GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA

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 thiolsinks 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.

GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA 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

GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA by AMPARO LIMA Major Professor: Committee: Scott A. Merkle 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 hyperaccumulate, 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 selenitecontaminated 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 fiveday 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 m 3 /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 Agrobacteriummediated 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 cocultivation 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.

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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 m 3 /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 residuerich 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 cocultivation 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 wildtype 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). MWL DNA Control (+) WT E-1 E-2 γ-ecs lines E-3 E-4 E-5 E-6 E-7 E-8 Blank MWL 439 bp Figure 3. PCR analysis of genomic DNA from putative γ-ecs-transformed and wildtype (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. ECS control (+) Blank E-1 E-2 E-3 E-4 E-5 E-6 E-7 E-8 WT 57 kd Figure 4. Western blot analysis of γ-ecs expression. Blots containing crude protein extracts from γecs-transgenic and untransformed plants (WT) were probed with anti- ECS monoclonal antibody and visualized using chemiluminescence. Arrow indicates purified ECS protein isolated from confirmed transgenic A. thaliana plants expressing the γ-ecs gene (57 kd). Sensitivity experiments indicated that levels of arsenate lower than 800 µm had little visible effect on leaf section survival, callus development, axillary shoot survival and adventitious root formation up to 8 weeks. Following 4 weeks of culture on 800 µm sodium arsenate, leaf sections began to bleach, leaves on the axillary shoots began to turn chlorotic and the bases of the axillary shoots darkened. Therefore we chose 800 µm sodium arsenate for the toxicity experiments. A month after being cultured on the medium supplemented with 800 µm arsenate, leaf sections from γ-ecs transgenic lines remained green and began to develop 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). A B 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.

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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. m 2. s 1 ) 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). 70 % Explants Producing Callus 60 50 40 30 20 10 0 SN1 SN4 SN-3 Jan-01 Jan-02 TREES 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). % Explants Producing Adventitious Shoots 20 15 10 5 0 SN-1 SN-4 SN-3 Jan-01 Jan-02 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 D Figure 9. Callus and adventitious shoot initiation from black willow inflorescence tissue. Shoot-forming callus development following thirty days of culture with thiadiazuron (Bar = 0.2 cm) (A). Adventitious shoot development following sixty days of culture in WPM basal medium (Bar= 0.2 cm) (B). Adventitious shoot elongation after 80 days of culture in WPM basal medium (bar = 0.5 cm) (C). Rooted axillary shoots, eleven months after culture initiation (D).