Anatomical Structure of Anatomical Structure of Transgenic Aspen Trees Transgenic Aspen Trees COST Action FP 0802 COST Action FP 0802 May 11 May 11 - - 13, 2009, Vienna, Austria 13, 2009, Vienna, Austria ************************************************************************************ Ilona Ilona Peszlen Peszlen 1 1 , , Balazs Balazs Horvath Horvath 1 1 , Perry Peralta , Perry Peralta 1 1 , , Bohumil Bohumil Kasal Kasal 2 2 & & Laigeng Laigeng Li Li 3 3 1 1 Wood & Paper Science, North Carolina State University, Raleigh, NC 2 2 Architectural Engineering, Pennsylvania Housing Research Center, University Park, PA 3 3 Institute of Plant Physiology and Ecology, Chinese Academy of Science, Shanghai, China
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Anatomical Structure of Anatomical Structure of Transgenic Aspen TreesTransgenic Aspen Trees
IlonaIlona PeszlenPeszlen11, , BalazsBalazs HorvathHorvath11, Perry Peralta, Perry Peralta11,,BohumilBohumil KasalKasal22 & & LaigengLaigeng LiLi33
1 1 Wood & Paper Science, North Carolina State University, Raleigh, NC2 2 Architectural Engineering, Pennsylvania Housing Research Center, University Park, PA
3 3 Institute of Plant Physiology and Ecology, Chinese Academy of Science, Shanghai, China
INTRODUCTION
Scanning electron micrographs of stem transverse sections of control (a.) and
transgenic line with reduced lignin content (b.) (Hu et al. 1999)
Enhanced stem and leaf growth in transgenic trees (A6, A9, A11, A8) with decreased lignin content (Hu et al.
1999).
a.
b.
OBJECTIVETo evaluate wood and fiber anatomy of wild type aspen and
transgenic aspen with different levels of lignin content and S:G ratios.
Novelty and SignificanceInvestigation of wood properties of transgenic trees to understand the
mechanism of lignification as it relates to anatomical structure and mechanical behavior of wood.
Long-term GoalsTo assess different wood quality traits of genetically engineered wood for specific applications including solid wood, fiber, composites, and energy
and thus enhance the utilization potential of the forest biomaterial.
MATERIALSMATERIALS
A total of A total of forty forty oneone--year old quaking aspen (year old quaking aspen (PopulusPopulus tremuloidestremuloides) ) clonesclones including wild type and three transgenic groups:including wild type and three transgenic groups:
T0 T0 -- Wild typeWild type aspen as control (Line 271)aspen as control (Line 271)
T1 T1 -- ReducedReduced lignin contentlignin content through transfer of an antisensethrough transfer of an antisense 44--CLCL gene gene controlled by a xylemcontrolled by a xylem--specific promoter (Lines 21, 23, 37)specific promoter (Lines 21, 23, 37)
T2 T2 –– ChangedChanged lignin structurelignin structure –– (increased (increased syringyl/guaiacylsyringyl/guaiacyl ratio)ratio) through through insertion of a senseinsertion of a sense CAld5HCAld5H gene (Lines 94, 96)gene (Lines 94, 96)
T3 T3 -- CombinationCombination of the two genes (Lines 72, 141)of the two genes (Lines 72, 141)
Five Five sample treessample trees from each transgenic line.from each transgenic line.
MATERIALSMATERIALS
Quaking Aspen(Populus Tremuloides)
Ptr WT
Wild-Type
271
Ptr 4CL
- 4CL
21 ; 23 ; 37
Ptr CAld5H
CAld5H
94 ; 96
Ptr 4CL/CAld5H
-4CL and CAld5H
72 ; 141
DecreasedLignin Content
Increased S/G RatioDecreased
Lignin Content& Increased S/G
1 -year-old
MATERIALSMATERIALS
One-year old wild type and transgenic aspen trees growing in the greenhouse.
MATERIALSMATERIALS
MATERIALSMATERIALS
Plant comparison between wild-type quaking aspen (PtrWT-271) andtransgenic aspen with increased S:G ratio (PtrCAld5H-96).
Ptr4CL-271 PtrCAld5H-96
MATERIALSMATERIALS
1 Actual lignin content measured on sample trees used in this study by Horvath et al. 2009b (unpublished);2 Adopted values from Li et al. (2003).
Ptr WT ( Wild‐type )
Ptr 4CL ( Reduced Lignin Content )
Ptr CAld5H ( Increased S/G Ratio )
Prt 4CL/CAld5H (Reduced Lignin Content, Increased S/G Ratio )
Sample Disks– Radial growth rate– Eccentricity of the stem
Microtome Sections– Quantitative Wood Anatomy with Image Analyzer
Fiber Maceration– Fiber Length and Coarseness (Fiber Quality Analysis)
Statistical Analyses– Determine Sources of Variation with SAS Enterprise Guide 4.1
METHODSMETHODS
k j iiji j i1k j i + ) (G L + G + D × + = Y εβμ
Anatomical PropertiesAnatomical Properties
Quantitative Wood Anatomy
– Vessel Number – Vessel Diameter– Vessel Lumen Area%
– Fiber Lumen Area % – Ray Area %– Total Cell Wall %
Fiber Diameter.
Fiber Lumen Area Fraction
Fiber Length– Average of 3000 fibers
– Fine: 0~0.2 mm
Fiber Coarseness– Weight per unit fiber length (mg/m)
Anatomical PropertiesAnatomical Properties
RESULTSRESULTS
1mm 1mm
Stem diameter comparison between wild-type quaking aspen (PtrWT-271) and transgenic aspen with increased S:G ratio (PtrCAld5H-96) using same magnification under stereo light microscope.
PtrWT-271 PtrCAld5H-96
RESULTSRESULTS
Cross sections of a wild-type quaking aspen (PrtWT-271) and a transgenic with both reduced lignin content and increased S/G ratio (Prt4CL/CAld5H-72) exhibiting differences in anatomical structure.
20µm 20µm
PtrWT-271 Ptr4CL/CAld5H-72
RESULTSRESULTS
Genetic groupsand lines/Properties
Ptr WT( Control )
Ptr 4CL( Reduced Lignin Content )
Ptr CAld5H( Increased S/G Ratio )
Prt 4CL/CAld5H(Reduced Lignin Content, Increased S/G Ratio )
271 21 & 23 & 37 94 & 96 72 * 141 *
Stem Diameter (mm) 9.3 a (8.2) 9.6 a (11.1) 7.4 b (9.8) 9.2 a (6.6) 9.6 a (8.2)
Vessel Number 72.1 bc (14.7) 76.5 b (20.0) 82.5 a (19.4) 58.8 d (13.5) 69.5 c (20.4)
Vessel Lumen Area Fraction (%)
20.7 c (12.0) 22.2 b (11.5) 22.0 b (9.6) 24.1 a (15.8) 21.9 b (13.3)
Vessel Lumen Diameter (μm)
27.4 cd (6.6) 27.9 bc (10.9) 26.5 d (9.4) 32.8 a (8.7) 28.9 b (10.7)
Fiber Lumen Area Fraction (%)
40.2 bc (7.3) 39.5 c (8.2) 37.8 d (11.2) 41.1 ab (5.9) 42.1 a (5.6)
Fiber Lumen Diameter (μm)
7.6 c (9.5) 7.7 c (11.6) 7.5 c (9.1) 9.6 a (8.3) 8.0 b (8.2)
Ray Number 5.8 a (22.5) 6.0 a (22.1) 5.9 a (23.4) 4.8 b (25.8) 5.4 a (17.5)
Ray Area Fraction (%) 10.4 a (24.9) 10.5 a (21.6) 11.0 a (24.5) 11.1 a (28.2) 10.3 a (19.3)
Cell Wall Area Fraction (%)
28.4 a (13.5) 27.9 a (16.3) 29.1 a (15.9) 23.6 b (17.7) 25.5 b (11.9)
Fiber Length FQA (mm) 0.48 b (2.7) 0.44 c (6.6) 0.44 d (4.7) 0.50 a (5.8) 0.46 b (5.0)
Fiber Coarseness FQA (mg/m)
0.12 a (15.9) 0.08 d (13.5) 0.10 b (13.8) 0.10 b (10.6) 0.09 c (9.2)
Radial growth and anatomical properties of wild-type and transgenic aspen groups using quantitative wood anatomy and fiber quality analysis
Notes: Results are presented as mean values. Common superscript letter(s) indicate(s) values are not differ significantly from each other as determined by Duncan multiple range test at α=0.01. Letter `a` shows the highest value(s). Values in parentheses are coefficient of variation in percentage. * Genetic line Ptr4CL/CAld5H-72 and genetic line Ptr4CL/CAld5H-141 were handled as separate genetic groups, because of the high lignin content differences within genetic group Ptr4CL/CAld5H.
RESULTS
SUMMARY & CONCLUSIONSSUMMARY & CONCLUSIONS
• Decrease in lignin content has no effect on the anatomical structure;
• Increase in the syringyl/guaiacyl ratio results in a slower diameter growth and formation of smaller but more numerous vessels;
• The combined effect of lignin content and S/G ratio changes on radial growth and cell morphology seems to be more complex and needs further investigation;
• All transgenics have significantly different fiber length and coarseness values than wild-type. However, these differences are very small and negligible from a practical point of view.
ACKNOWLEDGEMENT
• This project is being supported by the NationalResearch Initiative of the USDA-CSREES,grant number 2005-35504-16145.
• The plants and greenhouse facilities were provided by Dr. Vincent Chiang, North Carolina State University, Forest Biotechnology Group.
• FORBIRC Member Companies:Aracruz, Brazil; Arborgen, USA; Arauco, Chile; CMPC, Chile; FOSA, Uruguay; Mondi, South Africa; Oji Paper, Japan; Sappi, South Africa; VCP, Brazil; Weyerhaeuser, USA
ACKNOWLEDGMENTSACKNOWLEDGMENTS
• Richard Giles & Laszlo Horvath – Graduate Student
Department of Wood and Paper Science, NCSU
• Erik Mendoza – Undergraduate Student
Department of Wood and Paper Science, NCSU
• Szabolcs Koman – PhD Student, Research Assistant
Faculty of Wood Science, UWH, Hungary
Thank you for your attention!Thank you for your attention!
