GENETIC ENGINEERING OF POPULUS DELTOIDES … engineering of populus deltoides for arsenic phytoremediation and the ... for arsenic phytoremediation and the establishment of ... pathway

GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC

PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO

PROPAGATION SYSTEM FOR SALIX NIGRA

by

AMPARO LIMA

(Under the Direction of Scott Arthur Merkle)

ABSTRACT

Arsenic pollution is an environmental problem affecting the health of millions of

people worldwide. Unfortunately, conventional remediation technologies for this toxic

pollutant are costly and environmentally destructive. An alternative to conventional

remediation methods is phytoremediation, the use of plants to extract pollutants from

contaminated soil, water and air. Recent studies demonstrated that increasing the thiol-

sinks in transgenic plants by over-expressing the bacterial γ-glutamylcysteine synthetase

gene resulted in a higher tolerance and accumulation of arsenic. To further explore the

potential of transgenic plants to remove arsenate from polluted soil, we genetically

engineered eastern cottonwood (Populus deltoides) trees to over-express γ-ECS and, we

also established an in vitro propagation system for another phytoremediation candidate,

Salix nigra. Our results show that eastern cottonwood trees over-expressing the γ-ECS

gene were able to grow normally on toxic levels of arsenate. We also established an in

vitro regeneration system for Salix nigra from immature inflorescence explants.

INDEX WORDS: Phytoremediation, arsenate, γ-glutamylcysteine synthetase.




by

AMPARO LIMA

Biologo. Autonomous University of the State of Morelos. Mexico. 1999

A Thesis Submitted to The Graduate Faculty of The University of Georgia in Partial

Fulfillment of The Requirements for The Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2003

2003

AMPARO LIMA

All Rights Reserved




by

AMPARO LIMA

Major Professor: Scott A. Merkle

Committee: Jeffrey F.D. Dean C. Joseph Nairn Richard B. Meagher

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia May 2003

iv

TABLE OF CONTENTS

CHAPTER I

INTRODUCTION AND LITERATURE REVIEW …………………………..1

CHAPTER II

ENHANCED ARSENIC TOLERANCE OF TRANSGENIC EASTERN

COTTONWOOD PLANTS OVEREXPRESSING γ-GLUTAMYLCYSTEINE

SYNTHETASE………………………………………..…….…………………33

CHAPTER III

ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX

NIGRA……………………………………………………………………….…53

CHAPTER IV

CONCLUSIONS………………………………………………………………67

1

CHAPTER I

INTRODUCTION AND LITERATURE REVIEW

Arsenic Contamination

Over the past century, mining, agriculture, manufacturing and urban activities

have all contributed to extensive soil and water contamination (Cunningham et al., 1995).

High on the list of toxic pollutants affecting the health of millions of people worldwide is

arsenic (Nriagu, 1994). Arsenic is a naturally occurring element widely distributed on

the earth's crust, mainly existing as arsenic sulfide, metal arsenates or arsenites (Emsley,

1991). Arsenic contamination can be from natural or man-made sources. Natural

contamination results from the dissolution of naturally existent minerals/ores or soils and

up-flow of geothermal water (Emsley, 1991). Man-made pollution generates from most

industrial effluents, copper smelting, pesticides and atmospheric deposition (Nriagu,

1988). In the environment, arsenic combines with oxygen, chlorine, and sulfur to form

inorganic arsenic compounds (Nriagu, 1994). These toxic metalloids, classified as “group

A” human carcinogens, can cause skin lesions, lung, kidney and liver cancer, and damage

to the nervous system (U.S. EPA 1996: www.epa.gov/ogwdw/ars/arsenic.htm1).

In the United States, hundreds of superfund sites are listed on the National

Priority List as having unacceptably high levels of arsenic (www.epa.gov). The

processes currently being used to remediate contaminated soils are physical, chemical

and biological (Cunningham et al., 1995). These processes either decontaminate the soil

or stabilize the pollutant within. Decontamination reduces the amount of pollutants by

2

removing them. Stabilization does not reduce the quantity of pollutant at a site, but makes

use of soil amendments to alter the soil chemistry so as to sequester or absorb the

pollutant into the matrix, thereby reducing or eliminating environmental risks (Pignatello,

1989; Merian and Haerdi, 1992).

Traditional arsenic remediation methods include oxidation, co-precipitation,

filtration, adsorption, ion exchange and reverse osmosis. Unfortunately, managing

contaminated soils, sludge, and groundwater is costly and the resultant environmental

damage is very high (U.S. Army Toxic and Hazardous materials Agency, 1987). The

enormous costs and relative ineffectiveness of traditional remediation methods have

prompted the development of alternative remediation methods.

Phytoremediation

There are several species of plants that can survive on highly polluted sites. Most

survive by either avoiding toxic materials or by accumulating and sequestering them in

their tissues (Baker and Brooks, 1989; Hedge and Fletcher, 1996; Chaudhry et al., 1998;

Khan et al., 1998; Schnoor et al., 1995). Plants that use the latter mechanism are known

as hyper-accumulators. The following foliar concentrations have been suggested as a

threshold to define hyper-accumulation: 10,000 mg/kg for zinc, 1000 mg/kg for copper

and 100 mg/kg for cadmium (Reeves et al., 1995). The ability of some plants to hyper-

accumulate, and in some cases degrade, toxic compounds gave rise to an alternative

remediation method known as phytoremediation.

Phytoremediation uses plants to extract, sequester or detoxify pollutants from soil,

water and air (Rashkin, 1996). This innovative technology offers advantages over

3

conventional physical or chemical techniques. It is estimated that phytoremediation costs

can be between two- and four-fold less than existing remediation technologies (Meagher

and Rugh, 1996). In addition, this approach is an ecologically preferable method because

it reclaims soil in situ instead of permanently removing it to a storage site (Salt et al.,

1995). Although phytoremediation as a technology is still in its development stages, it

has become a rapidly expanding research area because of its promise for the remediation

of organic and inorganic pollutants.

Organic pollutants include polychlorinated biphenyls (PCBs), polycyclic aromatic

hydrocarbons (PAHs), nitroaromatics, and linear halogenated hydrocarbons (Meagher,

2000). In phytoremediation, the main goal is to completely mineralize these compounds

into relatively non-toxic constituents, such as carbon dioxide, nitrate, chlorine, and

ammonia (Cunningham et al., 1996). Using plants, organic pollutants can be remediated

through several biophysical and biochemical processes including absorption, transport

and translocation or hyper-accumulation, or transformation and mineralization (Meagher

2000).

Inorganic pollutants include toxic metals such as aluminum, arsenic, cadmium,

chromium, copper, lead, mercury, nickel, zinc, cesium, strontium and uranium (Salt et

al., 1998). Inorganic pollutants are immutable at an elemental level and cannot be

degraded or mineralized (Salt et al., 1998); thus, their remediation is difficult to achieve

(Meagher and Rugh, 1996). Plant-based phytoremediation strategies for inorganic

pollutants rely on plant roots to extract, vascular systems to transport, and leaves to act as

sinks to concentrate these pollutants (Dhankher et al., 2002).

4

Phytoremediation strategies for arsenic contaminated soils are not very common,

but the few existing studies show great promise for the potential applications of this

alternative remediation method.

Arsenic Phytoremediation

As previously mentioned, certain plant species have the capacity to extract

pollutants from soil or water through their normal root uptake of nutrients. The plants

then store these compounds in their cells or convert them into less toxic forms (Meagher

2000). To date, there is only one report of a plant with the ability to handle arsenic in this

manner. Pteris vittata, a fern indigenous to the southern parts of the U.S., has the

capacity to hyper-accumulate arsenic to very high levels (7500 ppm; Ma et al., 2001).

Unfortunately, the enzymes responsible for arsenic hyper-accumulation in this plant are

not yet available for manipulation into other plant species. Although specific arsenic

hyper-accumulation enzymes have not been isolated, increased tolerance and

accumulation of arsenic has been reported in plants over-expressing the bacterial enzyme

γ-glutamylcysteine synthetase (Dr. Yujing Li, Genetics Department, University of

Georgia, personal communication).

Gamma-glutamylcysteine synthetase (γ-ECS) forms part of a three-step enzymatic

pathway responsible for the synthesis of phytochelatins. In plants, heavy metal

detoxification often occurs through the chelation of metal ions by metal-binding ligands

(Cobbett, 2000). To date, a number of metal-binding ligands have been recognized and

among the most studied are the phytochelatins. They are members of a small class of

Cys sulfhydryl residue-rich peptides [γ-glumatylcysteine (γ-EC), glutathione (GSH) and

5

phytochelatins (PC)] that play an important role in the detoxification and sequestration of

thiol-reactive heavy metals (Noctor et al., 1998; Zhu et al., 1999b; Xiang et al., 2001).

These γ-EC containing peptides are derived from common amino acids in a three-step

reaction. (Zhu et al., 1999b) (Figure 1).

Gly γ-Glu-Cys Glu + Cys → γGlu-Cys → Gly- γGlu-Cys → Gly (γGlu-Cys)n

γECS GS PS

Figure 1. Phytochelatin synthesis pathway. Three enzymes constitute the phytochelatin

biosynthetic pathway: γ-glutamylcysteine synthetase (γECS), glutathione synthetase (GS)

and phytochelatin synthetase (PS).

The first step is catalyzed by the enzyme γ-glutamylcysteine synthetase and

results in the formation of γ-EC dipeptides. The product of this first reaction contributes

multiple dipeptide units to the phytochelatins, and it is believed to be the limiting step for

both GSH and PCs production in the absence of heavy metals (Noctor et al., 1998b).

Genetically engineered Arabidopsis thaliana plants over-expressing the Escherichia coli

γ-ECS gene under the control of a strong constitutive actin promoter (ACT2p) were

highly resistant to arsenic (300 µM) compared to wild-type plants (Dr. Yujing Li,

Genetics Department, University of Georgia, personal communication). These results

showed that manipulation of γ-ECS in plants may become a promising approach for

arsenic phytoremediation. In recent years, reports showing over-expression of bacterial

6

or animal transgenes to enhance the capacity of selected valuable phytoremediating

plants have become more common (Bizly et al., 1999; Bizily et al., 2000; Dhankher et

al., 2002; Doty et al., 2000; Guller et al., 2001; Hannink et al., 2001; Heaton et al., 1998;

Li et al., submitted; Pilon et al., 2003; Rugh et al., 1998; Rugh et al., 1996;Yamada et al.,

2002; Zhu et al., 1999). Fast-growing, high biomass-producing plants with profuse root

systems and high evapotranspiration rates would make excellent candidates for

phytoremediation. Poplar and willow trees possess many of these characteristics, making

them ideal candidates for use in phytoremediation process.

Poplar phytoremediation

Poplars (Populus spp.) are fast-growing trees with high transpiration rates and

wide-spreading root systems, which make them ideal to intercept, absorb, degrade and/or

detoxify contaminants, while reducing soil erosion (Harlow et al., 1999). In addition to

having a wide geographical distribution, they grow naturally in riparian areas. Thus,

poplars are particularly well suited for use on many potential remediation sites (Dix et al.,

1999). Populus species have been extensively studied, and have well-established

silvicultural, vegetative propagation, breeding, and harvesting protocols (Harlow et al.,

1999). In addition, poplars are amenable to tissue culture manipulation and genetic

engineering (Kang and Chun, 1997; Kim et al., 1996). All of these characteristics have

made poplars ideal candidates for genetic engineering for absorption, detoxification, and

/or degradation of environmental pollutants.

Poplars have been used to remove atrazine (Burken and Schnoor, 1997),

trichloroethylene (Newman et al., 1997), trinitrotoluene (Thompson et al., 1998), dioxane

7

(Kelley et al., 2000), and selenium (Pilon-Smits et al., 1998) from contaminated soils.

Trichlorethylene (TCE) is one of the most widespread environmental contaminants in the

United States (Westrick et al., 1984). Conventional remediation methods for this

compound are extremely costly and very slow (Travis and Doty, 1990). In 1998, Gordon

et al. reported the degradation of trichloroethylene to carbon dioxide and other non-toxic

metabolites by Populus trichocarpa x P. deltoides hybrids.

Thompson et al. (1998) examined the potential of the hybrid poplar, Populus

deltoides x P. nigra, for remediating sites contaminated with the highly explosive,

trinitrotoluene (TNT). Their results showed that while TNT was strongly bonded to the

root tissues, it was moderately translocated to the leaves and transformed into 4-amino-2,

6-dinitrotoluene and 2-amino-4, 6-dinotrotoluene.

Dioxane has also been widely used as a solvent, and is considered to be a

probable human carcinogen (http://www.epa.gov/ttn/atw/hlthef/dioxane.html). This toxin

is a persistent environmental pollutant that is difficult to remove from contaminated sites.

Kelley et al. (2000) showed that within 9 days, rooted cuttings of the hybrid Populus

deltoides x P. nigra were able to remove up to 54% of dioxane from contaminated soil.

Dioxane taken up by the poplars was transpired from leaf surfaces into the atmosphere,

where it could be dispersed and photodegraded.

Pilon-Smits et al. (1998) showed significant selenium volatilization rates from the

hybrid poplar, Populus tremula x P. alba. Volatilization rates were similar to Typha

latifolia, a species already being used for the cleanup of selenate- and selenite-

contaminated wastewater. The data from these studies showed that poplar trees could

take up and metabolize pollutants into less toxic forms.

8

Willow phytoremediation

The genus Salix, a member of the Salicaceae, is composed of approximately 300

species of trees and shrubs (Harlow et al., 1996). These different species are largely

scattered throughout the cooler regions of the Northern Hemisphere, although a few are

distributed in the tropical regions of Indonesia and South Africa, as well as southern

South America (Harlow et al., 1996). In North America, there are approximately 80

native Salix species, but only 30 of them attain tree size. They are fast-growing trees,

reaching maturity in 50 to 70 years (Harlow et al., 1996). Reproduction by seeds is

restricted because germination must occur on moist mineral soil soon after the seeds are

shed; however, propagation by sprouts and root suckers is excellent. These

characteristics have contributed to the use of willows in phytoremediation.

Perttu and Kowalik (1997) reported the use of Salix sp. as a vegetation filter.

Willow stands irrigated with municipal wastewater were shown to function effectively as

purification plants, while at the same time producing fuel wood. Corseuil and Moreno

(2001) reported the phytoremediation potential of weeping willow trees (Salix

babylonica) growing on aquifers contaminated with ethanol-blended gasoline. Rooted

cuttings from mature willows were exposed to different concentrations of ethanol.

Results indicated that ethanol concentrations were reduced by more than 99% in a five-

day period, and benzene concentrations were reduced by more than 99 % in a seven-day

period. These results suggested that deep-rooted willow trees were of practical use in

removing hydrocarbons from contaminated aquifers.

Robison et al. (2002), reported cadmium accumulation in five different willow

clones. Clones were grown under controlled conditions in pots of soil containing

9

different concentrations of cadmium, zinc, manganese and iron. Accumulation rates

varied among clones, ranging from 1.5 to 10 mg/kg. Shrub willows had significantly

higher leaf and stem concentrations of cadmium, manganese and zinc compared to tree

willows.

The published studies suggest that both poplar and willow trees have the capacity

to tolerate and accumulate pollutants, as well as the capacity to metabolize them into less

toxic forms. Of all the poplar and willow species used for phytoremediation, there are

two species in particular, Populus deltoides and Salix nigra, that show enormous

potential for phytoremediation, particularly in the southeastern U.S., where they are

natives. However, their use in this field has not been as common as the other species of

poplar and willows.

Eastern cottonwood (Populus deltoides)

Eastern cottonwood is the fastest growing native tree in North America (Fenner et

al., 1984), and it often occurs as a dominant or co-dominant component of floodplain and

bottomland hardwood forests (Curtis, 1959; Fitzgerald et al., 1975; Hosner and Mickler,

1963). Cottonwoods have high rates of biomass production (up to 10-30 m3/ha/year of

wood on a short rotation of six to eight years) and have extensive root systems (300,000

km/ha, Gordon et al. 1997). Cottonwood is easily established and propagated by rooted

cuttings, and are also amenable to tissue culture manipulation and genetic engineering

(Ernst, 1993; Kang and Chun, 1997; Saito 1980; Prakash and Thielges, 1988; Douglas

1984; Coleman and Ernst, 1989; Ho and Ray, 1985; Uddin et al., 1988; Koudier et al.,

1984; Savka et al., 1987; Kim et al., 1997; Han et al., 2000; Parsons et al., 1986; De

10

Block 1990; Wang et al., 1994; Charest et al., 1992; Hauchelin et al., 1997; Noon et al.,

2002).

Tissue culture and Genetic Engineering of Eastern Cottonwood.

In vitro propagation systems for eastern cottonwood have been studied since the

1980s (Chun et al., 1988). Eastern cottonwood tissue has a high degree of developmental

plasticity; adventitious shoots can be induced from in vitro cultured cambial tissue,

leaves, internodes and anthers (Saito, 1980; Prakash and Thielges, 1988; Douglas 1984;

Coleman and Ernst, 1989; Ho and Ray, 1985; Uddin et al., 1988).

The first in vitro regeneration of adventitious shoots was achieved via

organogenic callus derived from cambial tissue explants grown on callus induction

medium for eight months and then transferred to shoot induction medium. All explants

produced callus and shoots, with an average of 15 shoots per explant (Saito 1980).

Prakash and Thielges (1988) reported the establishment of adventitious shoot cultures

from leaves via organogenic callus. Calli were grown on MS medium (Murashige and

Skoog, 1962) supplemented with auxins and cytokinins, and shoot development was

induced from the calli with cytokinins. Douglas (1984) reported the formation of

adventitious shoots from internodes cultured in vitro on MS medium (Murashige and

Skoog, 1962) without exogenous plant growth regulators. Anatomical studies revealed

cell differentiation initiating from cambium and phloem cells. Douglas also found an

increase in bud and shoot production between internodes four and seven. This suggests

that endogenous plant growth regulators may be interacting with the tissue, resulting in a

gradient of potential organogenic response from the shoot tip downward. Coleman and

11

Ernst (1989) also induced adventitious shoots from internodes cultured on woody plant

medium (Lloyd and McCown, 1980) supplemented with benzyladenine, 2,4-

dichlorophenoxyacetic acid or zeatin. The greatest number of shoots obtained was from

the cultures growing on medium with zeatin. Further studies showed that stabilized shoot

cultures could be established and maintained by placing elongated adventitious shoot

segments on Driver and Kuniyuki (1984) medium supplemented with zeatin (Coleman

and Ernst, 1989).

Haploid plantlets regenerated from anther cultures demonstrated that the

developmental stage of the explants was a determining factor in the induction of haploid

callus (Ho and Ray, 1985; Uddin et al., 1988). Superior callus growth was achieved

when pollen grains were at the uninucleate stage (microspore stage of development) (Ho

and Ray, 1985; Uddin et al., 1988). Unfortunately, plants regenerated from the anther

cultures had a variety of ploidy levels (Ho and Ray, 1985).

The predominant gene transfer method for poplars has been Agrobacterium-

mediated transformation (Kim et al., 1997). Much of the work in this field has been

restricted to a few model hybrids (Parson et al., 1986; De Block 1990; Wang et al., 1994;

Charest et al., 1992; Heuchelin et al., 1997) and species of section Leuce (aspens and

white poplars), because of their ease of transformation (Han et al., 2000). To date, there

have been few reports demonstrating Agrobacterium-mediated transformation of eastern

cottonwood (Dinus et al., 1995; Han et al., 2000; Che et al., in press). Dinus et al (1995)

inoculated leaf sections of eastern cottonwood clone C-175 with Agrobacterium

tumefaciens strain LBA 4404. Three transformation efficiency factors were evaluated:

Pre-incubation treatment, exposure time and bacterial concentration. The results showed

12

that increasing the pre-incubation treatment resulted in higher transformation frequencies

and recovery of transgenic calli, primordia and shoots. However, regeneration of

transgenic plants was not reported. Han et al. (2000) compared stem and leaf sections as

explant sources for eastern cottonwood transformation, and found that stems were

markedly superior to leaf blades for regeneration of callus and shoots. Furthermore,

shoot regeneration was mainly observed from the vascular bundles of shoots, possibly

due to higher rates of contact between bacteria and host.

Even though eastern cottonwood possesses many characteristics that make it an

excellent candidate for phytoremediation, there is only one report in the literature of its

use in phytoremediation. Che et al. (in press) generated transgenic eastern cottonwood

trees for use in mercury phytoremediation. Transgenic plants expressing the mercuric ion

reductase enzyme were capable of growing in high concentrations of mercuric chloride

(25 µM), while wild-type plants were killed. Also, these plants were capable of

volatilizing 2-4 times more elemental mercury than wild-type plants. Other results

showed that eastern cottonwood trees expressing the organomercurial lyase enzyme were

able to root in media containing phenylmercuric acetate while the wild-type plants were

killed (Che et al., in prep.).

Black Willow (Salix nigra)

Black willow (Salix nigra) is small to medium size tree, ranging from 30 to 60

feet high in height, with a broad, irregular crown and a superficial root system (Harlow et

al., 1996). The tree grows on wet soils along the banks of streams and lakes, especially

in flood plains, where it is often found in pure stands associated with cottonwoods

13

(Harlow et al., 1996). Black willow is a fast-growing tree with a profuse root system and

high evapotranspiration rate (Persson and Lindroth, 1994). These deciduous trees have

been used commercially for pulp, charcoal and furniture manufacturing (Harlow et al.,

1996). Like other species of willow, black willows are easily established and propagated

from rooted cuttings (Harlow et al., 1996). To date, there are no de novo in vitro

propagation systems for black willows, but there are some reports of other species of

Salix that have been successfully propagated in vitro and genetically engineered.

Tissue Culture and Genetic Engineering of Salix spp

Some species of Salix have been micropropagated via axillary shoot

multiplication. Read et al. (1982) micropropagated Salix viminalis and Salix alba from

lateral buds gathered from the soft apical portion of young greenhouse stock plants.

Three auxins [indoleacetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-

dichlorephenoxy-acetic acid (2,4-D)], two cytokinins [kinetin (K) and benzyladenine

(BA)] and two types of media [woody plant medium (Lloyd and McCown, 1980) and MS

medium] were tested. Lower concentrations of auxins (<0.01 mg/L) combined with

cytokinins (K or BA) promoted callus formation, while the lack of auxin promoted shoot

formation. In another study, five Salix clones [(S. viminalis x S. purpurea (clone 077), S.

dasyclados Gigantea var. aquatica (clone 056), S. viminalis (clone 683), S. dasyclados

(clone 032), and S. caprea hybrid (clone L79-10)] were micropropagated in vitro from

the lateral buds of a 9-year old coppice plantation (Bergman et al., 1985). Different

levels of auxin (BA) were tested for their ability to promote shoot induction. Results

indicated that the optimum concentration was of BA was 0.5 µM. Salix carpea was

14

propagated in vitro from single node explants of field grown mature trees. Two different

media [SH medium (Schenk and Hilderbrandt, 1972) and ACM medium (Ahuja, 1983)]

and two different cytokinins (BA or K) were tested at different concentrations. The study

showed that the addition of plant growth regulators did not significantly increase shoot

production (Neuner and Beiderbeck, 1992). The hybrid Salix fragilis x S. lispoclados was

propagated in vitro from nodal cuttings of in vitro propagated seedlings. WPM

supplemented with different levels of BA was tested, and the concentration found to

produce a maximal increase in shoot proliferation was 0.2 mg/L (Agrawal and Gebhardt,

1994). Salix tarraconenesis was micropropagated in vitro from nodal segments of adult

trees growing in natural strands. Different levels of BA were tested to stimulate bud

break and shoot multiplication. WPM medium supplemented with a 4.9 µM BA

enhanced bud break, whereas lower concentrations (0.89 µM) promoted shoot

proliferation (Amo-Marcos and Lledo, 1995).

There are only two reports in the literature describing in vitro regeneration from

adventitious buds (de novo regeneration). Grönroos et al. (1989) reported somatic

embryogenesis of Salix viminalis from floral explants. Callus was initiated from pistils

and catkins on MS medium supplemented with BA and 2,4-D. Three types of callus

were regenerated: non-organogenic, rhizogenic and embryogenic. Unfortunately, only

one of the ten clones tested produced embryogenic callus, and complete plant

regeneration was not reported. Stoehr et al. (1989) induced callus formation and plant

regeneration from leaf explants of Salix exigua. Their results indicated that the greatest

callus growth resulted from WPM medium supplemented with 0.1 mg/L of BA and 0.5

15

mg/L 2,4-D. However, shoot proliferation was greatest for clones grown on MS medium

supplemented with the same concentrations of growth regulators.

Attempts to produce transgenic willow trees have not been completely successful.

Vahala et al. (1989) produced transformed calli of Salix viminalis; however, none of the

transclones were morphogenic. Salix lucida was putatively transformed via co-

cultivation of nodal segments with Agrobacterium, but analyses of the putative transgenic

plants failed to show the expected inserted DNA (Xing and Maynard, 1995).

Research Objectives

The project described in this thesis is divided into two independent research areas:

Genetic engineering and in vitro propagation.

The goal of the work in the first area was to create arsenic-resistant eastern

cottonwood trees by increasing thiol-sinks throughout the plant. To address this goal we

set two primary objectives: First, to generate transgenic eastern cottonwood trees

expressing the γ-ECS gene constitutively. Second, to perform toxicity assays to

determine the arsenic resistance of the transgenic plants.

The goal of the work in the second area was to establish a de novo in vitro

propagation system for Salix nigra. To achieve this goal we set one primary objective:

To determine if immature inflorescence explants had the potential to become competent

to generate adventitious shoots.

The following chapters describe the results of this project. Chapter II describes

how eastern cottonwood trees were engineered with the bacterial gene γ-glutamylcysteine

synthetase (γ-ECS), as well as their response to toxic levels of arsenate. Chapter III

16

presents the de novo in vitro propagation system established for black willow. Chapter

IV briefly summarizes the overall findings from this project and provides an overview of

the directions this work might follow in the project that will build upon this work.

17

Literature Cited

Agrawal, D.C. and Gebhardt, K. 1994. Rapid micropropagation of hybrid willow (Salix)

established by ovary culture. Plant Physiol. 143:763-765.

Ahuja, M.R. 1983. Somatic cell differentiation and rapid clonal propagation of aspen.

Silvae Genet. 32:131-135

Amo-Marco, J.B. and Lledo, M.D. 1995. In vitro propagation of Salix tarraconensis pau

ex Font Quer, an endemic and threatened plant. In Vitro Cell Dev. Biol.-Plant 32:42-46.

Baker, A.J.M. and Brooks, R.R. 1989. Terrestrial higher plants which hyper-accumulate

metallic elements- a review of their distribution, ecology and phytochemistry.

Biorecovery 1:81-126.

Bergman, L., Von Arnold, S., Eriksson, N. 1985. Effects of N6-benzyladenine on shoots

of five willow clones (Salix spp.) cultured in vitro. Plant Cell Tiss.Org. Cult..4: 135-144.

Bizily, S., Rugh, C.L., Summers, A.O. and Meagher, R.B. 1999. Phytoremediation of

methyl-mercury pollution: merB expression in Arabidopsis thaliana confers resistance to

organomercurials. Proc. Natl. Aca. Sci. 96:6808-6813.

Bizily, S., Rugh, C.L. and Meagher, R.B. 2000. Phytodetoxification of hazardous

organomercurials by engineered plants. Nature Biotech. 18:213-217.

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Burken, J.G. and Schnoor, J.L. 1997. Phytoremediation: uptake of atrazine and the role of

root exudates. J. Environ. Eng. 122:958-63.

Charest, P.J., Steward, D., and Budicky, P.L. 1992. Root induction in hybrid Populus by

Agrobacterium genetic transformation. Can. J. For. Res. 1:133-141.

Chaudhry, T.M., Hayes, W.J., Khan, A.G. and Khoo, C.S. 1998. Phytoremediation-

focusing on accumulator plants that remediate metal contaminated soils. Austra. J.

Ecotoxicol. 4:37-51.

Che, D., Meagher, R.B., Heaton, A.C.P., Lima, A., Rugh C.L., and Merkle, S.A.

Expression of mercuric ion reductase in eastern cottonwood (Populus deltoides) confers

mercuric ion reduction and resistance. Plant Biotechnology Journal (in press).

Chen, C.J., Chen, C.W., Wu, M.M., and Kuo, T.L. 1992. Cancer potential in liver, lung,

bladder and kidney due to ingested arsenic in drinking water. Br. J. Cancer. 66:888-892.

Chun, Y.W. Klopfenstein, N.B., McNabb, H.S., and Hall, R.B. 1988. Biotechnological

applications in Populus species. J. Kor. For. Soc. 77:467-43.

Cobbett, C.S. 2000. Phytochelatins and their role in heavy metal detoxification. Plant

Physiol. 123:825-832.

19

Coleman, G.D. and Ernst, S.G. 1989. In vitro shoot regeneration of Populus deltoides:

effect of cytokinin and genotype. Plant Cell Rep. 8:459-462.

Corseuil, H.X., and Moreno, F.N. 2001. Phytoremediation potential of willow trees for

aquifers contaminated with ethanol-blended gasoline. Wat. Res. 35 (12):3013-3017.

Cunningham, S.D., Anderson, T.A., Schwab, P. and Hsu, F.C. 1996. Phytoremediation of

soils contaminated with organic pollutants. Adv. Agronomy. 56:55-114.

Curtis, J. T. 1959. The vegetation of Wisconsin. Madison, WI: The University of

Wisconsin Press. pp 7116.

DeBlock, M. 1990. Factors influencing the tissue culture and the Agrobacterium

tumefaciens mediated transformation of hybrid aspen and poplar clones. Plant Physiol.

93:1110-1116.

Delhaize, E.P., and Ryan, R. 1995. Aluminum toxicity and tolerance in plants. Plant

Physiol. 107:315-321.

Dhankher, O.P., Li, Y., Rosen, B.P., Shi, J., Salt, D., Senecoff, J.F., Sashti, N.A. and

Meagher, R.B. 2002. Engineering tolerance and hyperaccumulation of arsenic in plants

by combining arsenate reductase and gamma-glutamylcysteine synthetase expression.

Nature Biotech.. 20(11):1140-1145.

20

Dinus, R.J., Stephens, C.J., and Chan, S. 1995. Agrobacterium tumefaciens-mediated

transformation of eastern cottonwood (Populus deltoides). In Proceedings of the

International Poplar Symposium: Poplar Biology and its Iimplications for Management

and Conservation. Seattle, WA, USA. p. 42.

Dix, M.E., Klopfenstein, N.B., Zhang, J.W., Workman, S.W., and Kim, M.S. 1996.

Potential use of Populus for phytoremediation of environmental pollution in riparian

zones. In: Klopfenstein, NB., Chun, YM., Kim, MS., and Ahuja, MRA. (eds).

Micropropagation, genetic engineering, and molecular biology of Populus. Gen. Tech.

Rep. RM-GTR-297, US, Dept.Agri-Fors. Serv., Fort Collins, Col. 206-211.

Doty, S.L., Shang,T.Q., Wilson, A.M., Tangen, J., Westergreen, A.D., Newman, L.A.,

Strand, S.E., and Gordon, M.P. 2000. Enhanced metabolism of halogenetad hydrocarbons

in transgenic plants containing mammalian cytochrome P-450 2E1. Proc. Natl. Acad. Sci.

USA 97(12):6287-6291.

Douglas, G.C. 1984. Formation of adventitious buds in stem internodes of Populus

species cultured in vitro on basal medium: influence of endogenous properties of

explants. J. Plant Physiol. 116:313-321.

Driver, J.A. and Kuniyuki, A.H. 1984. In vitro propagation of Paradox walnut rootstock.

HortScience. 19:507-509.

Emsley, J. 1991. The Elements. In: The Elements. Oxford University Press. NY, NY.

21

Ernst, S.G. 1993. In vitro culture of pure species non-aspen poplars. In: Ahuja, M.R. ed.

Micropropagation of woody plants. Dorrecht. Kluwer Academic Publishers. The

Netherlands. pp 195-208.

Fenner, P., Brady, W. and Patton, D. R. 1984. Observations on seeds and seedlings of

Fremont cottonwood. Desert Plants. 6(1):55-58.

Fletcher, J.S., and Hedge, R.S. 1995. Release of phenols by perennial roots and their

potential importance in bioremediation. Chemosphere. 31:3009-3016.

Fuente J.M., Ramirez-Rodriguez V., Cabrera-Ponce J.L., and Herrera-Estrella, L. 1997.

Aluminum tolerance in transgenic plants by alteration of citrate synthesis. Science.

276(5318):1566-1568.

Gordon, M., Choe, N., Duffy, J., Ekuan, G., Heilman, P., Muiznieks, I., Ruszaj, M.,

Shurtleff, B.B., Strand, S., Wilmoth, J. and Newman, L.A. 1998. Phytoremediation of

trichloroethelyne with hybrid poplars. Environ. Health Prespect.. 106 Supple 4:1000-

1004.

Grönroos, L., von Arnold, M. and Ericsson, T. 1989. Callus production and somatic

embryogenesis from floral explants of basket willow (Salix viminalis). J. Plant Physiol.

134:558-566.

22

Gullner, G., Komives, T. and Rennenber, H. 2001. Enhanced tolerance of transgenic

poplar plants overexpressing γ-glutamylcusteine synthetase towards chloroacetanilide

herbicides. J. Experimental Biol. 52 (358):971-979.

Han, K.H, Meilan, R., Ma, C. and Strauss S.H. 2000. An Agrobacterium tumefaciens

transformation protocol effective on a variety of cottonwood hybrids (genus Populus).

Plant Cell Rep. 19:315-320.

Hannink N, Rosser S.J., French C.E., Basran A., Murray J.A., Nicklin S., and Bruce N.C.

2001. Phytodetoxification of TNT by transgenic plants expressing a bacterial

nitroreductase. Nature Biotech. 19(12):1168-1172.

Heaton, A.C.P., Rugh, C.L., Wang W., and Meagher, R.B. Phytoremediation of mercury

and methyl-mercury polluted soils using genetically engineered plants. J. Soil Cont. 7(4):

497-509.




Harlow, W.M., Harrar, E.S., Hardin, J.W., and White F.M. 1996. Textbook of

Dendrology. McCraw Hill. 8th Ed. USA.

23

Hasselgren, K. 1988. Sewage sludge recycling in energy forestry. Proc. 5th International

Solid Waste Conference. Academic Press. NewYork, US. pp 189-197

Hedge, R.S. and Fletcher, J.S. 1996. Influence of plant growth stage and season on the

release of root phenolics by mulberry as related to development of phytoremediation

technology. Chemosphere 32:2471-2479.

Hauchelin, S.A., Harold., S.M., and Klopfenstein, N.B. 1997. Agrobacterium mediated

transformation of Populus x americana “Ogy” using the chimeric CaMV 35S-pin2 gene

fusion. Can J. For. Res. 27:1041-1048.

Ho, R.H., and Ray, Y. 1995. Haploid plants through anther culture in poplars. For. Ecol.

Mgmt. 13:133-142.

Hodson, R.W., Slater, F.M. and Randerson, P.F. 1994. Effects of digested sewage sludge

on short rotation coppice in the UK. Willow Vegetation Filters for Municipal Wastewater

and Sludges – A Biological Purification System. Proc. of a Study tour, Conference and

Workshop in Sweden. June. pp 113-118.

Hosner, J. F. and Minckler, L. S. 1963. Bottomland hardwood forests of southern Illinois-

regeneration and succession. Ecology. 44(1):29-41.

Kaiser, J. 1998. Toxicologists shed new light on old poisons. Science. 279:1850-1851.

24

Kang, H. and Chun, Y.W. 1997. Plant regeneration through organogenesis in poplar. In:

Klopfenstein, N.B. Chun, YW., Kim, MS., and Adhuja, MRA. (eds). Micropropagation,

genetic engineering and molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297,

US, Dept.Agri-Fors. Serv., Fort Collins, Col. pp 13-23.

Kelley, S.L., Alvarez, P.J.J., and Schnoor, J.L. 2000. Phytoremediation of 1,4-dioxane by

hybrid poplar trees. Water Environ. Research. 72(3):313-321.

Kim, M.S., Klopfenstein, N.B., and Chun, Y.W. 1997. Agrobacterium-mediated

transformation of Populus species. In: Klopfenstein, NB., Chun, YM., Kim, MS., and

Ahuja, MRA. (eds). Micropropagation , genetic engineering, and molecular biology of

Populus. Gen. Tech. Rep. RM-GTR-297, US, Dept.Agri-Fors. Serv., Fort Collins, CO.

pp. 51-59.

Kouider, M., Skirvin, R.M., Saladin, K.P., Dawson, J.O., and Jokela, J.J. 1984. A method

to culture immature embryos of Populus deltoides in vitro. Can. J. For. Res. 14:956-958.

Landberg, T. and Greger, M. 1996. Differences in uptake and tolerance to heavy metals

in Salix from unpolluted and polluted areas. Appl. Geochem. 11:175-180.

Larsen, P.B., Degenhardt, J., Stenzler, L.M., Howell, S.H. and Kochian, L.V. 1998.

Aluminum-resistant Arabidopsis mutant that exhibit altered patterns of aluminum

accumulation and organic acid release from roots. Plant Physiol. 117:9-18.

25

Leple, J.C., Brasileiro, A.C.M., Michel, M.F., Delmonte, F., and Jouanin, L. 1992.

Transgenic poplars: expression of chimeric genes using four different constructs. Plant

Cell Rep. 11:137-141.

Lloyd, G.B. and McCown, B.H. 1980. Commercially feasible micropropagation of

mountain laurel (Kalmia latifolia) by use of shoot-tip culture. Combined proceedings.

International Plant Propagators’ Society, Milltown, NJ. 30:421-437.

Ma, L.Q., Komart, K.M., Cong, T., Weihu, Z., Young, C. and Kennelley, E. 2001. A fern

that hyperaccumulates arsenic. Nature. 409:579.

Masheswari, N., Rajyalakshmi, K., Baweja, K., Dhir, S.K., Chowdhry, C.N., and

Maheshwari, S.C. 1995. In vitro culture of wheat and genetic transformation. Retrospect

and prospect. Crit. Rev. Plant Sci. 14(2):149-178.

Meagher, R.B. 2000. Phytoremediation of toxic elemental and organic pollutants. Curr.

Opin. Plant Biol. 3:153-162.

Meagher, R.B., and Rugh C.L. 1996. Phytoremediation of heavy metal pollution: Ionic

and methyl mercury. OECD Biotechnology for Water Use and Conservation Workshop.

pp 305-321.

26

Merian, E., and Haerdi, W. 1992. Metal compounds in environment and life. 4th

Interrelationship Between Chemistry and Biology. Northwood, US. pp 42-54.

Merkle, S.A. 1999. Application of in vitro culture for conservation of forest trees. In:

Plant Propagation and Conservation. Bowes, B (eds). Manson Publishing. London,

England. pp 119-130.

Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays

with tobacco tissue cultures. Phyisol. Plant. 15:473-497.

Neuner, H. and Beiderbeck, R. 1993. In vitro propagation of Salix carpea L. by single

node explants. Silvae Genet. 42 (6):308-310.

Newman, L.A., Strand, S.E., and Choe, N. 1997. Uptake and biotransformation of

trichloroethylene by hybrid poplars. Environ. Sci. Technol. 31:1062-1067.

Nielsen, K.H. 1994. Sludge fertilization in willow plantations. Willow Vegetation Filters

for Municipal Wastewater and Sludges – A Biological Purification System. Proceedings

of a Study Tour, Conference and Workshop in Sweden. pp 79-82.

Noctor, G., Arisi, A., Jouanin, L., Kuner, K., Rennenberg, H., and Foyer, C. 1998a.

Glutathione biosynthesis: metabolism and relationship to stress tolerance explored in

transformed plants. J. Expo. Bot. 49:623-647.

27

Noctor, G., Arisi, Ac., Jouanin, L., and Foyer, C.H. 1998b. Manipulation of glutathione

acid biosynthesis in the chloroplast. Plant Physiol. 118:471-478.

Noon, N., Leple, J.C and Pilate, G. 2002. Optimization of in vitro micro propagation and

regeneration for Populus x interamericana and Populus x euramericana hybrids (P.

deltoides, P. trichocarpa, and P. nigra). Plant Cell Rep. 20 (12):1150-1155.

Nriagu, J.O., and Pacyma, J.M. 1988. Quantitative assessment of worldwide

contamination of air, water, and soils by trace elements. Nature. 333:134-139.

Nriagu, E. 1994. Arsenic in the environment. Part I: Cycling and Characterization.

Nriagu (ed). John Wiley & Sons, Inc

Parsons, T.J., Sinkar, R.F., Steller, E.W., Nester, E.W. and Gordon, M.P. 1986.

Transformation of poplar by Agrobacterium tumefaciens. Bio. Tech. 4:533-536.

Persson, G. and Lindroth, A. 1994. Simulating evaporation from short-rotation forest:

variation within and between seasons. J. Hydrol. 156:21-45.

Pettru, K.L. 1992. Sludge, wastewater , leakage water, ash-a resource for energy forestry.

Energy forest as Vegetation Filter for Sludge, Wastewater, Leachates and Bioash. 47:7-

19.

28

Pignatello, J.J. 1989. Reaction and movement of organic chemicals in soils. Sawahney,

B.L., and Brown, K (eds). Soil Science Society of America. pp 45-80.

Pilon-Smits, E.A.H., Souza, M.P., Lytle, C.M., Shang, C., Lugo, T., and Terry, N. 1998.

Selenium volatilization and assimilation by hybrid poplar (Populus tremula x alba). J.

Exp. Bot. 49(328):1889-1892.

Pilon M., Owen J.D., Garifullina G.F., Kurihara T., Mihara H., Esaki N., and Pilon-Smits

E.A.H. 2003. Enhanced selenium tolerance and accumulation in transgenic Arabidopsis

expressing a mouse selenocysteine lyase. Plant Physiol. 131(3):1250-1257.

Prakash, C.S. and Thielges, B.A. 1989. Plantlets from leaf discs of Populus deltoides.

Poster abstract. In: Hanvoer, J.W. and Keathly, D.E. (eds). Genetic Manipulation of

Woody Plants. Plenum Press, New York. pp 482.

Perttu, K.L. 1989. Short-rotation forestry: an alternative energy source? Modelling

Energy Forestry. Growth, Water Relations and Economics. pp 181-186.

Punshon, T. and Dickinson, N. 1997. Acclimation of Salix to metal stress. New

Physiologist. 137 (2):303-314.

Rashkin, I. 1996. Plant genetic engineering may help with environmental cleanup. Proc.

Natl. Acad. Sci. USA 93:164-166.

29

Rauser, W.E. 1995. Phytochelatins and related peptides: structure, byosinthesis, and

function. Plant Physiol. 109:1141-1149.

Reeves, R.D., Baker, A.J. and Brooks, R.R. 1995. Abnormal accumulation of trace

metals by plants. Mining Environ. Management. 3:4-8.

Riddell-Black, D., Rowlands, C. and Snelson, A. 1995. Heavy metal uptake from sewage

sludge amended soil by Salix and Populus species grown for fuel. 14th Annual

Symposium on Current Topics in Plant Biochemistry, Physiology and Molecular

Biology. April 19-22, 1995. Columbia, Mo. pp 51-52.

Robison, B., Millis, T., Clothier, B., Green, S., and Fung, L. 2002. Cadmium

accumulation by willow clones used for soil conservation, stock fodder, and

phytoremediation. Australian Journal of Soil Research. 40(8):1331-1337.

Rugh, C.L., Senecoff, J.F., Meagher, R.B. and Merkle, S.A. 1998. Development of

transgenic yellow poplar for mercury phytoremediation. Nature Biotech. 16:925-928.

Rugh, C.L., Wilde, H.W., Stack, N.M., Thompson, D.M., Summers, A.O., and Meagher,

R.B. 1996. Mercuric ion reduction and resistance in transgenic Arabidposis thaliana

plants expressing a modified bacterial merA gene. Proc. Natl. Acad. Sci. USA 93:3182-

3187.

30

Saito, A. 1980. Medium for shoot formation from somatic callus tissues in Populus. J.

Jap. For. Soc. 62(7):270-272.

Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Viatchslav, D., and Ensley, B.D. 1995.

Phytoremediation: a novel strategy for the removal of toxic metals from the environment

using plants. Bio-Technology. 13:468-474.

Salt, D.E., Smith, R.D., and Raskin, I. 1998. Phytoremediation. Ann. Review. Plant

Physiol. Plant Mol. Biol. 49:643-668.

Savka, M.A., Dawson, J.O., Jokela, J.J., and Skirvin, R.M. 1987. A liquid culture method

for rescuing immature embryos of eastern cottonwood. Plant Cell Tiss. Org. Cult. 10:221-

226.

Schnoor, J., Light, L.A., McCutchenson, S.C., Wolfe, N.L. and Carreira, L.H. 1995.

Phytoremediation of organic and nutrient contaminants. Environ. Sci. Technol. 7:318-

323.

Schenck, R.U. and Hilderbrandt, A.C. 1972. Medium and techniques for induction and

growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50:199-

204.

31

Stoehr, M.U., Cai, M. and Zsuffa, L. 1989. In vitro plant regeneration via callus culture

of mature Salix exigua. Can. J. For. Res. 19:1634-37.

Travis, C.C., and Doty, C.B. 1990. Can contaminated aquifers at superfund sites be

remediated? Environ. Sci. and Technol. 24:1464-1466.

Thompson, P.L., Ramer, L.A., and Schnoor, J.L. 1999. Hexahydro-1,3,5-trinitro-1,3,5-

triazine translocation in poplar trees. Environ. Toxicol. Che. 18(2):279-284.

Uddin, M.R., Meyer, M.M. and Jokela, J.J. 1998. Plantlet production from anthers of

eastern cottonwood (Populus deltoides). Can. J. For. Res. 18:937-941.

U.S. Army Toxic and Hazardous Materials Agency. 1987. Heavy metal contaminated soil

treatment. Interim Technical Report. In: Heavy metal contaminated soil treatment.

Interim Technical Report. Roy F. Weston Inc., West Chester, Pennsylvania.

Vahala, T., Stabel, P. and Eriksson, T. 1989. Genetic transformation of willows (Salix

spp) by Agrobacterium tumefaciens. Plant Cell Rep. 8:55-58.

Wang, X., Newman, L.A., Gordon, M.P. and Strand, S.E. 1999. Biodegradation of carbon

tetrachloride by poplar trees: results from cell culture and field experiments. Fifth

International In-Situ and On-Site Bioremediation Symposium.. San Diego, Ca, USA.

5(6):133-138

32

Westrick, J.J., Mello J.W. and Thomas, R.F. 1984. The groundwater supply survey. J.

Am. Water Works Assoc. 5:52-59.

Xiang, C., Werner, B.L., Christense, E.M., and Oliver, D.J. 2001. The biological

functions of glutathione revisited in Arabidposis transgenic plants with altered

glutathione levels. Plant Physiol. 126(2): 564-574.

Xing, Z. and Maynard, C.A. 1995. Producing transgenic shining willow (Salix lucida)

shoots from stems segments via Agrobacterium tumefaciens transformation. In Vitro

Cell. Dev. Biol. 31:223.

Yamada T., Ishige T., Shiota N., Inui H., Ohkawa H. and Ohkawa Y. 2002. Enhancement

of metabolizing herbicides in young tubers of transgenic potato plants with the rat

CYP1A1 gene. Theor Appl Genet. 105(4):515-520.

Zhu, Y.L., Pilon-Smiths, E.A.H., Tarun A.S., Stefan, U.W., Jouanin, L. and Terry, N.

1999a. Cadmium tolerance and accumulation in Indian mustard is enhanced by

overexpressing γ-glumamylcystein synthetase. Plant Physiol. 121:1169-1177.

Zhu, Y.L., Pilon-Smits, E.A.H., Jouanin, L. and Terry, N. 1999b. Overexpression of

glutathione synthetase on Indian mustard enhances cadmium accumulation and tolerance.

Plant Physiol. 119(1):73-80.

33

CHAPTER II

ENHANCED ARSENIC TOLERANCE OF TRANSGENIC EASTERN

COTTONWOOD PLANTS OVEREXPRESSING γ-GLUTAMYLCYSTEINE

SYNTHETASE

Over the past century, mining, agriculture, manufacturing and urban activities

have all contributed to extensive soil and water contamination (Cunningham et al., 1995).

High on the list of toxic pollutants affecting the health of millions of people worldwide is

arsenic (Nriagu, 1994). Arsenic is a naturally occurring element widely distributed on

the earth's crust (Emsley, 1991). In the environment, arsenic combines with oxygen,

chlorine, and sulfur to form inorganic arsenic compounds (Nriagu, 1994). These

extremely toxic metalloids, classified as “group A” human carcinogens, cause skin

lesions, lung, kidney and liver cancer, and damage to the nervous system (US EPA 1996:

www.epa.gov/ogwdw/ars/arsenic.htm1). Traditional arsenic remediation methods include

oxidation, co-precipitation, filtration, adsorption, ion exchange and reverse osmosis.

Unfortunately, managing contaminated soils, sludge, and groundwater is costly and the

environmental damage is very high (U.S. Army Toxic and Hazardous Materials Agency,

1987). The enormous costs and the ineffectiveness of traditional methods have prompted

the development of alternative remediation techniques.

Phytoremediation is an alternative remediation method that uses plants to extract,

sequester or detoxify pollutants from soil, water and air (Rashkin, 1996). This innovative

34

technology offers advantages over conventional physical techniques. It is estimated that

phytoremediation costs are between two- and four-fold less than existing remediation

technologies (Meagher and Rugh, 1996). In addition, this approach is an ecologically

preferable method because it reclaims soil at the site by recycling it in a biologically safe

manner, instead of disposing of it at a storage site (Salt et al., 1995).

Eastern cottonwood (Populus deltoides) is a good candidate for phytoremediation

purposes for a number of reasons. First, it is a fast-growing, high biomass (up to 10-30

m3/ha/year of wood on a short rotation of six to eight years) producing tree with an

extensive root system (300,000 km/ha) (Fenner et al., 1984; Gordon et al. 1997). Second,

cottonwoods can be easily established and propagated by rooted cuttings (Harlow et al.,

1996). Third, they are amenable to tissue culture manipulation and genetic engineering

(Ernst, 1993; Kang and Chun, 1997; Saito 1980; Prakash and Thielges, 1988; Douglas

1984; Coleman and Ernst, 1989; Ho and Ray, 1985; Uddin et al., 1988; Koudier et al.,

1984; Savka et al., 1987; Kim et al., 1997; Han et al., 2000; Parsons et al., 1986; De

Block 1990; Wang et al., 1994; Charest et al., 1992; Hauchelin et al., 1997; Noon et al.,

2002). Finally, research has shown that species of this genus are capable of sequestering

pollutants or metabolizing them into less toxic forms. Hybrid poplars have been used to

remove atrazine (Burken and Schnoor, 1997), trichloroethylene (Newman et al., 1997),

trinitrotoluene (Thompson et al., 1998), dioxane (Kelley et al., 2000) and selenium

(Pilon-Smits et al., 1998) from contaminated soil.

To date, there is only one report of a natural arsenic hyper-accumulating plant.

Pteris vittata, a fern indigenous to the southern parts of the U.S., has the capacity to

hyper-accumulate arsenic to very high levels (Ma et al., 2001). Unfortunately, the

35

enzymes responsible for arsenic hyper-accumulation in this plant are not yet available for

manipulation into other plant species. Although specific arsenic hyperaccumulation

enzymes have not been isolated, increased tolerance and accumulation of arsenic has

been reported in plants over-expressing the bacterial enzyme γ-glutamylcysteine

synthetase (Dhankher et al., 2002).

Gamma-glutamylcysteine synthetase (γ-ECS) catalyzes the initial reaction in a

three-step enzymatic pathway involved in the synthesis of phytochelatins (Zhu et al.,

1999b) (Figure 2). Phytochelatins are member of a small class of Cys sulfhydryl residue-

rich peptides [γ-glumatylcysteine (γ-EC), glutathione (GSH) and phytochelatins (PC)]

that play an important role in the detoxification and sequestration of thiol-reactive heavy

metals (Noctor et al., 1998; Zhu et al., 1999b; Xiang et al., 2001)..

Gly γ-Glu-Cys Glu + Cys → γGlu-Cys → Gly- γGlu-Cys → Gly (γGlu-Cys)n γECS GS PS

Figure 2. Phytochelatin synthesis pathway. Three enzymes constitute the phytochelatin

biosynthetic pathway: γ-glutamylcysteine synthetase (γECS), glutathione synthetase (GS)

and phytochelatin synthetase (PSs).

γ-Gluamylcysteine synthetase produces γ-EC dipeptides for subsequent synthesis

of the phytochelatins, and is believed to be limiting step for both GSH and PCs

production in the absence of heavy metals (Noctor et al., 1998). Genetically engineered

Arabidopsis thaliana plants over-expressing the Escherichia coli gene γ-ECS from a

36

strong constitutive actin promoter (ACT2p) were highly resistant to arsenic (300 µM)

compared to wild-type plants (Dr. Yujing Li, Genetics Department, University of

Georgia, personal communication). These results show that γ-ECS manipulation in

plants may be a promising approach for development of systems to address arsenic

contamination by phytoremediation.

The main goal of the research reported here was to increase the arsenic tolerance

capacity of eastern cottonwood trees. Two primary objectives were set to achieve this

goal: 1) generate transgenic eastern cottonwood trees expressing the γ-ECS gene

constitutively; 2) perform toxicity assays to determine the levels of arsenic resistance of

transgenic trees in comparison to non-transformed controls.

Materials and Methods

Plant material and tissue culture. In vitro shoot cultures of eastern cottonwood (clone

C-175) were kindly supplied by Dr. H. D. Wilde (MeadWestvaco Corp., Summerville,

SC). These cultures were maintained on Driver and Kuniyuki Walnut (DKW) medium

(Driver and Kuniyuki, 1984) in GA-7 vessels (Magenta Corp.) at 25° C under a 16 hr

photoperiod (100 µmol·m-2·s-1).

Gene construct and bacteria culture. The modified bacterial γ-ECS gene construct,

pBINACT2/γ-ECS, was kindly provided by Dr. Yujing Li (Genetics Department,

University of Georgia). It contained the E. coli γ-ECS gene driven by a strong

constitutive actin promoter (ACT2p), polyadenylation sequences, and the nptII gene,

conferring kanamycin resistance, driven by the CaMV 35S promoter. pBINACT2/γ-ECS

was electroporated into Agrobacterium tumerfaciens strain C5851 (GIBCO/BRL). Prior

37

to plant transformation, the A. tumefaciens carrying the γ-ECS gene was grown overnight

(O.D.600 ≈ 0.9) at 28°C on liquid YEP medium [(10g/L Bacto-peptone (DIFCO

Laboratories), 10 g/L yeast extract, 5 g/L sodium chloride)], in the presence of 50 mg/L

kanamycin, 25 mg/L gentamycin and 50 mg/L rifampicin.

Plant transformation and regeneration. Preliminary experiments were conducted to

test different variables that could affect transformation frequency. The variables tested

were A. tumefaciens initial culture optical density (O.D.600 of 0.7, 0.8, 0.9 and 1.4), liquid

inoculation times (5, 10, 15, 100, and 120 minutes) and the effect of acetosyringone [0 or

200mM (Sigma)]. Following these preliminary experiments, we adopted the protocol

detailed below, which produced all the γ-ECS transclones that were part of this study.

Young leaves of eastern cottonwood (≈1cm in length) were isolated from

proliferating in vitro shoot cultures, and a total of two hundred leaf sections (5 x 5 mm)

were cut and held in Agrobacterium induction medium (10 mM galactose and 0.25mg/L

MES, pH5.0) to prevent tissue desiccation. The bacterial culture, previously grown

overnight, was adjusted with Agrobacterium induction medium to an O.D. 600 ≈ 0.3. Leaf

sections were immersed in the adjusted bacterial culture and shaken at 100 rpm for 90

minutes. After incubation, leaf sections were blotted dry with filter paper and transferred

to semi-solid shoot induction medium [DKW medium supplemented with 1 mg/L

naphthaleneactic acid (NAA) and 1 mg/L benzylaminopurine (BA)]. Ten leaf sections

were cultured per 100 mm petri plate on a total of 20 plates. After three days of co-

cultivation in the dark at 25° C, leaf sections were washed three times in sterile distilled

water for five minutes, shaking at 200 rpm. After the washes, leaf sections were blotted

dry and transferred to DKW selection medium containing 1 mg/L NAA, 1 mg/L BA, 50

38

mg/L kanamycin and 400 mg/L Timentin (Smithkline Beechman Pharmaceuticals) to

kill residual bacteria. Cultures were maintained at 25° C with a 16 hr photoperiod and

transferred onto fresh selection medium every two weeks. For plantlet regeneration,

adventitious shoots arising from leaf disk explants and reaching 1 cm in length were

excised and transferred into in GA-7 vessels (Magenta Corp.) containing 100 ml of

semisolid rooting medium (basal DKW medium) supplemented with 50 mg/L

kanamycin.

Genomic DNA analysis. Genomic DNA-PCR (polymerase chain reaction) analysis was

used to identify the γ-ECS transgene among the kanamycin-resistant lines obtained.

DNA for PCR was extracted from leaf tissues following the Extract-N-Amp plant DNA

isolation protocol (Sigma). The PCR primers used were sense primer (ECS-49F), 5’-

TGA CGC ACA AAT GGA TTA CTA C-3’, and antisense primer (ECS-930R), 5’-AAC

AGA TAA GGA ATG ACC CAA C-3’. The PCR products were separated by

electrophoresis in buffer (TAE 1X) on a 1% agarose gel, stained with ethidium bromide,

and detected under ultraviolet light.

Western Blot Analysis. Western blot analysis was used to examine the expression of

γ−glumatylcysteine synthetase in transgenic eastern cottonwood plantlets. Leaves from

transgenic lines and wild-type plantlets were collected in Eppendorf tubes, ground in

liquid nitrogen and resuspended in 2X SDS-PAGE sample buffer (100mM Tris-HCL pH

6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10mM β-mercaptoethanol and

0.2% bromophenol blue). The mixture was centrifuged for ten minutes at 10,000 rpm.

Supernatants were transferred into a new tube and boiled for five minutes. Protein

samples were separated on a 10% SDS-PAGE gel (Laemmli, 1970). Resolved proteins

39

were electroblotted onto a nitrocellulose membrane (Amersham Pharmacia Biotech)

using a Trans–Blot (BIO-RAD) according to the manufacturer’s instructions. Blots were

probed with ECS-specific monoclonal antibody, Mab ECS (Li et al., 2001), followed by

a secondary polyclonal sheep antimouse IgG conjugated with horseradish peroxidase

(Amersham Pharmacia). Signals were visualized using chemiluminescence (ECL

Western Blotting Analysis System, Amersham Lifesciences).

Toxicity assays. Two experiments were conducted to assess the arsenate resistance of

the γ-ECS eastern cottonwood clones generated. The first toxicity experiment tested the

relative callus induction capacities of leaf sections isolated from the γ-ECS transclones

and from wild-type plantlets. First, to establish the sensitivity of wild-type eastern

cottonwood leaves to arsenate, we tested the ability of leaf sections to survive and

produce callus on medium with sodium arsenate. Leaf sections (5 x 5 mm) from wild-

type plants were cultured, nine per plate, in 100 mm plastic Petri plates containing 25 ml

of semi-solid shoot induction medium supplemented with nine different concentrations

(0, 100, 200, 300, 400, 500, 600, 700 and 800 µM) of sodium arsenate. Plates were

incubated in the light at 25° C for eight weeks and scored based on their color and ability

to produce callus. Following the sensitivity assay, leaf sections (5 x 5 mm) were isolated

from each of the eight γ-ECS transclones and from wild-type plants and cultured, nine per

plate, in 100 mm plastic Petri plates containing 25 ml of shoot induction medium with or

without 800 µM sodium arsenate. Plates were incubated in the light at 25° C for four

weeks and scored for color and callus induction.

The second toxicity experiment tested the relative abilities of axillary shoots from

the γ-ECS transclones and the wild-type to survive and produce adventitious roots on

40

rooting medium supplemented with arsenate. First, as with the leaf sections, a sensitivity

assay was conducted, in which wild-type axillary shoots were cultured, nine per vessel, in

GA-7 vessels (Magenta Corp.) containing rooting medium (basal DKW medium)

supplemented with nine different sodium arsenate concentrations (0, 100, 200, 300, 400,

500, 600, 700 and 800 µM) per treatment. Axillary shoots were evaluated after 8 weeks

for stem and leaf color and ability to form adventitious roots. Following the sensitivity

assay, nine axillary shoots from each of three selected γ-ECS lines (E-1, E-2 and E-3) and

from the wild-type were cultured in GA-7 vessels containing 100 ml of rooting medium

(basal DKW medium) with or without 800 µM sodium arsenate. Vessels were

maintained in the light at 25° C for six weeks before scoring the explants for leaf and

stem color and ability to form adventitious roots.

Statistical Analysis. To determine whether the over-expression of γ-ECS in eastern

cottonwood trees significantly increased their arsenate resistance, contingency table

analysis (Ott 1993) was performed on the rooting data collected from the axillary shoot

toxicity experiment described above.

Results

The pBINACT2/γ-ECS construct was used to transform eastern cottonwood leaf

sections via Agrobacterium-mediated transformation. A total of 19 independent

kanamycin-resistant shoots were isolated and transferred to basal DKW medium

containing 50mg/L kanamycin for rooting. Genomic DNA-PCR analysis that of the 19

kanamycin resistant plantlets assayed, 8 had the expected 439 base pair γ-ECS PCR

product (Figure 3). No product was observed with DNA from wild-type plants. Based

41

on the original 200 explants inoculated in the experiment, the overall transformation

frequency was 0.4 %.

Leaf samples of all PCR positive γ-ECS lines were assayed for γ-glutamylcysteine

synthetase protein. Western blotting demonstrated that all eight γ-ECS lines contained a

protein of the same molecular mass (57 kD) as that from confirmed transgenic γ-ECS

Arabidopsis thaliana plants provided by Dr. Yujing Li (Genetics Department, University

of Georgia; Figure 4). No γ-ECS band was detected in wild-type plant extracts or in

protein extracts from Agrobacterium tumefaciens carrying the γ-ECS gene (data not

shown).

MW

L

Bla

nk

MW

L

WT

E-1

E-2

E-3

E-4

E-5

E-6

E-7

E-8 DN

A C

ontro

l (+)

439 bp

γ-ECS lines

Figure 3. PCR analysis of genomic DNA from putative γ-ECS-transformed and wild-

type (WT) eastern cottonwood leaves. The expected 439 bp γ-ECS product for the

genomic DNA-PCR is seen in lanes E-1 through E-8 (transformed eastern cottonwoods)

and in the DNA positive control (PCR product generated from the pBINACT2/γ-ECS

42

construct). DNA extracted from wild-type eastern cottonwood leaves and a water blank

were included as negative controls.

57 kD

ECS

cont

rol (

+)

Bla

nk

E-1 -2

-3

-8 T -7

-6

-5

-4

Figure 4. Western blot analysi

extracts from γECS-transgenic a

ECS monoclonal antibody and

purified ECS protein isolated from

γ-ECS gene (57 kD).

Sensitivity experiments in

little visible effect on leaf sectio

and adventitious root formation u

sodium arsenate, leaf sections be

chlorotic and the bases of the a

sodium arsenate for the toxicity

medium supplemented with 800

remained green and began to de

E E E WEEEEs of γ-ECS expression. Blots containing crude protein

nd untransformed plants (WT) were probed with anti-

visualized using chemiluminescence. Arrow indicates

confirmed transgenic A. thaliana plants expressing the

dicated that levels of arsenate lower than 800 µM had

n survival, callus development, axillary shoot survival

p to 8 weeks. Following 4 weeks of culture on 800 µM

gan to bleach, leaves on the axillary shoots began to turn

xillary shoots darkened. Therefore we chose 800 µM

experiments. A month after being cultured on the

µM arsenate, leaf sections from γ-ECS transgenic lines

velop callus (Figure 5A), while the leaf sections from

43

wild-type plantlets showed no evidence of callus and appeared chlorotic (Figure 5B).

After 30 days on medium containing 800 µM arsenate, wild-type adventitious shoots did

not form roots and their leaves appeared chlorotic (Figure 6A). The γ-ECS shoots

appeared similar to those maintained on medium with no arsenate and adventitious roots

began to appear 21 days after initial culture (Figure 6B). The difference between the γ-

ECS lines and the wild-type plants in their abilities to produce adventitious roots in

medium with 800 µM arsenate was statistically significant (p < 0.001).

B A

Figure 5. Leaf sections cultured one month on shoot induction medium containing 800

µM arsenate. Leaf sections from γ-ECS transformed eastern cottonwood plantlets began

to form callus 30 days after initiation (A). Leaf sections from wild-type eastern

cottonwood plantlets were chlorotic and bleached after 30 days on arsenate (B).

44

A B

Figure 6. Transgenic eastern cottonwood expressing γ-ECS and wild-type shoots

cultured on rooting medium containing 800 µM arsenate. Wild-type shoots darkened at

the base, failed to develop adventitious roots and leaves became chlorotic (A). Transgenic

γ-ECS shoots developed roots approximately after 15 days of culture and leaves remained

dark green (B).

Discussion

The goal of the current study was to genetically engineer eastern cottonwood trees

to over-express the E. coli γ-ECS gene and enhance their resistance to arsenate by

increasing the thiol-sinks throughout the plant. To achieve this, eastern cottonwood trees

were transformed via Agrobacterium-mediated transformation. The results indicated

that transgenic eastern cottonwood plants over-expressing γ-ECS were significantly more

tolerant of arsenate than wild-type plants. Similar results have been reported for A.

thaliana plants. Dhankher et al., (2002) engineered A. thaliana to over-express γ-ECS,

and the transgenic plants were highly tolerant of arsenate and mercuric ions. In another

study, over-expression of γ-ECS increased the herbicide resistance of transgenic hybrid

45

poplar, Populus tremula x P. alba (Gullner et al., 2001). Prior to this study, the use of

transgenic eastern cottonwood trees for arsenic phytoremediation had never been

reported.

The increase in arsenic tolerance in γ−ECS eastern cottonwoods may be explained

by an elevation in glutathione and phytochelatin levels. The concentration of these

peptides was not measured, however, one study showed increased concentrations of

glutathione and phytochelatins in hybrid poplars engineered to over-express the bacterial

of γ-ECS showed (Noctor et al., 1998a). These metal binding peptides have high affinity

for arsenite (Schmoeger et al., 2000), the reduced form of arsenate. Arsenate has been

shown to be naturally reduced in plant roots to arsenite (Pickering et al., 2000).

In a recent report (Dhankher et al., 2002), A. thaliana plants were engineered to

co-express γ-ECS and a bacterial arsenate reductase (ArsC). Plants co-expressing these

two enzymes had a higher resistance to arsenate than either wild-type plants or

engineered Arabidopsis plants expressing only γ-ECS. The increased arsenic resistance

was achieved by altering the electrochemical state of arsenic, reducing arsenate to

arsenite, which has a strong affinity to thiol-groups. Future work with eastern

cottonwood may involve the re-transformation of the lines produced in this work with the

ArsC gene, to determine whether co-expression of these two enzymes further enhances

the tree’s arsenic resistance.

46

Literature Cited

Burken, J.G. and Schnoor, J.L. 1997. Phytoremediation: uptake of atrazine and the role of

root exudates. J. Environ. Eng. 122:958-63.

Charest, PJ., Steward, D., and Budicky, P.L. 1992. Root induction in hybrid Populus by

Agrobacterium genetic transformation. Can. J. For. Res. 1:133-141.

Coleman, G.D. and Ernst, S.G. 1989. In vitro shoot regeneration of Populus deltoides:

effect of cytokinin and genotype. Plant Cell Rep. 8:459-462.

Cunningham, S.D., Anderson, T.A., Schwab, P. and Hsu, F.C. 1996. Phytoremediation of

soils contaminated with organic pollutants. Adv. Agronomy. 56:55-114.

DeBlock, M. 1990. Factors influencing the tissue culture and the Agrobacterium

tumefaciens mediated transformation of hybrid aspen and poplar clones. Plant Physiol.

93:1110-1116.




Nature Biotech. 20(11):1140-5.

47

Douglas, G.C. 1984. Formation of adventitious buds in stem internodes of Populus

species cultured in vitro on basal medium: influence of endogenous properties of

explants. J. Plant Physiol. 116:313-321.

Driver, J.A. and Kuniyuki, A.H. 1984. In vitro propagation of Paradox walnut rootstock.

HortScience. 19:507-509.

Emsley, J. 1991. The Elements. In: The Elements. Oxford University Press. NY, NY.

Ernst, S.G. 1993. In vitro culture of pure species non-aspen poplars. In: Ahuja, M.R. ed.

Micropropagation of woody plants. Dorrecht. The Netherlands: Kluwer Academic

Publishers. Pp. 195-208.

Fenner, P., Brady, W. and Patton, D. R. 1984. Observations on seeds and seedlings of

Fremont cottonwood. Desert Plants. 6(1):55-58.

Gordon, M., Choe, N., Duffy, J., Ekuan, G., Heilman, P., Muiznieks, I., Ruszaj, M.,

Shurtleff, B.B., Strand, S., Wilmoth, J and Newman, L.A. 1998. Phytoremediation of

trichloroethelyne with hybrid poplars. Environ. Health Perspect. 106(4):1000-1004.

Gullner, G., Komives, T. and Rennenber, H. 2001. Enhanced tolerance of transgenic

poplar plants overexpressing γ-glutamyl synthetase towards chloroacetanilide herbicides.

J. Exp. Biol. 52 (358): 971-979.

48




Harlow, W.M., Harrar, E.S., Hardin, J.W., and White F.M. 1996. Textbook of


Hauchelin, S.A., Harold., S.M., and Klopfenstein, N.B. 1997. Agrobacterium mediated

transformation of Populus x Americana “Ogy” using the chimeric CaMV 35S-pin2 gene

fusion. Can J. For. Res. 27:1041-1048.

Ho, R.H., and Ray, Y. 1995. Haploid plants through anther culture in poplars. For. Ecol.

Mgmt. 13:133-142.

Kang, H. and Chun, Y.W. 1997. Plant regeneration through organogenesis in poplar. In:

Klopfenstein, N.B. Chun, YW., Kim, MS., and Adhuja, MRA. (eds). Micropropagation,

genetic engineering and molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297,

US, Dept.Agri-Fors. Serv., Fort Collins, CO. pp. 13-23.

Kelley, S.L., Alvarez, P.J.J., and Schnoor, J.L. 2000. Phytoremediation of 1,4-dioxane by

hybrid poplar trees. Water Environ. Research. 72(3):313-321.

49

Kim, M.S., Klopfenstein, N.B., and Chun, Y.W. 1997. Agrobacterium-mediated

transformation of Populus species. In: Klopfenstein, NB., Chun, YM., Kim, MS., and

Ahuja, MRA. (eds). Micropropagation , genetic engineering, and molecular biology of

Populus. Gen. Tech. Rep. RM-GTR-297, US, Dept.Agri-Fors. Serv., Fort Collins, CO.

pp. 51-59.

Kouider, M., Skirvin, R.M., Saladin, K.P., Dawson, J.O., and Jokela, J.J. 1984. A method

to culture immature embryos of Populus deltoides in vitro. Can. J. For. Res. 14:956-958.

Laemmli, U.K. 1970. Cleavege of structural proteins during the assembly of

bacteriophage T4. Nature. 227:680-685.

Li, Y., Kandasamy, M.K., and Meagher, R.B. 2001. Rapid Isolation of Monoclonal

Antibodies. Monitoring enzymes in the phytochelatin synthesis pathway. Plant Physiol.

127:711-719.



Meagher, R.B., and Rugh C.L. 1996. Phytoremediation of heavy metal pollution: Ionic

and methyl mercury. OECD Biotechnology for Water Use and Conservation Workshop.

pp 305-321.

50

Newman, L.A., Strand, S.E., and Choe, N. 1997. Uptake and biotransformation of

trichloroethylene by hybrid poplars. Environ. Sci. Technol. 31:1062-1067.

Noctor, G., Arisi, A., Jouanin, L., Kuner, K., Rennenberg, H., and Foyer, C. 1998a.

Glutathione biosynthesis: metabolism and relationship to stress tolerance explored in

transformed plants. J. Exp. Bot. 49:623-647.

Noon, N., Leple, J.C. and Pilate, G. 2002. Optimization of in vitro micro propagation

and regeneration for Populus x interamericana and Populus x euramericana hybrids (P.

deltoides, P. trichocarpa, and P. nigra). Plant Cell Rep. 20(12):1150-1155.

Nriagu, E. 1994. Arsenic in the environment. Part I: Cycling and Characterization.

Nriagu (ed). John Wiley & Sons, Inc.

Parsons, T.J., Sinkar, R.F., Steller, E.W., Nester, E.W., and Gordon, MP. 1986.

Transformation of poplar by Agrobacterium tumefaciens. Bio. Tech. 4:533-536.

Pilon-Smits, E.A.H., de Souza, M.P., Lytle, C.M., Shang, C., Lugo, T., and Terry, N.

1998. Selenium volatilization and assimilation by hybrid poplar (Populus tremula x

alba). J. Exp. Botany. 49(328):1889-1892.

51

Prakash, C.S. and Thielges, B.A. 1989. Plantlets from leaf discs of Populus deltoides.

Poster abstract. In; Hanvoer, JW and Keathly, DE (eds). Genetic Manipulation of Woody

Plants. Plenum Press, New York. p. 482.



Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Viatchslav, D., and Ensley, B.D. 1995.

Phytoremediation: a novel strategy for the removal of toxic metals from the environment

using plants. Bio-Technology. 13:468-474.

Saito, A. 1980. Medium for shoot formation from somatic callus tissues in Populus. J.

Jap. For. Soc. 62 (7):270-272.

Savka, M.A., Dawson, J.O., Jokela, J.J., and Skirvin, R.M. 1987. A liquid culture method

for rescuing immature embryos of eastern cottonwood. Plant Cell Tiss. Org. Cult. 10:221-

226.

Schmoger, M.E.V., Oven, M., and Grill, E. 2000. Detoxification of arsenic by

phytochelatins in plants. Plant Physiol. 122:793-801.

Thompson, P.L., Ramer, L.A., and Schnoor, J.L. 1999. Hexahydro-1,3,5-trinitro-1,3,5-

triazine translocation in poplar trees. Environ. Toxicol. Che. 18(2):279-284.

52

Uddin, M.R., Meyer, M.M., and Jokela, J.J. 1998. Plantlet production from anthers of

eastern cottonwood (Populus deltoides). Can. J. For. Res. 18:937-941.




Wang, X., Newman, L.A., Gordon, M.P. and Strand, S.E. 1999. Biodegradation of carbon

tetrachloride by poplar trees: results from cell culture and field experiments. Fifth

International In-Situ and On-Site Bioremediation Symposium. San Diego, Ca, USA.

5(6):133-138

Xiang, C., Werner, B.L., Christense, E.M., and Oliver, D.J. 2001. The biological

functions of glutathione revisited in Arabidposis transgenic plants with altered

glutathione levels. Plant Physiol. 126(2):564-574.

Zhu, Y.L., Pilon-Smits, E.A.H., Jouanin, L. and Terry, N. 1999b. Overexpression of

glutathione synthetase on Indian mustard enhances cadmium accumulation and tolerance.

Plant Physiol. 119 (1):73-80.

53

CHAPTER III

ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX

NIGRA

The genus Salix, of the family Salicaceae, is composed of approximately 300

species of trees and shrubs. These species are largely scattered throughout the cooler

regions of the Northern Hemisphere, although a few are distributed in the tropical regions

of Indonesia and South Africa, as well as in southern South America. In North America,

there are approximately 80 native Salix species, only 30 of which attain tree size. They

are generally fast-growing trees, reaching maturity in 50 to 70 years. Reproduction by

seeds is restricted because germination must occur on moist mineral soil soon after the

seeds are shed. However, propagation by sprouts and root suckers occurs readily. In

Europe, willows have been used as vegetation filters for the purification of sewage,

wastewaters and sludges (Perttu, 1992; Hasselgren, 1998; Hodson et al., 1994; Nielsen,

1994). They have the ability to accumulate heavy metals such as cadmium, zinc, copper

and nickel (Riddle-Black et al., 1995; Landberg and Greger, 1996).

Black willow (Salix nigra) is small- to medium-size tree, ranging from 30 to 60

feet high with a broad, irregular crown and a superficial root system. It is a fast-growing,

deciduous tree that grows mainly on wet banks of streams and lakes. Commercially it is

used for pulp, charcoal and furniture manufacturing (Harlow et al., 1996). Like other

species of willow, black willow is easily propagated from rooted cuttings, but in vitro

propagation has not been reported for the species. Other species of

54

willows have been successfully micropropagated via axillary shoot multiplication (Read

et al., 1982; Bergman et al., 1985; Neuner and Beiderbeck, 1992; Agrawal and Gebhardt,

1994; Amo-Marco and Lledo, 1995) and organogenic callus (Grönroos et al., 1989;

Stoehr et al., 1989).

The main objective of this study was to establish a successful de novo plant

regeneration system for mature Salix nigra trees via organogenesis from immature

inflorescences.

Materials and Methods

Explant collection and preparation. Plant material was collected from a natural

population of black willows growing at Oconee Forest Park in Athens, GA. In January,

2002, 30 dormant buds were collected from each of two mature trees (SN1 and SN4).

The same number of buds was collected from SN1 again in January, 2002, but tree SN3

provided buds in place of SN4 the second year. All buds were surface-sterilized using

the following sequence of treatments: 70% ethanol for four minutes, 20% Roccal (10%

alkyldimethyl benzyl ammonium chloride; L&R Products) for five minutes, 20% Clorox

(5.25% sodium hypochlorite) with five drops of Tween 20 (per 100 ml Clorox) for fifteen

minutes, sterile water rinse for three minutes, 0.01 M HCl rinse for three minutes, three

3-minute sterile water rinses, 0.5% Captan (Micro Flo) for five minutes and three

additional 3-minute sterile water rinses. Following sterilization, bud scales and bracts

covering the staminate inflorescences were removed aseptically. After excision from the

bud, ten inflorescences were isolated from each tree, and were cut transversely to yield

three sections that were placed on the medium.

55

Medium and culture initiation. Woody plant medium (WPM, Lloyd and McCown,

1980) supplemented with 0.1mg/L thidiazuron (TDZ), 2% sucrose and 0.3% Phytagel

(Sigma) was tested for its effectiveness for stimulating callus induction and

morphogenesis from the inflorescence explants. The medium was sterilized by

autoclaving at 121° C at 1 kg.cm-2 for 25 minutes, and poured into 60 x 15mm plastic

Petri plates. One inflorescence, divided into three segments, was cultured per plate.

Cultures were maintained in darkness at 25°C and transferred to fresh medium every 30

days. Callus induction was visually assessed every 30 days and calli with visible shoot

primordia were transferred to basal WPM medium (shoot elongation medium). Cultures

producing adventitious shoots were transferred to basal WPM medium and maintained

under a 16 hr photoperiod (100 µmol·m-2·s-1) at 25° C, with transfer to fresh medium

every 30 days. After 8 weeks on basal medium, cultures were transferred to GA-7

vessels (Magenta Corp.) containing 100 ml of semi-solid basal medium, to allow further

elongation.

Axillary shoot multiplication. Adventitious shoots 2 cm in length or longer were

excised from calli, cut into 1 cm segments and placed on WPM medium supplemented

with 0.1 mg/L zeatin. The cultures were maintained under a 16 hr photoperiod at 25° C

and transferred to fresh medium every 30 days.

Rooting of shoots. Axillary shoots that were 5 cm in length or longer were excised and

rooted ex vitro in Peat-Lite (Fafard) potting mix in Hillson-type Roottrainers (Spencer-

Lemaire). While rooting, shoots were maintained in a Plexiglas humidifying chamber at

100% relative humidity under cool white fluorescent lights (120 µmol.m2.s1) and a 16 hr

photoperiod.

56

Results

Callus began to develop after 30 days on callus induction medium (WPM

supplemented with 0.01 mg/L TDZ). Callus developed initially from the cut surface at

the base of the inflorescence, then expanded outward and upward. Generally, calli were

hard and yellow at the base. However, a cluster of creamy white promeristemoids

developed on top of this callus. (Figure 9A). Results from the January 2001 initiation

showed a callus initiation frequency of 43.3 % for trees SN-1 and 56.6% for tree SN-4

(Figure 7). The January 2002 initiation resulted in similar frequencies, with tree SN1

producing callus at a frequency of 66.6% and SN-3 explants producing callus at a

frequency of 60% (Figure 7).

010203040506070

SN1 SN4 SN-3

Jan-01Jan-02

TREES

% E

xpla

nts P

rodu

cing

Cal

lus

Figure 7. Callus initiation frequencies for inflorescence explants from 3 black willow

trees cultured in 2001 and 2002. Averages represent 30 inflorescence explants.

57

After 60 days on shoot elongation medium (basal WPM), most calli were green

and adventitious shoots grew mainly from the periphery of the callus (Figure 9B).

January 2001 cultures had an adventitious shoot induction frequency of 10 % for tree SN-

1 and 13.3% for tree SN-4 (Figure 8). Adventitious shoot induction frequencies for the

2002 explants were 6.6% for tree SN-1 and 16% for tree SN-3 (Figure 8).

Following five monthly transfers to fresh medium, adventitious shoots had fully

developed and reached approximately 2 cm in length (Figure 9C).

0

5

10

15

20

SN-1 SN-4 SN-3

Jan-01Jan-02

% E

xpla

nts P

rodu

cing

A

dven

titio

us S

hoot

s

TREES

Figure 8. Adventitious shoot formation frequencies for inflorescence explants from 3

black willow trees cultured in 2001 and 2002. Averages represent 30 inflorescence

explants.

After approximately 60 days on shoot propagation medium, axillary shoots had

reached 5 cm in length. Shoots were excised and transferred to potting mix for rooting in

a humidifying chamber. Over 85% of the shoots successfully rooted (data not shown).

58

To date, shoots propagated in vitro and maintained under greenhouse conditions have had

a survival rate of 100 % (Figure 9D).

A B

C

Figure 9. Callus and adventitious shoot in

Shoot-forming callus development followi

= 0.2 cm) (A). Adventitious shoot develo

basal medium (Bar= 0.2 cm) (B). Advent

in WPM basal medium (bar = 0.5 cm) (C

culture initiation (D).

D

itiation from black willow inflorescence tissue.

ng thirty days of culture with thiadiazuron (Bar

pment following sixty days of culture in WPM

itious shoot elongation after 80 days of culture

). Rooted axillary shoots, eleven months after

59

Discussion

In this study, an adventitious shoot regeneration system for Salix nigra was

established from immature inflorescence explants isolated from dormant buds. Most

studies published to date reporting plant regeneration from immature inflorescence tissue

of woody plants describe plant regeneration via somatic embryogenesis (Merkle et al.

1997; Gingas 1991; Grönroos et al., 1989; Rout and Lucas, 1995; Teixeira et al. 1994;

Verdeil, et al. 1994). Reports of shoot-forming callus derived from inflorescence

explants, similar to that described here, are rare. Holme and Peterson (1996) established

an in vitro propagation system for the grass crop, Miscanthus x ogiformis, which

generated four different callus types, one of which was described as a shoot-forming

callus, similar to the promeristemoid-producing callus described in this study.

Once adventitious shoots were regenerated from the black willow inflorescence

explants, rapid clonal propagation was achieved via axillary shoot multiplication.

Multiple axillary shoots were induced on stem segments of approximately 4 month-old

adventitious shoots cultured in WPM medium with 0.45 µM zeatin. On average, each

stem segment generated eight axillary shoots in three months (data not shown). Clonal

propagation allowed us to create a clone bank, consisting of 14 different culture lines

representing all 3 ortets from which explants were collected. In the case of Salix nigra,

there was no need to apply plant growth regulators to stimulate ex vitro rooting. The

survival rates reported here (85%) show that axillary shoot multiplication followed by ex

vitro rooting is an effective propagation approach for black willow.

One of the most serious problems encountered during the micropropagation of

Salix species has been fungal contamination. Salix tarrarconensis cultures from nodal

segments of mature trees had a contamination frequency of 54.4% (Amo-Marco and

60

Lledo, 1996), and approximately 69% of the single nodal cultures of Salix carpea were

reported lost to contamination (Neuher and Beiderbeck, 1993). We believe that our

system had low levels of fungal contamination because the dormant buds could be

stringently surface-disinfected without affecting the viability of the inflorescence tissues

inside. Contamination in our experiments was less that 5%, a significantly lower rate

than those reported for Salix tarraconensis and Salix carpea (Amo-Marco and Lledo,

1996; Neuher and Beiderbeck, 1993).

As mentioned previously, willows have been used as vegetation filters because

they have the capacity to accumulate certain heavy metals (Perttu, 1992; Hasselgren,

1998; Hodson et al., 1994; Nielsen, 1994; Riddle-Black et al., 1995; Landberg and

Greger, 1996). They are fast-growing trees capable of high biomass production, possess

an extensive root system and have high evapotranspiration rates (Persson and Lindroth,

1994). All of these characteristics make willows excellent candidates for

phytoremediation processes, a relatively new remediation method that uses plants to

clean up polluted soil, water or air (Rashkin, 1996). Unfortunately, willows can only

tolerate and accumulate certain quantities of toxic metals (Riddell-Black et al., 1995).

Through genetic engineering, heavy metal resistance genes could be introduced into

selected clones of black willow, thereby increasing their capacity to metabolize and

detoxify toxic metals. Immature inflorescence explants might be especially suitable for

genetic transformation because they contain meristematic cells that may be particularly

amenable for transformation (Barcello et al., 1994). The propagation system described

here offers the potential to use immature inflorescences as targets for A. tumefaciens

mediated genetic transformation.

61

Reports of de novo plant regeneration systems for willow species are scarce, even

though willows were one of the first woody plants to be cultured in vitro (Gautheret

1938). Our results are further evidence that immature inflorescence explants of woody

angiosperms are a potentially valuable source of cells that can become morphogenic and

thus useful for in vitro propagation.

62

Literature Cited

Agrawal, D.C. and Gebhardt, K. 1994. Rapid micropropagation of hybrid willow (Salix)

established by ovary culture. Plant Physiol. 143:763-765.

Amo-Marco, J.B. and Lledo, M.D. 1995. In vitro propagation of Salix tarraconensis pau

ex Font Quer, an endemic and threatened plant. In Vitro Cell Dev. Biol.-Plant 32:42-46.

Barcelo, P., Hagel., C., Becker, D., Martin, A. and Lorz, H. 1994. Transgenic cereal

(tritodeum) plants obtained at high efficiency by microprojectile bombardment of

inflorescence tissue. Plant J. 5(4):583-592.

Bergman, L., Von Arnold, S., Eriksson, N. 1985. Effects of N6-benzyladenine on shoots

of five willow clones (Salix spp.) cultured in vitro. Plant Cell Tiss. Org. Cult.4:135-144.

Harlow, W.M., Harrar, E.S., Hardin, J.W. and White F.M. 1996. Textbook of



Solid Waste Conference. Academic Press. 189-197. NewYork.

Gautheret, R. 1938. Transplanting cambial tissue cultures of Salix carpea. C.R. Acad.

Sci. 206:125-127.

63

Gingas, V.M. 1991. Asexual embryogenesis and plant regeneration from male catkins of

Quercus. HortScience. 26:1217-1218.

Grönroos, L., von Arnold, M and Ericsson, T. 1989. Callus production and somatic

embryogenesis from floral explants of basket willow (Salix viminalis). J. Plant Physiol.

134:558-566.


Solid Waste Conference. Academic Press. NewYork. pp. 189-197

Hodson, R.W., Slater, F.M. and Randerson, P.F. 1994. Effects of digested sewage sludge

on short rotation coppice in the UK. Willow Vegetation Filters for Municipal Wastewater

and Sludges – A Biological Purification System. Proceedings of a Study Tour,

Conference and Workshop in Sweden. Pp. 113-118.

Holme, B.I. and Peterson, KK. 1996. Callus induction and plant regeneration from

different explant types of Miscanthus x ogiformis Honda “Giganteus’. Plant Cell Tiss.

Org. Cult. 45:43-52.

Landberg, T. and Greger, M. 1996. Differences in uptake and tolerance to heavy metals

in Salix from unpolluted and polluted areas. Appl. Geochem. 11:175-180.

64

Lloyd, G.B. and McCown, B.H. 1980. Commercially feasible micropropagation of

mountain laurel (Kalmia latifolia) by use of shoot-tip culture. Combined Proceedings.

International Plant Propagators’ Society, Milltown, NJ. 30:421-437.

Merkle, S.A., Bailey, R.L., Pauley, B.A., Neu, K.A., Kim, M.K., Rugh, C.L. and

Montello, P.M. 1997. Somatic embryogenesis from tissues of mature sweetgum trees.

Can. J. For. Res. 27:959-964.

Neuner, H. and Beiderbeck, R. 1993. In vitro propagation of Salix carpea L. by single

node explants. Silvae Genet. 42(6):308-310.

Nielsen, K.H. 1994. Sludge fertilization in willow plantations. Willow Vegetation Filters

for Municipal Wastewater and Sludges – A Biological Purification System. Proceedings

of a Study Tour, Conference and Workshop in Sweden. pp. 79-82.



Persson, G. and Lindroth, A. 1994. Simulating evaporation from short-rotation forest:

variation within and between seasons. J. Hydrol. 156:21-45.

65

Pettru, K.L. 1992. Sludge, wastewater , leakage water, ash-a resource for energy forestry.

Energy forest as Vegetation Filter for Sludge, Wastewater, Leachates and Bioash. 47:7-

19.

Read, P.E., Garton,S., Louis, K.A., and Zimmerman, E.S. 1982. In vitro Propagation of

Species for Bioenergy Plantations. Proc. 5th Intl. Cong. Plant Tissue and Cell Culture. pp.

757-758.

Riddell-Black, D., Rowlands, C. and Snelson, A. 1995. Heavy metal Uptake from

Sewage Sludge Amended Soil by Salix and Populus Species Grown for Fuel. 14th Annual

Symposium on Current Topics in Plant Biochemistry, Physiology and Molecular

Biology. April 19-22, 1995. Columbia, MO. pp. 51-52.

Rout, J.R. and Lucas, W.J. 1996. Characterization and manipulation of embryogenic

responses from in vitro cultured immature inflorescens of rice. Planta. 198:127-138.

Stoehr, M.U., Cai, M. and Zsuffa, L. 1989. In vitro plant regeneration via callus culture

of mature Salix exigua. Can. J. For. Res. 19:1634-37.

Teixeira, J.B., Sondahl, M.R., and Kirby, E.G. 1994. Somatic embryogenesis from

immature inflorescence of oil palm. Plant Cell Rep. 13:247-250.

66

Verdeil, J.L., Huet, C., Grosdemange, F., and Buffard-Morel, J. 1994. Plant regeneration

from cultured immature inflorescences of coconut: evidence of somatic embryogenesis.


67

CHAPTER IV

CONCLUSIONS

Arsenic pollution is an environmental problem affecting the health of millions of

people worldwide. Managing contaminated soils, sludge, and groundwater with current

remediation technologies is costly and the resultant environmental damage is very high

(U.S. Army Toxic and Hazardous materials Agency, 1987). The enormous costs and

limited effectiveness of traditional remediation techniques have prompted efforts to

develop alternative remediation methods, such as phytoremediation.

To date, only one plant, a fern indigenous to the southern U.S., has been reported

to have the capacity to hyper-accumulate arsenic to very high levels (Ma et al., 2001).

Unfortunately, the enzymes responsible for arsenic hyper-accumulation are not yet

available for transfer into other plant species. However, recent studies have demonstrated

that transgenic plants expressing a bacterial γ-glutamylcysteine synthetase gene (γ-ECS)

showed a higher tolerance and accumulation of arsenic by increasing the thiol-sinks for

this metal throughout the plant (Dhankher et al., 2002).

The present study was divided into: genetic engineering and tissue culture. In this

first section, was examined the potential enhancement of arsenic tolerance in eastern

cottonwood (Populus deltoides) by introducing a bacterial gene γ-glutamylcysteine

synthetase (γ-ECS). In the second section, immature inflorescence explants of black

willow (Salix nigra), another potential candidate forest species for use in

68

phytoremediation, were tested as a source of competent cells for generating adventitious

shoots. With such a regeneration system in hand, black willow could be engineered with

arsenic resistance genes.

The E. coli gene for γ-ECS was introduced into P. deltoides, a fast-growing, high

biomass-producing tree, via A. tumefaciens-mediated transformation. Leaf sections

isolated from transgenic γ-ECS plantlets produced callus on shoot induction medium in

the presence of 800 µM arsenate, a treatment that inhibited callus induction in wild-type

leaf explants. Transgenic shoots expressing γ-ECS cultured on root induction medium

containing 800 µM arsenate showed normal growth and rooted, while wild-type shoots

were chlorotic and did not grow. These results indicated that transgenic eastern

cottonwood plants over-expressing γ-ECS were highly tolerant to arsenate compared to

wild-type plants.

S. nigra is also a fast-growing, high biomass-producing tree, but unlike P.

deltoides, no in vitro regeneration system had been reported for it until now. In this

study, an in vitro regeneration system for Salix nigra was established from immature

inflorescence explants. In addition, rapid clonal propagation was obtained via axillary

shoot multiplication. These results established an efficient and reliable de novo

propagation system, an essential requirement for the production of transgenic black

willow trees.

In summary, this work has demonstrated the potential application of genetic

engineering to improve a forest tree for arsenic phytoremediation, and established an in

vitro propagation system for another potential candidate species for phytoremediation.

69

Literature Cited




Nature Biotech. 20(11):1140-5.






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