Physiological Studies of the Halophyte Salicornia Bigelovii: a Potential Food and Biofuel Crop for Integrated Aquaculture-Agriculture Systems

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Physiological Studies of the Halophyte Salicornia bigelovii: A Potential Food and Biofuel Crop for Integrated Aquaculture-Agriculture Systems

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Authors Martinez Garcia, Rafael

Publisher The University of Arizona.

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PHYSIOLOGICAL STUDIES OF THE HALOPHYTE Salicornia bigelovii: A POTENTIAL FOOD AND BIOFUEL CROP FOR INTEGRATED AQUACULTURE- AGRICULTURE SYSTEMS

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

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

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.

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

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

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

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

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.

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).

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).

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).

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.

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 .

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.

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

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.

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.,

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.

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 25-35 oC 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 Ca 2+ and Mg 2+ . 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).

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.

Figure 1.1.Water use and growth parameters of Salicornia bigelovii plants grown along a salinity gradient in drying soils.

Water Use Per Pot Soil Moisture Per Pot

600 0.5

A B 500

) 0.4 -1 400 0.3

300

0.2 200 Water (ml) Water Use

Soil Soil Moisture (g g 0.1 100

0 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Salinity (M NaCl) Salinity (M NaCl)

Root and Shoot Dry Weights Water Use Efficiency

2.5 2.5 7

C D )

6 -1 2.0 2.0 O)

2 5 H

1.5 -1 1.5 4 Roots 3 1.0 Shoots 1.0

Dry WeightDry (g) 2

0.5 (gdw WUE kg 0.5 1 Water Content(g gdw

0.0 0.0 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Salinity (M NaCl) Salinity (M NaCl)

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).

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

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

10 Sodium Potassium Calcium Magnesium 5 Cation Content (% dry dry Cationweight) Content(%

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Salinity (M NaCl)

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 Ca 2+ 4,11 4.123 0.067

Shoot Mg 2+ 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.

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 Ca 2+ contributed more than Mg 2+ (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.

+ + 2+ 2+ NaCl Concentration (M) Na K Ca Mg

0 31.9 19.8 45.9 2.4

0.09 59.8 7.6 30.4 2.3

0.18 63.6 6.2 24.7 5.6

0.35 68.5 5.1 19.4 7.1

0.53 71.4 4.3 19.7 4.6

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.

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

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.

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.

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).

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 21-37 oC 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)

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.

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 4 0 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.

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

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 (F 1,49 = 0.11 P= 0.74) or among irrigation salinities (F 2,49 =

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.

400

330

260

190 DryMass(g)

120

50 5 15 30

Salinity (ppt)

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 (F 2,12 =20.22,

P=0.0001) and plant line (F 1,12 =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 54

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 (F 2,12 =5.06, P=0.0255), but not between the Salicornia bigelovii lines (F 1,12

=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

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 (F 2,49

=34.77, P=0.0000) but not between varieties (F 1,49 =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.

1400

1100

800 Plant Osmolarity Plant

500 5 15 30

Salinity (ppt)

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 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 CO 2, 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).

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.

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.

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.

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