seawater from
different sources are available for salt-tolerant crops to grow (Pielke et al., 1993; Hodges
et al., 1993; Glenn et al., 1994a; 1994b; 1998a; 1999). The availability of commercial
crops capable of growing in brackish and seawater could increase agriculture products
worldwide (Riley et al., 1997; Qadir and Oster, 2004; Rogers et al., 2005; Ayars et al.,
2006; Masters et al., 2007). In addition halophytes could be produced on the plentiful
salinized lands around the globe. It has been proposed that if commercial crops capable
of growing on brackish water and seawater were available, the world's irrigated acreage
could potentially be doubled (Glenn et al., 1991a; 1992a).
Numerous studies over the past thirty years have demonstrated the technical
feasibility of using seawater and other saline water supplies for irrigation. Guidelines for
managing salinity have been developed for conventional crops (Ayars et al., 2006) and
extended to halophytes irrigated with higher salinity water (Watson and O'Leary, 1993;
Glenn et al., 1997, 1998a, 1999; Brown and Glenn, 1999; Noaman and El-Haddad, 2000;
El-Haddad and Noaman, 2001). Halophytes can maintain productivity at salinities up to
60-80 g/l in their root zone (approximately twice seawater salinity) (Glenn et al., 1997;
44
1999). Yields of 10-20 ton/ha of dry biomass and 2 ton/ha of seed have been produced by
halophytes irrigated with hypersaline seawater (40 g/L) in small-plot trials in a coastal
desert environment (Glenn and O'Leary, 1985; Glenn et al., 1991a).
There is a growing interest in oilseed halophytes as bioenergy crops for producing
liquid biofuels due to its environmental benefits and the fact that it is a renewable source
of energy. These liquid biofuel can theoretically replace or supplement petroleum fuel
supplies, reducing the U.S. dependence on foreign oil, and reducing net carbon dioxide
emissions by replacing fossil fuels with renewable sources. However, a drawback to
conventional biofuel crops is that they require the diversion of farmland, pastures, and
rangelands from food to fuel production. A promising avenue is the production of
biofuels from halophyte crops (Glenn et al., 1999; Hendricks and Bushnell, 2008;
Rozema and Flowers, 2009), as they can be produced on land that is not suitable for
conventional agriculture. Recent efforts have resulted in the development of many
processes to convert vegetable oil into a suitable form for use as fuel (Ranganathan et al.,
2008). In addition oil-yielding crop plants are an important component of economic
growth in the agricultural sector. The oilseeds containing unusual fatty acids are
industrially important for their use as biofuel (Eganathan et al., 2006).
Of particular interest is Salicornia bigelovii Torr. (Chenopodiaceae), a small-
seeded, annual saltmarsh plant, with demonstrated potential as a saline water crop for
45
coastal deserts (Glenn et al., 1998a). Currently, Salicornia bigelovii is commercially
cultivated as a minor specialty vegetable for U.S. and European fresh produce markets; it
is perhaps the world's first terrestrial crop produced exclusively on seawater. It is also a
potential oilseed, forage, and biomass crop, promising carbon sequestrations plant. The
biomass has been evaluated as a source of pressboard and it has been produced under
seawater irrigation in several large projects over the past two decades (Glenn et al.,
1992b; 1992c). Feeding trials have demonstrated the value of the oil, seed meal, and
forage as animal feed ingredients (Swingle et al., 1996). Furthermore, Salicornia
bigelovii yields of seed and biomass equate or exceed freshwater oilseed crops such as
soybean and sunflower and its oil content is similar to safflower oil in fatty acid
composition (>70% linoleic acid, a desirable polyunsaturated oil for human consumption)
(Glenn et al., 1991a). S. bigelovii seeds contain 26 to 33 percent oil (Glenn et al., 1991a).
Also S. bigelovii seed and oil yield have shown improvement through selective breeding
(Zerai et al., 2010)
Among the many varieties evaluated by Zerai et al. (2010) we selected two that
were the most promising, lines from Texas and Florida. We conducted green house trials
in order to evaluate the biomass production, seed yield, seed composition and osmotic
adjustment of these two Salicornia bigelovii Torr. (Chenopodiaceae) lines (Texas and
Florida) under three different irrigation salinities.
46
Materials and Methods
Germplasm selection
We selected two varieties of Salicornia bigelovii Torr. from the University of
Arizona, Environmental Research Laboratory (ERL) collection; SATX (variety of
Salicornia fromTexas) seeds from a salt marsh near Galveston, Texas, and SAFL (variety
of Salicornia from Florida) seeds from the Gulf of Mexico near Florida. SATX and
SAFL were compared in a greenhouse experiment for growth, seed yield, and tissue
osmolarity with regard to irrigation salinity. Salicornia bigelovii is a North American C4,
annual salt marsh plant with green, succulent, jointed stems that ultimately form terminal
fruiting spikes in which seeds are borne. It accumulates very high levels of sodium and
grows poorly in low-sodium cultures (Ayala and O’Leary, 1995). It can complete its
entire life cycle on undiluted seawater (Glenn et al., 1985). The native range of
Salicornia bigelovii is coastal salt marshes in North America and the Caribbean, where it
germinates in winter or spring, flowers in summer, and sheds seeds in the fall. S. bigelovii
is normally an outcrossing species but is capable of self-pollination if no other source of
pollen is available. S. bigelovii is capable of high biomass and seed yields under
agronomic conditions (Glenn et al., 1991a).
47
Greenhouse growth experiments
An experiment was conducted in a greenhouse at the Environmental Research
Laboratory of the University of Arizona in Tucson, Arizona during summer and fall of
2008. The greenhouse has approximately 80% light transmission and is evaporatively
cooled. Diurnal temperatures typically ranged from 21-37oC during the development of
the experiment.
Figure 2.1. Picture of the mature Salicornia bigelovii plants irrigated with three different
salinities (5, 15 and 30 ppt of NaCl)
48
Effect of irrigation salinity
The experiment was conducted from June 1 to December 13, 2008. Seeds were
germinated in trays with fresh water then transplanted to 2-L pots. Each pot was lined
with plastic mesh sheeting to prevent soil drainage during irrigation and filled with 1600
g of soil. The soil was a mixture of medium-textured, river-washed sand and a peat-
based, potting mix (Sunshine Mix No. 1, Sun Gro Horticulture, Bellevue, Washington).
The spacing of the pots, approximately 0.5 m, was similar to what would be found in
agronomics settings. This was so the total biomass and seed yields could be expressed on
per area basis.
The experiment lasted 27 weeks during which plants were irrigated with water at
one of three salinities; of 5 ppt, 15 ppt, or 30 ppt. Salinities of the irrigation water were
achieved through the use of commercially available salts for artificial seawater (Marine
Enterprises International). Commercial soluble fertilizer was added (Miracle Gro™)
(20% N -20% P - 20% K, plus trace metals) (Scotts Miracle-Gro, Inc., Marysville, Ohio)
to the irrigation water at 0.165 g/L. Irrigation flow was 0.5 gallons/hour at three times per
day via a drip irrigation system with submersible pumps and timers. Flowering began in
September and continued into October. Plants began to ripen and senesce in October and
were harvested in October and November.
49
At harvest, sample of plant tissue were collected and frozen for later tissue
analysis. Plants were then placed individually in paper bags and air dried. The dry mass
of each plant was determined by weighing them on an electronic balance. Seeds were
obtained following methods in Glenn et al. (1991a). We milled the dry plants in a
hammer mill (Jacobson, Machine Works Inc.), and the chaff was screened on a series of
agricultural screens. Air from a blower was used to separate chaff and seed material. For
each plant, one g of the seed and remaining chaff was weighed and the seeds counted by
hand. This was used to calculate the number of seeds produced per plant. Remaining
chaff content was determined (%) and subtracted to determine the accurate number of
seeds per g of sample. Then, for each plant, groups of twenty seeds were weighed with an
analytical balance. This allowed calculation of the seed mass, both individually and
collectively per plant, in grams. The seeds were pooled by variety and treatment and
stored at 40 C for further analysis. Proximate analysis was performed on seed samples in
order to determine oil, protein, moisture, carbohydrate and ash content, these analyses
were conducted by Litchfield Analytical (Litchfield, MI).
The tissue osmolarity was determined for each of the frozen shoot samples. One
gram of tissue was ground immediately while being defrosted. The tissue was then
thawed in a mortar that was nestled in ice and ground with a pestle. Tissues were ground
in 1mL of distilled water. Osmolarity was measured for a 10 µl sample of the tissue
extract with a Wescor Model 5130C (Logan, Utah,USA) Vapor Pressure Osmometer
50
calibrated with 0.29 and 1.0 M standards. The tissue osmolarity was expressed in mM
Kg-1
per plant.
Figure 2.2. Salicornia bigelovii irrigated with three salinities (5, 15 and 30ppt of NaCl)
in the green house of the Environmental Research Laboratory.
51
Figure 2.3. Procedures to obtain dry weight and seed cleaning of the Salicornia bigelovii
growth experiment at the Environmental Research Laboratory. (A) drying of S. bigelovii
to obtain dry biomass. (B) milling of dry biomass of S. bigelovii in order to obtain seeds
A
B
52
Statistics
Height, biomass growth, tissue osmolarity and seed yield of SATX and SAFL
were compared among treatments with a two way Analysis of Variance in which varieties
and salinities were categorical variables. Statistical analysis were carried out using
Statistix 9 software (Tallahassee, Fl).
Results
Biomass production
There were no significant differences in mean final biomass of plants (mean=
213.90± 8.36) among lines (F1,49 = 0.11 P= 0.74) or among irrigation salinities (F2,49 =
0.73 P=0.48) and final values ranged from 183 to 228g (Fig. 2.4). Based on the size of
the crop canopy, the rate of total biomass production was approximately 12,000 kg/Ha.
53
Figure 2.4. Final dry mass (g) per plant of Salicornia bigelovii plants grown in 2-L- pots
with irrigation salinites of either 5, 15, or 30ppt.
Seed analysis and yield
The mean dry mass of individual seeds differed among both salinity (F2,12 =20.22,
P=0.0001) and plant line (F1,12 =15.57, P=0.0019) with a significant interaction (F2,12
=6.74, P=0.0109) between these variables. The maximum seed individual weight was
found at the treatment 15ppt in the Texas line with a value of 1.5 x10-2
g and the
minimum seed individual weight was found at 5ppt irrigation salinity in the Texas line
with a value of 0.45 x10-2
g. For both lines, the highest seed weights were found in the
5 15 30
50
120
190
260
330
400
D
ry M
ass (
g)
Salinity (ppt)
54
SA
plants irrigated with saline water at 15 ppt, and the effect of salinity on seed weight was
more pronounced in the line from Texas (Figure 2.5).
Figure 2.5. Mean dry mass (g) of individual seeds from two varieties (one from Texas
and one from Florida) of Salicornia bigelovii plants grown in pots irrigated with saline
water at either 5, 15, or 30ppt.
The total seed yields (g) per plant differed significantly among irrigation
treatments (F2,12 =5.06, P=0.0255), but not between the Salicornia bigelovii lines (F1,12
=3.33, P=0.0936). The maximum seed yields were obtained in the treatments at 15ppt
for both Texas and Florida lines with values of 53 g and 28 g respectively. The minimum
seed yields were found at the 5ppt treatment in both Texas and Florida lines with values
55
of 13 g and 6 g respectively (Figure 2.6). In addition, the color of the seeds differed
among treatment groups (Figure 2.7).
Figure 2.6. Seed yield (grams) per plant in greenhouse trials with two varieties (one from
Florida and one from Texas) of Salicornia bigelovii grown along a salinity gradient
56
Figure 2.7. The color and size of Salicornia bigelovii seeds from two lines (Texas and
Florida) irrigated with three different salinities ( 5, 15 and 30ppt)
Shoot tissue osmolarity
The adjustment in osmoregulation was directly proportional to the increase in
salinity irrigation (this means that plants at 30 ppt irrigation salinity had a higher
osmoregulation). Tissue osmolarity was significantly differed in relation to salinity (F2,49
=34.77, P=0.0000) but not between varieties (F1,49 =1.74, P=0.1929) of Salicornia. The
media tissue osmolarity values were above 1100 mM kg-1
at 30ppt and below at 5ppt.
(Figure 2.8). The highest tissue osmolarity value was 1192 mM kg-1
found at the
57
treatment 30ppt in the Florida line. The lowest osmolarity value was 749.6 mM kg-1
found at the treatment 5ppt in the Texas line.
Figure 2.8. Shoots tissue osmolarity (mM kg-1
) of Salicornia bigelovii plants grown at
three different salinity irrigation (5, 15 and 30ppt). Pooled samples.
Discussion
Biomass production in relation to irrigation salinity
From these studies we estimated a production rate of 12 dry Ton/Ha; this is
similar to what other researchers found in field and greenhouse studies using S. bigelovii.
For example, Grattan et al. (2008) obtained 9 Ton/Ha of S. bigelovii from production in a
greenhouse over a range of salinities from 32.6 to 52 dS m-1
(16.3 to 28.6ppt), using
5 15 30
500
800
1100
1400
Pla
nt O
sm
ola
rity
Salinity (ppt)
58
hyper-saline drainage water from irrigation and seawater in the San Joaquin Valley,
California. Glenn et al. (1997) obtained 13 Ton/Ha with seawater irrigation (35ppt), at
irrigation volumes ranging from 46–225% of potential evaporation in a coastal area in
Puerto Penasco, Sonora, Mexico. Abdal (2009), reported production of S. bigelovii of 11
Ton/Ha based seawater irrigation in sandy soils of coastal areas in Kuwait over a similar
cycle duration.
The biomass production of Salicornia observed is within the ranges reported for
other irrigated halophyte crops and also compares favorably to that of coventional forage
crops. Aronson et al. (1988) evaluated seven Atriplex species and obtained a biomass
production of 12.6 to 20.9 Ton/Ha with half and full strength seawater irrigation
respectively. Gallagher (1985) obtained yields of 5.2 to 9.5 Ton/Ha of the saltgrass
Distichlis spicata under seawater (30 ppt) irrigation in Delaware, USA. By comparison
Wade et al. (1994) reported yields of 16 and 15 Ton/Ha for the two conventional forage
crops Sudan grass and alfalfa in commercial fields in Yuma County, Arizona.
Salicornia bigelovii is considered to be highly salt tolerant (Ayala and O’Leary,
1995; Ayala et al., 1996), and we found that S. bigelovii grew well at the three salinities
tested (5ppt,15ppt, and 30ppt). Other studies (Glenn et al., 1999; Subbaroa et al., 2003;
Tester and Davenport, 2003; Flowers and Colmer, 2008) have shown that; in general Na+
stimulates growth in halophytes up to a point, followed by declines in growth with further
increases in salinity. However, in our case we did not observe any reduction of
59
production at either the lowest (5ppt) or highest (30ppt) salinity in our trials. This
suggests that S. bigelovii could be grown successfully over an even wider range of
salinities. Our results show that irrigated production of Salicornia bigelovii would be
suitable in a wide range of coastal environments from estuarine to marine.
Osmoregulation
Salicornia bigelovii is well adapted to growing in saline areas, it is a short,
leafless, succulent, intertidal hydrophyte with both a high transpiration rate and a
relatively open canopy through which surface evaporation can take place, it requires high
volumes of irrigation water, and, as a result, salts will tend to accumulate in the vicinity
of the roots (Glenn et al., 1997). S. bigelovii is a C4 plant, with higher water use
efficiency than C3 plants because of the higher assimilation rates of CO2, and this
contributes to increased water use. In addition S. bigelovii is an osmoconformer and can
increase the osmolarity of its tissue in response to increases in salinity. In general, S.
bigelovii and other salt-marsh species accumulate much higher levels of Na+ in their
tissues, as Na+ is abundant in the saline and sodic soils of their natural habitats (Flowers
et al., 1977; Bradley and Morris, 1991; Glenn et al., 1997; 1999; Parida and Das, 2005).
60
Seed production
Even though there were no differences among the treatments in biomass
production, seed production was affected by both plant variety and irrigation salinity.
Seed production in S. bigelovii is affected directly by the number and sizes of spikes
produced as well as degrees of maturation. Greater number of spikes result in higher seed
yields. In our trial, seed production was 11% of the total biomass produced. The sizes of
seed spikes were similar to those reported by Glenn et al. (1991a) from fields at Kino
Bay, Mexico, where they found seed spikes of 10-15cm long from irrigated S. bigelovii in
a field experiment. In addition, our biomass seed yields ( 1.3 Ton/Ha) were also
comparable to yields obtained by Glenn et al. (1991a), who reported an average of 2
Ton/Ha of seeds produced under seawater (40ppt) irrigation. Similar seed yields were
also found by Glenn et al. (1997) in a later field experiment using seawater irrigation in
Puerto Penasco, Sonora, Mexico. Also, Bashan et al. (2000) obtained a seed yield of 1.8
Ton/Ha in a greenhouse setting with seawater irrigation of S. bigelovii in Baja California,
Mexico. These comparisons lend confidence in the extrapolation of our results to field
production.
61
CONCLUSIONS AND RECOMMENDATIONS
The general objective of this study was to understand the physiological aspects in
the culture of Salicornia bigelovii under different environmental conditions. The specific
objectives were; to determine the drought tolerance and salt stress of Salicornia bigelovii
in drying soils, analyzing water use efficiency and optimal growth. Salicornia bigelovii
plants were tolerant to high salinity but exhibited little drought tolerance. Optimal growth
and water use efficiency coincided at 0.35 – 0.53 M NaCl. Both growth and water use
efficiency were affected by salinity. Na+, the primary cation involved in osmotic
adjustment of this species, apparently stimulates growth by mechanisms apart from its
role as an osmoticum.
Others specific objectives of this study were; to evaluate the production seed yield
and osmotic adjustment of two Salicornia bigelovii Torr. (Chenopodiaceae) lines (Texas
and Florida).We found that the halophyte Salicornia bigelovii grows well at salinities
from 5 to 30 ppt with suitable levels of seed production and biomass in many areas,
particularly in arid regions adjacent to the sea or with conditions similar to the sea. In
addition agronomic development of S. bigelovii for biofuels production or other porpoises
would be suitable using waters of a variety of salinities and could become a potential
bionergy crop without negatively affecting conventional crop production.
62
Future studies should be conducted in order to determine if other varieties of
Salicornia bigelovii will exhibited the same tolerance to salinity and drought with the
same optimal growth and water use efficiency, other varieties can have different
tolerance due to different adaptation to different environmental factors in their endemic
areas. In the present dissertation we compared two Salicornia varieties; Salicornia Texas
and Florida, Salicornia Texas showed little to no differences when compared with
Salicornia Florida, although further studies are needed to facilitate a better variety with
the desire characteristics and for the determined environment. Studies which evaluate
these same results obtained in this study at field levels are needed in order to assess
drought and salt tolerance. At field levels environmental factors such as temperature can
affect water evaporation thus irrigation regimes will be higher, water salinity can also be
affected at field levels, having salinity fluctuations by seasons or sometimes daily, due to
several environmental factors.
63
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