CHAPTER-3

Materials and Method

3.1. Study site

Study site falls between latitude, 30˚ 20‟40” N; longitude, 77˚ 52‟12” E and an
altitude of 640.08 m asl. Climatic data of 2006 were obtained from Division of Forest
Ecology and Environment, Forest Research Institute, Dehradun, Uttarakhand (Annexure I).
Maximum and minimum rainfall occurred during the month of August (489.6 mm) and
November (1.1mm) respectively. Total annual precipitation was 1968 mm. Mean air
temperature of the year was 21.2˚ C with a mean maximum of 26.5˚ C (July) and mean
minimum of 12.5˚ C (January) in the year 2006.

3.2. Sampling
The study material for wood anatomical studies was collected from four-year-old
provenance trial of Eucalyptus tereticornis Sm. established at the campus of Forest Research
Institute, Dehradun (Uttarakhand), India. In the trial, 13 provenances (91 families) of
Eucalyptus tereticornis Sm. had been planted in Randomized Block Design in eight
replicates with four plants in a treatment. The details of the provenances, there source and
families is given in Table 1.

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Table 1. Details of the different provenances of Eucalyptus tereticornis established at
FRI, Dehradun.
S.No Provenances Number Origin
/Seed lot No. of
families Locality Latitude Longitude

1. 13399 6 Orobay to Emo, Papua New 080 57‟ 1480 28‟

Guinea (PNG)
2. 13418 1 Sirinumu Sogeri Plat, PNG 090 30‟ 1470 26‟
3. 17762 4 Warwick, Queensland, 280 15‟ 1520 05‟
Austraslia (QLD)
4. 17768 5 Yurammie, SF, New South 360 49‟ 1490 45‟
Wales (NSW)
5. 17770 4 Buckenbowra SF, NSW 350 40‟ 1500 07‟
6. 18732 10 Selection flat SF559, NSW 290 10‟ 1520 58‟
7. 19315 11 Credition SF, QLD 210 07‟ 1480 31‟
8. 20468 9 Cardwell, QLD 180 10‟ 1450 58‟
9. 20469 5 Mitchell River MT Molloy, 160 44‟ 1450 20‟
QLD
10. 20470 9 Mill stream archer creek, QLD 170 39‟ 1450 21‟
11. 20471 9 Helenvale, QLD 150 48‟ 1450 15‟
12. 20472 7 Walsh River, QLD 170 20‟ 1450 18‟
13. 20474 11 Burdekin River, QLD 190 48‟ 1460 4‟

Forty seven (47) plus trees were selected using basic index method for studying
genetic variations. The plus trees were harvested in 2006, at 15 cm above the ground to
produce coppice shoots for propagating them clonally. Then plus trees were selected
randomly for within tree variation using random number table. Systematic sampling was
made for the collection of wood samples from the plus trees. At the time of coppice, discs
were collected from three uniform heights i.e. (i) base (ii) 4 m (iii) 8m to analyze intra-tree
variation. Each disc was further divided into three directions viz. north, north-east and south–
west. Each direction will further be divided into three pith to periphery radial locations.

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 PLATE. I

T7 T8 T9
T1 T2 T3

T4 T5 T6 T10 T11 T12

T10

T13 T14 T15 T19 T20

T10 T10
T21

T17 T18 T22 T23 T24

T16

Discs at different heights of selected plus trees of Eucalyptus tereticornis of different

provenances.

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 PLATE. II

T25 T26 T27 T31 T32 T33

T28 T29 T30 T34 T35 T36

T10

T37 T38 T39

T43
T44 T45

T40 T41
T42

T47
T46

Discs at different heights of selected plus trees of Eucalyptus tereticornis of different

provenances.

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 To study the range of various physical and anatomical parameters of the wood
among the plus trees, the following methodologies have been adopted

3.3. Physical properties

3.3.1. Specific gravity: Forty seven plus trees of Eucalyptus tereticornis of 13 provenances
(91 families) were selected for the study. Intra-tree vertical and horizontal (peripheral and
radial) variations were studied in three randomly selected progenies out of 47 progenies
selected for the study. A total of 27 samples (1 tree x 3 discs (height) x 3 direction
(peripheral) x 3 radial locations) were considered for intra- tree variation for each out of 3
selected progenies. Further, to standardized sample size for 47 plus trees, 15 plus trees were
randomly selected. A total of 405 samples (15 tree x 3 discs x 3 direction x 3 locations) were
considered for inter- and intra-tree variation. The directional variations were non-significant
so not considered for further analysis of inter- tree variations of 47 plus trees. A total of 564
samples (47 tree x 3 discs x 4 locations) were considered for inter-tree variation.

3.3.2. Determination of specific gravity: Specific gravity of each sample (block) was
determined by the gravimetric method using single pan balance. The green volume of the
blocks was measured by water displacement after blocks were fully soaked in water.
Thereafter, samples were kept for drying in hot air oven at 103 ± 2°C till the constant weight
and oven dry weight was recorded (Purkayastha et al., 1979). Basic density of wood sample
was calculated as the ratio of oven dry weight and volume of saturated wood sample.
Specific gravity was determined by using the following formula:

Specific gravity=Basic density of wood sample / Density of water

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 PLATE. III

8m

4m

Base

Sampling for within tree variations

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3.3.3. Anatomical properties

The anatomical features that were assessed include frequency, size and distribution of
various cell types viz. vessels, parenchyma, rays and fibers.

3.3.4. Microtomy

Blocks of size 1 cm3 were taken from each height level and radial position. Each
block was softened by boiling in water for 10-15 minutes. Cross, radial and tangential
sections of 20 µm thickness were prepared using Reichert microtome.

3.3.5. Staining procedure

Twenty micron thick transverse, radial and tangential sections were differentially
stained using saffranin and haematoxilin for 20 minutes. The stained sections were washed in
acetone-xylene of 1:1 ratio for 10 minutes to ensure complete dehydration. Finally they were
mounted in DPX mountant to prepare the permanent slides (size 75mm X 25mm, thickness
1mm) and covered by cover slips.

3.3.6. Microscopy

For microscopic examination, 20 µm thick cross, radial and tangential, microtome

sections were used and observations were taken. Tissue proportion, vessel frequency (mm-2)
and tangential diameter were taken from the cross section of wood. Cross, tangential and
radial sections were used to describe the structure of the wood.

3.3.7. Fibril angle

The macerated wooden elements were thoroughly mixed and spread on a glass slide,
followed by teasing the material on the slide with the help of needles. The observations for
fibril angle were taken under Polarized Microscope.

3.4. Maceration

Small radial chips from the sample blocks were macerated to determine vessel
element length, fiber length, fiber diameter and wall thickness as per Schultz's method (50%

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Nitric acid and a pinch of Potassium chlorate). The macerated chips were kept in sunlight for
two to four days till the chips turns to milky white. The chips were then washed with two to
three times water and shake and few drops of saffranin were added. The macerated wood
elements were thoroughly mixed and were spread on a glass slide and observations were
taken under a compound microscope for vessel element length and fiber length, diameter,
wall thickness.

3.4.1. Observations

From the macerated material, observations for fiber length, fiber diameter, wall
thickness and vessel element length were taken. Twenty five measurements from unbroken
cells were taken for fiber length, fiber diameter, wall thickness, vessel element length and
vessel element diameter from each sample block (IAWA, 1989). The average tangential
diameter of the vessels was determined from twenty five measurements from cross-sections
taken along radial direction.

Tissue proportions like fibers, vessels, parenchyma and rays were determined in cross
section. The total score of each cell type was obtained by moving the slide from one end to
the other both in tangential and radial direction.

3.4.2. Pulp and paper ratios

Anatomical data obtained was used to determine Runkel Ratio (2 x cell wall
2 2
thickness/ lumen diameter), shape factor (outer fiber diameter – lumen diameter / outer
fiber diameter 2 + lumen diameter 2) and fiber-length: diameter ratio (Singh et al., 1991).

3.5. Chemical Analysis of the Samples

Chemical composition of the plant gives an idea of how feasible the plant is as raw
material for papermaking. The fibrous constituent is the main important part of the plant.
Since plant fibers consist of cell walls, the composition and amount of fibers is reflected in
the properties of cell walls (Hartley, 1987; McDougall et al., 1993). Cellulose is the principal
component in cell walls and fibers (Taiz and Zeiger, 1991; Philip, 1992; Cassab, 1998). The
amount and composition of the cell wall compounds differ among plant species and even
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among plant parts and they affect the pulping properties of plant material (McDougall et al.,
1993). Some of non woody fiber plants contain more pentosans (over 20%), holocellulose
(over 70%) and less lignin (about 15%) as compared with hardwoods (Hunsigi, 1989). They
have also higher hot water solubility, which is apparent from the easy accessibility of
cooking liquors. The low lignin content in non woody plants lowers the requirement of
chemicals for cooking and bleaching (Hunsigi, 1989). The exact standards that were followed
for chemical analysis are presented in Table 2.

Table 2: Standards followed for chemical analysis.

S.No. Standard Methods TAPPI No.

1. Holocellulose Useful method – 249

2 Klason Lignin T 222 om – 02

Water Content

Two gram air dried dust of both non-destructured and destructured samples were
placed in pre weighted weighing bottles. The weighing bottles with the dust particles were
then kept in oven at 105°C for overnight. Then the weighing bottles were removed and placed
in the desiccators for cooling it to room temperature before reweighing. The following
formula was used to calculate the moisture content (dry-weight) of sample.

(W2 - W1)  100

Dryness (%) =

Weight of sample

W2 - Stands for weight of weighing bottle + sample.

W1 - Stands for weight of empty weighing bottle.

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Holocellulose

Two and half gram (O.D.) extractive-free samples from non destructured and
destructured samples were placed into 250 ml flasks with small watch glass covers. The
samples were then treated with 80 ml of distilled water, 0.5 ml of cold glacial acetic acid, and
one gram of NaClO2.The flasks were then placed into a water bath maintained between 70 °C
- 80°C. Every hour for three hours 0.5 ml of cold glacial acetic acid and 1 g of NaClO2 were
added and the contents of the flasks were stirred constantly. At the end of three hours, the
flasks were cooled until the temperature of the flasks was reduced to 25 °C. The contents of
the flasks were filtered into G-2 glass crucibles of known weight followed by recycling. The
residues were washed with acetone. The crucibles were then oven-dried at 105±2°C, then
cooled in desiccators, and weighed until a constant weight was reached. The following
formula was used to determine the holocellulose content in samples

(W2 - W1)  100

Holocellulose % =

O.D. weight of sample

W2 - Stands for weight of crucible + sample.

W1 - Stands for weight of empty crucible.

Klason Lignin

1 g oven-dried extractive-free non destructured and destructured samples dusts were

placed in 100 ml beakers. 15 ml of cold sulfuric acid (72%) was added slowly in each beaker
while stirring and mixed well. The reaction proceeded for two hours with frequent stirring.
When the two hours had expired, the specimens were transferred by washing it with 560 ml
of distilled water into 2,000 ml flasks, diluting the concentration of the sulfuric acid to three
percent. The flasks were placed on hot plates for four hours. The flasks were then removed
from the hot plates and the insoluble materials were allowed to settle. The contents of the
flasks were filtered by vacuum suction into G-3 glass crucibles of known weight. The
residues were washed with distilled water and then oven-dried at 105±2oC. Crucibles were
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then cooled in desiccators and weighed until a constant weight was obtained. The following
formula was used to obtain the klason lignin content in samples:

(W2 - W1)  100

Lignin (%) =

O.D. weight of sample

W2 - Stands for weight of crucible + sample.

W1 - Stands for weight of empty crucible.

3.6. Statistical analysis

3.6.1. Design for wood quality parameters

The factorial experiment with three factors will be used for the experiments for the
wood quality parameters viz. fiber length, fiber diameter, fiber wall thickness, vessel element
length, vessel element diameter and specific gravity:

3.6.2. Intra- tree variations for randomly 3 selected trees

Direction - vertical, direction – horizontal

Vertical with 3 levels

Horizontal peripheral direction with 3 levels

Horizontal radial direction with 3 levels

All factors fixed

3.6.3. Inter-tree variations

Progeny, direction - vertical, direction - horizontal.

The levels are:

Trees: 47

i. Vertical with 3 levels

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ii. Horizontal

a). Radial with 4 levels

All factors are fixed.

The vertical sections are three in number whereas horizontal radial with 4 numbers.

Variations were studied tree - wise, vertical direction and horizontal direction.

The data obtained were statistically analyzed using SPSS (version 10.0) Multivariate
analysis to test the within- (both vertical and horizontal) and inter-tree (progeny) variations.
Multivariate Tests namely Pillai's Trace, Wilks' Lambda, Hotelling's Trace and Roy's Largest
Root were used to test multivariate analysis for test of significance. Finally, variance ratio (F)
test was used for the test of significance. Duncan test (subsets) was used for grouping of
different trees based on wood element‟s dimension and specific gravity. The cluster analysis
was done for all trees on the basis of wood traits viz. fiber-length, -diameter, wall-thickness,
Runkel Ratio, Shape factor, length/ diameter, chemical properties, and growth parameters
using SPSS (10.0). Pearson correlation was also determined among different wood traits and
also with growth parameters in all trees of Eucalyptus tereticornis to understand the inter-
relationship among different wood traits and with the growth parameters.

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