1

Comparison of genetic and physiological traits of diploid progenitors and modified 1

polyploid lines of tree species 2

3

4

Harshi K. GamageA, Peter PrentisA,B, Andrew LoweA,B, Malcolm LamontC, Susanne SchmidtA 5

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A School of Integrative Biology, The University of Queensland, Brisbane, QLD 4072, Australia. 7

B School of Earth and Environmental Sciences, University of Adelaide, North Terrace, 8

Adelaide, SA 5005, Australia. 9

C Bioadapt Pty. Ltd., 981 Mount Tambourine, Oxenford Rd, Wongawallan, QLD 4210, 10

Australia. 11

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Keywords: Agathis, Elaeocarpus, genome stability, leaf anatomy, Paulownia, photosynthesis, 13

polyploidy, tree breeding 14

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Abbreviations: AFLP, Amplified Fragment-Length Polymorphism; A/Ci, CO2 response curve; 17

Amax, maximum rate of net photosynthesis; Ci, internal CO2 content of leaves; ETR, electron 18

transport rate; Fv/Fm, quantum yield, Jmax, maximum electron transport rate; LNUE, leaf 19

nitrogen use efficiency; Vcmax, maximum carboxylation rate. 20

21

b

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Abstract. We examined unmodified (diploid) progenitors and modified (putative polyploid) 21

lines of tree species Agathis robusta, Elaeocarpus grandis, and Paulownia tomentosa that were 22

generated by a new laboratory procedure. Potential advantages of polyploid compared with 23

diploid plants include higher growth rate and greater physiological performance and 24

environmental resilience. Unmodified progenitors and modified clone lines were subjected to 25

three sets of investigations: (i) verification of polyploidy with flow cytometry, (ii) verification 26

of genome stability using AFLP - a total genomic marker, and (iii) characterisation of plant 27

properties, including leaf anatomy, physiology and growth. Nuclear DNA content in cells of 28

Elaeocarpus and Paulownia clones (the two species successfully tested) were elevated, 29

consistent with individuals experiencing genome duplication. Variable results occurred in 30

Paulownia clone lines, where one line actually showed a reduction in genomic content. All 31

clone lines of Agathis, Elaeocarpus, and Paulownia had high genomic stability demonstrating 32

that mass clonal production should result in phenotypically stable clone lines. However, most 33

clone lines tested had slightly divergent genotypes possibly resulting from slight genome 34

rearrangements and indicating that all clone lines have resulted from an independent 35

polyploidisation process. The different genotypes also signify that the polyploidisation process 36

may create novel genetic variation. Under non-limiting growth conditions in the glasshouse, 37

selected Agathis and Paulownia clones (the two species investigated) had overall larger and 38

thicker leaves with greater stomatal aperture, different biochemical properties, increased 39

photosynthetic rates, and in the case of Agathis, significantly higher biomass production than 40

diploid parents. The physiological and genetic differences between diploid and polyploid clone 41

lines are consistent with those observed in previous studies. Thus, the new procedure for 42

manipulating nuclear DNA content of trees offers opportunities to develop tree species as 43

timber crops with improved performance and environmental resilience. 44

3

Introduction 45

A fundamental problem associated with the development of tree species for forestry is the time 46

required to produce a marketable crop. Many commercial tree species have rotation lengths in 47

excess of 15-50 years. Although improvements in the rate of plant growth have been achieved 48

through conventional tree breeding, this process is extremely time-consuming as most forest 49

trees reach reproductive maturity only after 8-20 years (Lantz 2000). Thus a single breeding 50

cycle for tree improvement takes between 8-20 years, and an even longer period is required 51

before significant increases in growth are achieved (Lantz 2000). Consequently, there is a need 52

to examine non-conventional breeding and genetic improvement practices to speed up breeding 53

cycle and rotation length of tree species. 54

Recently, accelerated breeding techniques have been developed which can substantially reduce 55

the breeding cycle of some promising timber trees. These methods include flower induction 56

techniques and genetic engineering (Zobel and Talbert 1984). In the case of flower induction 57

techniques a 20-year breeding cycle can be reduced to 16 years in loblolly pine by accelerating 58

flowering in the greenhouse (Zobel and Talbert 1984). Genetic engineering has received a 59

considerable amount of attention but few examples of application in forest trees are available 60

(Lantz 2000). It is unlikely that genetic engineering will result in a significantly improved 61

growth rate for many forestry species, but genetic engineering is thought to be useful for 62

transferring pest resistance to susceptible species (Carraway et al. 1994). One overlooked 63

technique that holds great promise to accelerate the growth rate of tree species is the induction 64

of polyploidy or genome doubling. Polyploidy has been and continues to be a pervasive force in 65

plant evolution (Abbott and Lowe 2004, Adams and Wendel 2005). The prominence of 66

polyploidy in flowering plants is possibly due to extensive modifications of the genome and/or 67

transcriptome, creating cascades of novel gene expression patterns, regulatory interactions and 68

4

new phenotypic variation (Stebbins 1971, Levin 1983, Soltis and Soltis 1993, Wendel 2000, 69

Ramsey and Schemske 2002). Consequently, polyploid plants tend to be more successful than 70

their diploid relatives, often having wider distribution, greater habitat amplitude and colonising 71

ability (Rothera and Davy 1986). Novel changes to the genome or transcriptome allow 72

polyploids to grow in a greater range of environments and to have increased tolerance to 73

drought and resistance to pests than diploid progenitors (Stebbins 1971, Levin 1983, Ramsey 74

and Schemske 2002). The advantages of polyploids over diploids have resulted in the selection 75

of polyploids for use in agriculture and horticulture, and almost all currently cultivated crop 76

species are either newly synthesised or paleopolyploids (Ezura et al. 1993, Ortiz and Vuylsteke 77

1995, Osborn 2003, Shoemaker et al. 2006). 78

Polyploidy can produce dramatic changes in chlorophyll content and photosynthetic rates 79

(Joseph et al. 1981, Baer and Schrader 1985, Warner and Edwards 1993) and result in faster 80

growth rates (Dermen 1940, DeMaggio et al. 1971). In some polyploid plants however, 81

increasing ploidy levels were not associated with higher photosynthetic or growth rates 82

(Curkrova and Auratovsakova 1968, Austin et al. 1982, Joseph et al. 1981, Warner and 83

Edwards 1987). Therefore it is not possible to generalise the effects of polyploidy across a wide 84

range of species as taxon specific effects often occur. 85

In this study, we compared diploid parent plants with putative polyploid clone lines of 86

Australian tree species Agathis robusta, Elaeocarpus grandis, and Chinese tree species 87

Paulownia tomentosa. The putative polyploids were generated by a new laboratory procedure. 88

Three sets of investigations were performed: (i) verification of polyploidy using flow 89

cytometry, (ii) verification of genome stability using AFLP, a total genomic marker, and (iii) 90

characterisation of plant properties, including leaf anatomy, morphology, physiology and 91

growth. 92

5

Materials and Methods 93

Plant material 94

Diploid parents and modified clones of tree species Agathis robusta (C. Moore ex F. Muell) 95

Bailey (Araucariaceae), Elaeocarpus grandis F. Muell (Elaeocarpaceae), and Paulowina 96

tomentosa (Thunb) Steud. (Scrophulariaceae) were examined for genetic, anatomical, 97

physiological and growth traits. Agathis robusta (Kauri pine) is a native Australian tree species 98

which has been harvested from natural forests for timber. Agathis robusta is an attractive 99

softwood timber suited to many indoor uses, including cabinet work (Holliday 2002). 100

Elaecocarpus grandis (blue quandong) is a fast-growing endemic Australian tree; the timber is 101

pale yellow, soft and light and has numerous uses (Holliday 2002). Trial plantations of both 102

Agathis and Elaeocarpus exist in Queensland and New South Wales, Australia (Vize et al. 103

2005). The Chinese tree species Paulownia tomentosa (princess tree) is a naturalised tree 104

species in Australia that rapidly colonises disturbed areas (Olson and Carpenter 1985). 105

Paulownia tomentosa grows to 20 m height within 8-10 years. Wood of Paulownia has a light 106

colour and low density and is used for the manufacture of solid wood products such as 107

furniture, doors, and pulp to produce fine papers. 108

109

Proof of ploidy and genome stability 110

Flow cytometry 111

Plant material for proof of ploidy and genome stability was obtained from parent plants and 112

clone lines grown together either in controlled environment facility or in a naturally lit 113

glasshouse. A piece of young leaf material (approximately 0.5 cm2) was cut with a razor blade 114

into small sections which were added to 1 mL nuclear extraction buffer solution (solution A, 115

Partec GmbH, Münster, Germany) for 60–90 s, incubated for 1–2 min, filtered through a 50 116

6

mm mesh filter (cell trics, Partec GmbH, Münster, Germany), and processed in a staining buffer 117

(solution B, Partec GmbH, Münster, Germany) containing RNAase and propidium iodide (PI) 118

for 10-30 min. Samples were analysed using a BD LSRII flow cytometer (BD Biosciences, 119

USA), equipped with a 20 mV argon gas laser exciting at 488 nm, a quartz–air objective and a 120

high-quality red-sensitive photo-multiplier. For each parental and clone line sample, two or 121

three preparations were made. 122

123

Amplified Fragment-Length Polymorphism (AFLP) analysis 124

Total cellular DNA was extracted from 0.1 g of plant material per sample according to the 125

Qiagen plant 96 kit (Qiagen, GmbH, Hilden). DNA samples were diluted with 0.5 x TE buffer 126

to obtain concentrations between 10 and 20 ng DNA µL-1. AFLP restriction/ligation was 127

performed following the protocol of Prentis et al. (2004). AFLP PCR was performed following 128

the method of Zawko et al. (2001), using four to six primer pairs for each species: E-AAG/M-129

AG, E-AAG/M-GA, E-ACA/M-AG, E-ACA/M-GA, E-ACG M-AG and E-ACG/M-GA, where 130

the selective EcoRI primer was hex labeled. The fluorescent labeled amplified products were 131

analysed by gel electrophoresis (5% acrylamide gels), using a Gelscan GS2000 (Corbett 132

Research). At an individual locus, bands of similar size and intensity were considered to be 133

homologous as has been shown in previous studies (Rieseberg 1996, O'Hanlon and Peakall 134

2000). Statistical analyses using variation in AFLP phenotypes were based on the assumptions 135

that AFLP markers are dominant and that no co-migration of fragments occurred. A dice 136

similarity matrix was used to compare genome stability and proof of parent among the different 137

parental and clone lines within species using SPSS version 14 (SPSS Inc., Chicago, IL, USA). 138

139

Leaf traits and plant growth of Agathis and Paulownia 140

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Plants of diploid and clone lines of Agathis robusta and Paulownia tomentosa approximately 141

10-15 cm tall were grown from October 2005 to April 2006 in a naturally lit glasshouse 142

ventilated with electrical fans in Brisbane, Australia. Four and three replicate plants of parent 143

and clone were used for the experiments, respectively. In January 2006, experiments with 144

additional Paulownia tomentosa plants (parent, clone 3, clone 7, and clone 15) commenced; 145

plants were approximately 10-15 cm tall and three replicate plants of parent and each clone line 146

were studied. All plants were potted in 7 liter pots with Californian potting mix (mixture of 147

sand, peat, and slow release fertiliser). Plants were daily watered during the experiment to 148

ensure adequate water supply. Foliar traits and growth measures were performed during the 3 to 149

6 month period, and all plants were destructively harvested in April 2006 and oven dried at 85 150

ºC for the estimation of total biomass gain and allocation. 151

152

Leaf anatomy 153

To determine leaf anatomy and cell dimensions of Agathis and Paulownia, a young fully 154

expanded leaf was used to determine leaf anatomy from three plants per each parent and 155

modified clone. 1 x 0.5 cm cross sections were taken from the middle portion of the lamina 156

across the midrib, and immediately fixed in 70 % FAA (5 : 5 : 90 v:v of formalin: acetic acid: 157

alcohol). The leaf strips were dehydrated in an ethyl alcohol series and embedded in separate 158

wax blocks (Ruzin 2000). Cross sections were cut from each strip at 8 µm thickness with a 159

rotary microtome and mounted on a glass slide. The tissue was then stained with safranin and 160

fast green (Ruzin 2000). One slide for each parent and clone was prepared. In each slide, three 161

measurements from three different leaf sections were made of leaf blade thickness, upper and 162

lower epidermal cell thickness, and palisade cell layer thickness. Each slide was measured in 163

different positions that avoided the midrib region. Cross sections were photographed using a 164

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digital camera attached to a light microscope. Measurements of cell dimensions were made 165

using Spot Advance software version 4.04 (Spot software BV, Amsterderm, Netherlands). 166

To examine stomatal size of parents and clones of Agathis and Paulownia, we measured 167

stomatal aperture length. Leaf sections (1 x 1 cm) were taken in the middle portion of the 168

lamina from the same leaf that was used for cross sections. One leaf section for each parent and 169

clone of Agathis and Paulownia species was taken. Each section was incubated in a 50 ºC oven 170

in 5 % sodium hydroxide to clear leaf pigments. Sections were then stained with 0.5 % aqueous 171

toluidine blue solution and mounted on viewing slide (Ashton et al. 1999). For each section, 172

three stoma aperture lengths were measured in different fields of view on the abaxial side of the 173

leaf using Spot Advance software. 174

175

176

Leaf morphology: size and elongation rate 177

Leaf area of all leaves that had developed over the 3 to 6 month experiment was determined 178

using a leaf area meter (Delta-T Digital Image Analysis System, 1990, Delta-T Devices Ltd., 179

Version 1.12, Cambridge, UK). At the onset of flowering after 8 weeks, Paulownia produced 180

smaller leaves than prior to flowering. To obtain an average leaf size per plant, the leaf area of 181

leaves below the flowering shoot was divided by the number of leaves used for the area 182

measurement. This procedure ensured that the differences found in leaf size between the parent 183

and clones were irrespective of differences in flowering time. Leaf area was determined for 184

three plants of each parent and clone of Agathis and Paulownia. Specific leaf area was 185

calculated by dividing a plant’s total leaf area per plant by the total leaf dry mass. Leaf 186

elongation rate was determined by measuring leaf length (leaf base to leaf tip) with a ruler from 187

the first appearance of the leaf until leaf elongation ceased. One leaf each was measured at three 188

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plants from each parent and clone. To estimate leaf expansion rate, a linear regression of leaf 189

length against the number of days after leaf appearance was performed (Sugiyama 2005). 190

191

Biochemical and physiological properties of leaves 192

To determine leaf chlorophyll content, three leaf discs of 3 mm diameter were taken from base, 193

center, and tip from same leaves that were used earlier for photosynthetic measurements. Leaf 194

discs were used for measuring total chlorophyll (chlorophyll a and b). Leaf discs were 195

immediately frozen in liquid nitrogen and were ground with buffered 80% aqueous acetone 196

(Porra et al. 1989). Samples were centrifuged and chlorophylls extracted. Absorbance of 197

chlorophyll a and b was measured at 663 nm and 646 nm, respectively with a 198

spectrophotometer. Total chlorophyll content was calculated according to Porra et al. (1989) 199

equations. For measuring soluble protein content of leaves, three leaf discs of 3 mm in diameter 200

were taken from base, centre and tip from the same leaves that were used earlier for 201

photosynthetic measurements. Leaf discs were immediately frozen in liquid nitrogen and were 202

ground with buffered phosphate solution (Bradford 1976). Soluble protein was determined 203

using the Biorad Protein kit (BioRad laborataries, Inc., Hercules, CA, USA) with bovine serum 204

albumen (BSA) as the protein standard. 205

Net photosynthesis and stomatal conductance were measured on three plants for each parent 206

and clone of Agathis and Paulownia using a portable infrared gas-exchange system with red-207

blue light source in the measuring head (LI-6400, Licor Inc., Lincoln, Nebraska, USA). Agathis 208

plants had been grown for 6 months in the glasshouse, while Paulownia plants had been grown 209

for 3 months in the glasshouse when the physiological measurements were performed in early 210

March 2006. A young fully expanded and undamaged leaf was selected from each plant for 211

measurements. Leaf temperature was maintained 30 ± 1 ºC and water vapor pressure deficit 212

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was between 1.0–1.2 kPa during the photosynthesis and stomatal conductance measurements. 213

Reference CO2 and H2O were maintained at 370 ± 1 ppm and 20 ± 2 ppm, respectively. Net 214

CO2 fixation and stomatal conductance were measured at high light intensity (2000 µmol m-2 s-215

1). All measurements were performed in the morning hours between 7.00 and 11.30 when 216

photosynthetic activity was highest. 217

Rates of CO2 fixation in relation to different internal leaf CO2 concentrations (A/Ci curves) 218

were measured for 3 plants from the parent and one clone of Agathis and Paulownia species 219

using Li-Cor 6400 infrared gas-exchange system. Three measurements per leaf were made at 220

each of the following CO2 concentrations inside the cuvette: 50, 100, 150, 200, 300, 400, 600, 221

900, 1200, and 1400 ppm. Leaves were kept for 5 minutes in each CO2 concentration level until 222

the photosynthetic rate was stable. Variables of the A/Ci curves (maximum carboxylation rate = 223

Vcmax, maximum electron transport rate = Jmax, and maximum rate of net photosynthesis = 224

Amax), for each parent and clone of Agathis and Paulownia species were calculated according to 225

Pury and Farquhar (1997) equations. Paulownia clone 7 was used for A/Ci curves since this 226

clone had the fastest growth rate of the three Paulownia clones under the experimental 227

conditions. Measurements were made on the same leaves that were used for net CO2 fixation 228

measurements. All measurements were performed between 7.00 and 11.30. During the 229

measurements, leaf temperature was 30 ± 1 ºC and leaf-air vapour pressure deficit was between 230

1.0 - 1.2 kPa inside the sample cuvette. Photosynthetic photon flux density within the cuvette 231

was 2000 µmol m-2 s-1. 232

To determine the instantaneous nitrogen use efficiency of leaves, leaf samples were taken from 233

the youngest fully expanded mature leaf from 3 plants for parent and clone of Agathis and 234

Paulownia species. Samples were oven dried at 60 ºC, ground using a vibrator ball mill (Retsch 235

MM-2, Haan, Germany), and analysed for total nitrogen content according to Rayment and 236

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Higginson (1992) using a combustion analyzer (LECO CNS 2000, LECO Corporation, MI, 237

USA). Instantaneous leaf nitrogen use efficiency (LNUE, µmols CO2 m-2 s-1 per % leaf N) was 238

calculated for each parent and clone as the ratio of maximum photosynthetic rate and mean 239

foliar nitrogen percentage: 240

LNUE = Amax / % leaf N 241

where Amax is the mean net photosynthetic rate at saturated light for each parent and clone (see 242

details above) and % N is the mean foliar nitrogen percentage on a dry weight basis. 243

Chlorophyll fluorescence was measured with the Plant Efficiency Analyzer (PEA, Hansatech 244

Instruments Ltd., England) to examine efficiency of photosystem II as a proxy for plant health. 245

One fully expanded mature leaf of clones and parents was darkened for 30 minutes using leaf 246

clips to determine chlorophyll fluorescence parameters of dark-adapted leaves. Quantum yield 247

(Fv/Fm) was obtained where Fv = Fm – Fo (Fv, variable fluorescence; Fm, maximum 248

fluorescence, Fo, initial fluorescence) (Schreiber et al. 1986). All measurements were taken 249

between 7.00 - 8.00 am on a single day during summer. 250

Maximum electron transport rates (ETR) were measured via rapid light response curves which 251

were performed on the same leaves as for the other photosynthesis measurements using a pulse-252

amplitude-modulated photosynthesis yield analyzer (Mini-PAM, Walz, GmbH, Effeltrich, 253

Germany). A rapid light response curve consists of the fluorescence responses to 9 increasing 254

actinic irradiances of 10 s duration interspersed with saturating light pulses (White and 255

Chitchley 1999). The same leaf and time of the measurements was used as for the other 256

fluorescence measurements. 257

258

Plant growth 259

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We measured plant height (from the root collar to the tip of the apical shoot) at the beginning 260

and at the end of the experiment for 3 plants from each parent and clone of Agathis and 261

Paulownia. At the end of the experiment, seedlings were taken from pots, roots were washed, 262

and root collar diameter (stem diameter) was measured using digital vernier calipers. Seedlings 263

were oven dried at 85 ºC until constant weight was gained. Shoot dry mass and dry mass 264

allocation to different plant parts was measured. Dry mass allocation to each tissue (flowers, 265

leaves, shoots, and roots) was calculated by dividing the dry mass of the tissue by the total dry 266

mass of the whole plant. 267

268

Statistical analysis 269

To test for differences in foliar traits and growth responses between parent and clones of 270

Paulownia, one-way analysis of variance was performed using MINITAB Version 12 (Minitab 271

Inc., PA, USA). All data were log transformed prior to analysis to meet the assumption of 272

ANOVA with the exception of proportional biomass allocation ratios. Traits that were 273

significant at P<0.05 level were further tested for multiple comparisons using Tukey’s test for 274

Paulownia. For Agathis differences in foliar and growth traits between parent and clone were 275

examined using a two sample t-test. Proportional biomass allocation ratios were Arcsine-Square 276

root transformed and significant differences were examined using Wilk’s test in MANOVA. 277

278

279

Results 280

Genome Content 281

Flow cytometry results demonstrated that Elaeocarpus clones had ~ 27 % more nuclear DNA 282

content (135-140) relative to their parents (110, Table 1) as measured by linear fluorescence. 283

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The genome content of Paulownia parents and clone lines was varied. Paulownia clone line 7 284

had 25 % less genome content (75) compared to clone line 15 which had similar nuclear DNA 285

content to the parent line (100, Table 1 ), while clone line 3 had 35 % more nuclear DNA 286

content (135 ) than clone 15 and the parent line, and 80 % more nuclear DNA content than 287

clone 7. It was not possible to produce consistently reliable flow results for Agathis robusta. 288

289

Genome stability 290

To accurately assess genome stability and genome re-organisation during the polyploidisation 291

process, over 450 (range 476-564) AFLP fragments were generated for each species across the 292

different parent and clone lines. AFLP analysis of four to six replicates of each line showed that 293

clone lines of the three studied species had extremely high genome stability ranging from 100 294

to 97 % for Elaeocarpus and Paulownia respectively (Table 1). Interestingly, AFLP data also 295

indicated that there has been slight genome re-organisation during the polyploidisation process 296

in all three tested species. The highest level of genome re-organisation was found in Paulownia 297

where genotypes of the different clone lines had a mean difference of 15 % in fragment 298

sharing(Table 1). The average dissimilarity in AFLP phenotypes among the Agathis clone lines 299

was 7 %(Table 1), while little genome re-organisation occurred in Elaeocarpus as clone lines 300

only differed by an average of 3 % in AFLP phenotypes (Table 1). 301

302

Leaf anatomy 303

Thickness of leaf blade, upper epidermis, palisade mesophyll, lower, epidermis, and stomatal 304

aperture length were compared between selected clones and parents of Agathis and Paulownia 305

(Fig. 3). For Paulownia, we selected clone 7 for leaf anatomical analysis since this clone line 306

was the most vigorous of all Paulownia clones. Paulownia clone 7 had a statistically 307

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significantly thicker lower epidermis (t = 4.76, p = 0.018; Fig. 3 C, D) compared with the 308

parent. Stomatal aperture length was significantly greater in clones of Agathis (t = 3.90, p = 309

0.03; Fig. E, F) and Paulownia species (t = 4.78, p = 0.041, Fig. 3 G, H) than parents. There 310

were no statistically significant differences in other anatomical features between parents and 311

clones but clones had a trend of greater thickness in leaf blade, epidermis, and palisade 312

mesophyll due to their greater cell elongation. 313

314

Leaf morphology 315

Agathis and Paulownia clones had relatively larger leaf size and greater total leaf area, but 316

lower specific leaf area than parent plants. However, these traits were statistically significantly 317

higher only for Agathis clone (leaf size: t =17.75, P = 0.003; Fig. 4 A, B; total leaf area: t = 318

16.74, p = 0.004; specific leaf area: t = 4.03, P = 0.03; Fig. 5). Leaf elongation rate (LER) was 319

significantly higher in clones than in parents (Agathis: t = 3.12, p = 0.048; Paulownia: t = 4.02, 320

p = 0.032; Fig. 5). 321

322

323

Biochemical and physiological properties of leaves 324

Biochemical properties including total chlorophyll content, soluble protein, and leaf nitrogen 325

use efficiency (LNUE) were determined for parents and clones (Table 1). Total chlorophyll 326

content was significantly higher in Paulownia clone 7than in other clones and parent (F = 6.38, 327

P = 0.016). While there was a trend for greater soluble protein content in leaves of clones than 328

parents in both species, this was not statistically significant. LNUE was significantly higher in 329

the Agathis clone than parent (t = 4.24, p = 0.024). 330

331

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Agathis clone and Paulownia clones 7 and 15 had a significantly higher net photosynthesis 332

rates (net CO2 fixation) than parents (Agathis: t = 6.63, p = 0.022; Paulownia: F = 8.18, P = 333

0.008; Fig. 6). Stomatal conductances were significantly greater in Agathis clone and 334

Paulownia clone 7 than parents (t = 4.24, p = 0.013; F = 5.2, P = 0.028 respectively; Fig. 6). 335

336 Paulownia clone 7 was used for the A/Ci curves since it had the best growth off all clones under 337

the experimental conditions. With increasing intercellular CO2 concentration, photosynthetic 338

rate increased for both parent and clones of Paulownia and Agathis species (Fig.7). A/Ci curves 339

for A. robusta showed that the clone had a significantly higher maximum carboxylation and 340

electron transport rates (t = 3.44, P = 0.04; t = 3.56 P = 0.04, respectively) than the parent while 341

no significantly differences were observed in Paulownia clone and parent (Table 2). 342

343

All plants had similar quantum yield values ranging from 0.80 – 0.85 indicative of healthy 344

plants (data not shown). The trend in higher maximum electron transport rate (ETR) was 345

observed in clones of Agathis and Paulownia compared with parent plants; however, maximum 346

ETR was statistically significant only in Paulownia clone 7 (F = 4.59, P = 0.038; Fig. 7). 347

Plant growth 348

After three months of growth in the glasshouse, Paulownia clone 7 had a greater height 349

increment than clone 3 (F = 4.53, P = 0.039). We observed similar height increments of the 350

other Agathis and Paulownia clones as parents (data not shown). Root collar diameter 351

increment was similar in clones and parents, and stem diameter parents and clones ranged 352

between 7.1 to 7.5 mm (Agathis) and 14.8 to 17.7 mm (Paulownia) (data not shown). 353

Paulownia clones 7 and 15 had significantly higher shoot biomass than clone 3 (Fig. 8 C). Total 354

biomass gain was significantly greater only when comparing Paulownia clone 7 (193.4 g) and 355

16

clone 3 (148.12 g; F = 4.056; P = 0.044). The Agathis clone had significantly greater shoot 356

biomass (t = 6.82, p = 0.021) and total biomass (t = 7.55, p = 0.004) than the parent. 357

Determining biomass allocation to above and below ground plant parts showed that with the 358

exception of Paulownia clone 3, all other clones had somewhat lower biomass allocation to 359

roots than parents. However, biomass allocation was not statistically significantly different 360

between parents and clones (Fig. 8 B, D). 361

362

Discussion 363

We found consistent differences in nuclear DNA content, leaf properties and growth in diploid 364

progenitors and newly generated lines of three tree species, which indicate that the new lines 365

are polyploid. Nuclear DNA content in cells can be used as an indication of polyploidy since 366

polyploids possess a higher DNA content per cell as a result of increased chromosome number 367

relative to their dipoloid progenitors (Warner and Edwards 1989, Blakesley et al. 2002). Flow 368

cytometry results of this study showed that the polyploidisation process had increased the 369

amount of nuclear DNA in the cells of Elaeocarpus and Paulownia clones in most cases. In this 370

study however, the maximum increase in nuclear DNA content was 35 % relative to parent 371

lines, and clone line 7 actually had 25 % less nuclear DNA content. This indicates that either a 372

large amount of the doubled genome is lost soon after the polyploidisation process or that 373

ploidy reversion may occur during development in polyploids. Most angiosperms genomes 374

have undergone chromosomal duplication (polyploidisation) events during their evolution, but 375

over time have lost large amounts of redundant genomic material (diploidisation) to revert to a 376

near diploid genome (Bowers et al. 2003, Langkjaer et al. 2003). For example in Arabidopsis 377

only 30 % of duplicated genes have been retained since polyploidisation (Bowers et al. 2003) 378

while yeast species Saccharomyces cerevisiae retained only 15% of duplicated genes (Wolfe 379

17

and Shields 1997). Therefore, the partial diploidisation of the genome of the three test species 380

may explain a nonlinear increase in nuclear DNA content of polyploidy lines. Alternatively, 381

ploidy reversion may be occuring during development of polyploid plants in the field. In a 382

study of synthetically synthesized polyploid bananas greater than 35 % of tetraploid individuals 383

reverted to diploid chromosome numbers in two years under field conditions (Hamill et al. 384

1992). To examine which of these hypotheses is most correct, ploidy verification should be 385

performed immediately after polyploidy induction and validated throughout the developmental 386

cycle to confirm if ploidy reversion is occuring or how quickly diploidisation of the genome 387

can happen. 388

389 Polyploidy can also be verified by increased organ size (Osborn et al. 2003), and by the 390

presence of increased stomata relative to their diploid progenitors - the so-called gigas effect 391

(Li et al. 1996, Beck et al. 2003). Selected clone lines of Paulownia and Agathis investigated 392

here produced relatively larger and thicker leaves and stomata with greater aperture than the 393

parental lines (discussed below), indicating that the clone lines are polyploids. 394

395

AFLP results showed that the clone lines of Agathis, Elaeocarpus, and Paulownia had 396

extremely high genomic stability demonstrating that mass clonal production programs should 397

produce phenotypically stable clone lines. Consistent with these results, induced polyploidy in 398

Tragopodon (Pires et al. 2004a) and Spartina (Baumel et al. 2002) exhibited high genome 399

stability. Polyploids however, do not always exhibit stable genomic compositions (Soltis 2005). 400

For example, induced tetraploid Brassica napus exhibited extensive genomic changes after 401

polyploidisation relative to their diploid parents (Song et al. 1995). Genomic investigations of 402

synthetically derived polyploids have revealed rapid and widespread genomic changes in a 403

18

number of plant groups (Han et al. 2003; Pires et al. 2004b). Therefore, slightly divergent 404

genotypes among most of the different clone lines tested in this study are not surprising and 405

indicate that all of the clone lines result from an independent polyploidisation process. Different 406

genotypes signify that polyploidisation can create novel genetic variation in clone lines, which 407

may be amenable to both natural and artificial selection for desirable traits. In other studies, 408

observed genomic changes have resulted in phenotypic changes on which selection can act 409

(Adams and Wendel 2005). In fact, in newly synthesised Brassica polyploids genomic change 410

has been associated with flowering time divergence (Pires et al. 2004b). Consequently, newly 411

derived genetic variation in different polyploid clone lines may produce differences in 412

phenotypes that allow them greater ecological tolerance relative to their parents and the 413

substrate for artificial selection programs in these species. 414

415

Under the experimental conditions which provided non-limiting growth conditions, leaves of 416

Agathis and Paulownia clone lines differed anatomically and morphologically from parents 417

having greater leaf thickness, leaf and stomata sizes. The observed differences in leaf anatomy 418

and morphology of clones and parents provide a structural basis for increased light interception, 419

for example via larger leaves (Givnish 1987). Further, thicker leaves can be better protected 420

from herbivores, pathogens and mechanical damage, and can have reduced water loss and 421

damage from overheating (Reich et al. 2003, Givnish 1987). Our initial results are consistent 422

with previous studies showing differences in leaf structure of polyploid and diploid plants. A 423

polyploid birch genotype (Betula papyrifera) had lower stomatal density and thicker upper and 424

lower epidermis and tolerated greater water deficit than diploid genotypes (Li et al. 1996). 425

Different leaf properties may have been fundamental for the occurrence of polyploid and 426

diploid birch genotypes in more extreme mountain habitat and more benign mesic habitat, 427

19

respectively (Li et al. 1996). Similarly, citrus polyploids had thicker leaves and greater drought 428

tolerance than diploids (Barrett 1974, 1992), highlighting the importance of leaf structure for 429

habitat differentiation and environmental resilience of polyploid plants. Further studies will 430

establish whether the observed leaf properties here result in increased environmental resilience 431

of clones compared with parents. 432

433

Differences of the studied clones and parents were not restricted to anatomical and 434

morphological differences, but also comprised leaf biochemistry and physiology. Compared 435

with parents, leaves of selected modified clones had higher chlorophyll content, nitrogen use 436

efficiency, carboxylation rates, and electron transport rates. The differences of parents and 437

clones in our study are consistent with previous studies of diploid and polyploid plants 438

(Cukrová and Avratovščuková 1968, Randall et al. 1977, Joseph et al. 1981, Warner et al. 439

1987). However, varying effects of polyploidy have been reported. Warner and Edwards (1989) 440

detected similar chlorophyll content in leaves of Atriplex confertifolia with different ploidy 441

levels. Leaf chlorophyll concentration of naturally occurring polyploid grass species Festuca 442

arundinacea increased from tetraploid to hexaploid but decreased from octaploid to decaploid 443

(Joseph et al. 1981). Similarly, photosynthetic rate per unit leaf area decreased or did not 444

change in Triticum species with different ploidy levels (Jellings and Leech 1984). The trend in 445

increasing chlorophyll content and/or photosynthetic rate per unit leaf area in polyploid plants 446

varied among taxa. An explanation could be that depending on taxa and ploidy level, amount of 447

DNA per cell, size of cells, and number of cells per unit leaf area can vary (Leech et al. 1985, 448

Warner and Edwards 1988, Ashton and Berlyn 1994). For example, if the increase in 449

photosynthetic capacity per cell is proportionally greater than the decrease in number of cells 450

per unit leaf area, photosynthesis will increase on an area basis (Warner et al. 1987). Thus, the 451

20

higher photosynthetic rates of Agathis and Paulownia clones are likely to be associated with an 452

increased photosynthetic rates per cell compared with parents. 453

We found that the Agathis clone produced more shoot biomass after six months of growth than 454

the parent. There was also a trend (although not statistically significant) for a greater stem 455

diameter of Agathis and Paulownia clones relative to parents. The differences in growth are 456

likely to be associated with differences in leaf traits of clones and parents, as leaf characteristics 457

are correlated with plant growth (Kamaluddin and Grace 1992, Lambers et al. 1998). Since 458

growth is a product of the plant’s net photosynthetic rate and leaf area (Lambers et al. 1998), 459

the observed faster leaf growth would allow clones to gain biomass more rapidly relative to 460

parents. Biomass gain and allocation provide useful information about the success of a plant in 461

the environment (Poorter 2001, King 2003). Compared with parents, Agathis and Paulownia 462

clones exhibited a trend of greater proportional biomass allocation to shoots, which will be 463

investigated further in a longer term study. If confirmed, the observed trend for greater shoot 464

biomass allocation of clones than parents under non-limiting nutrient and water supply could 465

point to greater phenotypic plasticity of clones. 466

467

In conclusion, we found consistent differences between modified polyploid clones and parent 468

plants of Agathis and Paulownia. The observed differences in leaf traits and growth between 469

diploid progenitors and polyploid lines can be explained by differences in the content of nuclear 470

DNA. Further research will establish how polyploid clones fare under natural conditions and 471

when exposed to environmental stresses; and more detailed research on genetic traits will be 472

linked to plant performance. The study highlights that targeted polyploidisation of trees could 473

transform tree breeding, choice of tree species for silviculture, and forest production systems. 474

475

21

Acknowledgements 476

This work was made possible by Bioadapt Pty. Ltd., Mount Tambourine, Australia, who 477

provided plant material and funding. 478

479

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630

27

Table 1. Nuclear DNA content obtained from flow cytometry and genome stability within 630

clone lines and genomic re-organization of clones determined from AFLP profiles for Agathis, 631

Eleaocarpus, and Paulownia. NA= data not available. 632

633

Genome content Dissimilarity to other Genome stability 634

(linear fluorescence) clone lines of same within clone lines 635

species (%) (%) 636

Agathis 637

Parent NA 4 99 638

Clone 1 NA 7 98 639

Clone 2 NA 9 99 640

Clone 3 NA 9 99 641

Elaeocarpus 642

Parent 110 1 100 643

Clone 1 140 4 100 644

Clone 2 135 4 100 645

Paulownia 646

Parent 100 12 99 647

Clone 3 135 17 95 648

Clone 7 75 16 96 649

Clone 15 100 15 98 650

651

652

653

28

Table 2. Leaf properties (total chlorophyll content, soluble protein content, leaf nitrogen use 653

efficiency; LNUE) of Agathis and Paulownia species. Data are averages and standard errors (in 654

parentheses) of three plants of each parent and clone of Agathis and Paulownia. Means marked 655

by different letters within a species are significantly different at P<0.05 level. 656

657 658 Chlorophyll Soluble protein LNUE 659 (µg chl. a and b cm-2) (µg protein cm-2) (µmol CO2 m-2 s-1 / % N) 660 Agathis 661 Parent 43.3 (6.6)a 6.33 (0.71)a 1.36 (0.11)b 662 Clone 46.4 (3.4)a 7.26 (0.36)a 1.89 (0.07)a 663 Paulownia 664 Parent 41.4 (0.24)b 15.4 (0.91)a 5.35 (0.53)a 665 Clone 3 42.9 (0.05)b 16.6 (0.49)a 6.18 (0.35)a 666 Clone 7 44.2 (0.84)a 19.0 (0.48)a 6.36 (0.34)a 667 Clone 15 42.1 (0.38)b 17.9 (0.86)a 7.01 (0.40)a 668

669

29

Table 3. Photosynthetic CO2 response curve (A/Ci) variables for Agathis and Paulownia. 669

Amax, maximum photosynthetic rate; Vcmax, maximum carboxylation rate; Jmax, maximum 670

electron transport rate. Data are averages of three curves for each parent and clone of 671

Paulownia and Agathis species with standard errors in parentheses. Means marked by different 672

letters are significantly different at the P<0.05 level within a species. 673

674 Amax Vcmax Jmax 675 (µmol CO2 m-2s-1) (µmol CO2 m-2s-1) (µmol electrons m-2s-1) 676 Agathis 677 Parent 9.1 (0.7)a 37.5 (3.7)b 55.1 (3.1)b 678 Clone 11.4 (1.0)a 59.0 (5.1)a 71.6 (4.4)a 679 Paulownia 680 Parent 31.2 (1.4)a 125 (6.0)a 163 (7.5)a 681 Clone 7 33.5 (0.6)a 140 (6.2)a 177 (2.7)a 682 683 684

685

30

685

686

687

688

689

690

691

692

693

694

695

696

697

698 Figure 4. Typical leaves of parents and clones of Agathis (A, parent; B, clone) and Paulownia 699

species (C, parent; D, clone) after 6 months and 3 months growth in the glasshouse, 700

respectively. Mean leaf size and standard errors from 3 plants is shown; means marked by 701

different letters are significantly different at the P<0.05 level. 702

703

a

A

B

D

C 10 cm

C

D

Leaf size = 669±44.3a cm2

Leaf size = 730±103a cm2 5 cm

A

B

Leaf size = 11.7±0.58b cm2

Leaf size = 37.7±1.31a cm2

31

703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743

Figure 5. Leaf elongation rate, specific leaf area, and total leaf area of parents and clones of 744 Agathis (A, B, C) and Paulownia (D, E, F, ) after 6 and 3 months of growth in the glasshouse, 745 respectively. Data are mean values from three plants for each parent and clone. Bars indicate 746 one ± standard error of the mean. Means marked by different letters are significantly different 747 at the P<0.05 level. 748

749

a a a a

F a

b

C

Tota

l lea

f are

a/ p

lant

(cm

2 ) Sp

ecifi

c le

af a

rea/

pla

nt

(cm

2 g-1 le

af)

a

b

B

a

a

a a

E

b

a A a

b

D Le

af e

long

atio

n ra

te (c

m d

-1)

32

749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 Figure 6. Differences in net photosynthetic rate and stomatal conductance of parents and 781

clones of Agathis (A and B) and Paulownia (C and D) measured using Li-Cor 6400 infrared gas 782

analyser. Data are mean values from three plants for each parent and clone. Bars indicate one ± 783

standard error of the mean. Means marked by different letters are significantly different at 784

P<0.05 level. 785

786 787

788

Net

CO

2 fix

atio

n (µ

mol

CO

2 m-2

s-1)

a

b

A

C

a

b b

a

Stom

atal

con

duct

ance

(m

ol H

2O m

-2s-1

)

a

b

b b

a

b

B

D

33

788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821

822

823

Figure 7. Photosynthesis versus intercellular CO2 concentration (A/Ci) and electron transport 824

rate as determined with rapid light response curves for parents and clones of Agathis (A and B) 825

and Paulownia (C and D). Each curve is the average of three plants. Bars indicate one ± 826

standard error of the mean. 827

A. robusta

Net

CO

2 fix

atio

n (µ

mol

CO

2 m-2

s-

1 )

Intercellular CO2 concentration (µmol mol-1)

Light intensity (µmol m-2s-1)

A

C

Elec

tron

trans

port

rate

(µ

mol

ele

ctro

ns m

-2s-1

)

D

B

34

828 829 830 831 832 833 834 835 836 837 838 839 840 841 842

843

844

845

846

847

848

849

850

Figure 8. Shoot biomass (g dry weight) and proportional biomass allocation to plant parts for 851

parents and clones of Agathis (A, B) and Paulownia (C, D) after 6 months and 3 months 852

growth, respectively. Data are mean values of three plants. Bars indicate one ± standard error of 853

the mean. Means marked by different letter are significantly different at P<0.05 level within a 854

species. 855

856

Shoo

t bio

mas

s/ p

lant

(g) a

b

A

a ab a

b

C

Prop

otio

nal b

iom

ass a

lloca

tion

Flowers Leaves Shoots Roots

D

Parent

B

Clone