CHAPTER 3

MATERIALS AND METHODS

The studies contained in this thesis were carried out in to two
segments; the first one deals with the fields and social effects on the
determination of farm forestry trends/attitudes with the farmers in three zones of
the Punjab. The second was on genetic and molecular characterization of three
farm forestry trees. These studies were made over a period of three consecutive
years.

3.1

Trends/Attitudes of Farm Forestry

The objectives of this aspect were to identify the farmers with believes that

influence them to be engaged in Farm Forestry or not and whether they can plan
any decision for the next five years. This empirical research was carried out in
three randomly selected divisional head quarters, representing three distinct
ecological zones in the Punjab province of Pakistan. For this purpose, a
comprehensive questionnaire (Appendix-I) was prepared and a number of
farmers of varying land holdings were interviewed in each zone. The
questionnaire included seven salient outcomes believes/ statements three salient
referents and seven salient control believes. The control believers included those
related to land tenure, inability to protect trees from damage, availability of barren
land, market factors and confirming the constraints of others.

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3.1.1 Selection/Categories of Farmers

Farmers were classified into various categories, based upon land
holdings, there occupation and association with farm forestry (called FF) and
those not engaged in farm forestry (called NFF) as female house holders play
very small role in the contest of this study, key decision makers in respect of tree
planting on the farm are dominantly the man who were recognized as holding
land. The following criteria were:

Farmers having land holding 25 acres and greater.

Farmers with land holding 10 to 24 acres.

Farmers having land holding less than 10acres.

3.1.2 Assessment of Believes

The two components of attitudes; Belief strength and outcome evaluation
were each measured using a five point Likert Scale (Likert 1931) ranging from:
Strongly agree (5) to strongly disagree (1) for belief strength, for out come, it
was Very good (5) to very bad (1). The two components of Subjective norms
(SN) were also measured on five point scale ranging from strongly agree to
strongly disagree, and for normative belief and motivation as Very much (5) to
not at all (1). Similarly the two components of Perceived behavioral control (PBC)
were measured on 5-1 scale, Strongly agree that the factors would be
hindrance to planting trees and Strongly disagree for control believes, as well
as for power of control believes.

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3.1.3 Evaluation of Results

The data was analyzed using SPSS. As the variables were measured on
ordinal scale; median and IQR, instead of mean and standard deviation were
used as measures of central tendency and dispersion. Significance of differences
between FF and NFF was observed by Mann-Whitney U Test. In the first phase
open interviews were taken with seven FF farmers and six NFF farmers from
each zone. for statements of believes in relation to outcomes of planting trees on
there farm in the next five years, along with their behaviors, limitations, freedom,
etc. Statements recorded from multiple interviews were regarded as salient to be
used for the interview in the second phase. In the second phase: 34 FF farmers
and 30 NFF farmers were interviewed. The samples were selected in three
stages depending upon land holding. The list of the farmers in each category was
drawn up from sixteen union councils and from District Officers of Agriculture;
eight farmers in each of the two categories were selected at random.

3.2

Description of the Study area

These studies were conducted in the three distinct ecological zones in the

Punjab which were selected at random (Fig. 1). Three divisional head quarters;
Lahore, Bahawalpur and Faisalabad representing each zone are described
ecologically and climatically as below:

Lahore Zone

Bahawalpur Zone

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Faisalabad Zone

Figure-1. The Map of the Punjab Province (Pakistan

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3.1.1 Lahore Zone

Lahore is a divisional head of different Districts of the Punjab. Gujranwala

and Kasur Districts form its boundaries in the North and South respectively, while
Amritsar (India) and Sheikhupura districts are respectively situated in the East
and West across the river Ravi. It is situated at an altitude about 213 meters (698
feet) above sea level, with the temperature ranging from 9C to 37C. The annual
precipitation limits are 160mm to 440mm. Regarding its topography, this district
has vast plain grounds dissected by river, canal and narrow water channels.

Lahore

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Figure-2. The map of Lahore (Punjab)

Meteorological Data

Table-1

Monthly minimum and maximum temperature (C)

during study period (Lahore)

Month
January
February
March
April
May
June
July
August
September
October
November
December

Minimum
Temperature C
2004-05
2005-06
9.6
8.4
12.2
14.7
19.1
16.1
23.3
22.7
26.1
27.4
27.2
26.8
27.7
27.6
26.9
26.9
26.2
24.9
19.5
21.6
15.0
16.1
10.7
9.9

Maximum
Temperature C
2004-05 2005-06
18.1
19.7
23.3
26.7
31.4
26.8
36.4
35.7
38.8
39.5
37.0
37.8
36.7
35.0
34.2
34.1
35.3
33.2
29.9
31.6
27.0
26.0
21.9
20.5

Table-2 Mean monthly rainfall (mm) during the study period (Lahore)
Year
Month

2004-2005

2005-2006

January
February

38.2
13.2

64.2
55.6

March

0.0

64.2

April

12.1

11.4

May

42.0

1.7

June

72.9

32.0

July

144.0

223.5

August

143.5

129.0

September

11.2

69.8

October

5.8

0.8

November

4.0

-1.0

December

8.3

0.0

Year Total

495.2

652.2

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3.1.2 Bahawalpur Zone

Bahawalpur is a District Headquarter of different districts. Bahawalnagar

and Rahimyar Khan District form its boundaries in the East and West
respectively, while Lodhran and Vehari districts are situated in the North across
the river Sutlej, which flows from North-East to North-West. On the South and
South-East the district forms the international Border with India. District
Bahawalpur is spread over an area of 24,830 square kilometers. District
Bahawalpur has very hot and dry climate in summer, dry and cold in winter. The
maximum temperature rises to 48C and minimum temperature falls to 7C and
dust storms are frequent during the summer. The average annual rain-fall in the
district is about 200 mm. The district may be divided into three areas. These are
the riverain area, the irrigated area and the desert area. The riverain area lies
close to the river Sutlej, which flows in the North along its boundary with Lodhran
and Vehari districts. The area is inundated during floods. The irrigated tract is
higher than the riverain area. Most of the land has been brought under
cultivation. The desert area is known as Cholistan. It lies to the south and East of
the irrigated tract and North of the desert of Bikanir and Jaisalmir in India. It
extends along the entire Eastern boundary of Bahawalnagar district in the North
and Rahimyar Khan district in the South. The surface of the desert consists of a
succession of sand dunes rising in places to a height of 150 meters. It is covered
with the vegetation peculiar to the sandy tracts.

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Bahawalpur

Figure-3. The map of Bahawalpur (Punjab)

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Meteorological Data

Table-3 Monthly minimum and maximum temperature (C)

during study period (Bahawalpur)
Month
January
February
March
April
May
June
July
August
September
October
November
December

Minimum
Temperature C
2004-05
2005-06
7.8
6.2
9.5
11.9
16.2
14.5
22.1
21.0
26.6
28.5
28.8
27.6
28.9
30.0
27.6
27.6
25.0
25.1
23.1
21.0
12.7
14.7
10.8
8.3

Maximum
Temperature C
2004-05 2005-06
20.4
20.7
25.9
27.1
34.2
29.1
40.5
38.7
42.1
43.6
42.4
42.0
40.7
41.0
37.9
37.7
37.1
36.7
33.1
34.5
30.8
28.4
25.1
22.8

Table-4 Mean monthly rainfall (mm) during the study period (Bahawalpur)
Year
Month

2004-2005

2005-2006

January
February

22.4
1.0

4.0
45.1

March

0.0

28.0

April

3.0

1.0

May

1.0

4.0

June

11.4

0.0

July

0.0

43.0

August

63.0

0.0

September

1.0

72.0

October

1.0

0.0

November

0.0

0.0

December

46.0

0.0

Year Total

146.8

197.3

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3.1.3 Faisalabad Zone

The Faisalabad district is bounded on the North and West by Hafizabad,

Jhang and Toba Tek Singh districts respectively. In the East it touches
Sheikhupura district and in the South it is bounded by the river Ravi, across
which lies Okara districts. The district is spread over an area of 5856 Square
kilometers. The district has extremely hot and cold climate. The maximum
temperature during the summer touches 48C and the minimum temperature
recorded is 4C. The monsoons visit the area from July to September, while
winter rains occur during January to March. The average rain fall in the District is
about 400 mm. The soil is fertile but some areas are affected by salinity and
water logging.

Faisalabad

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Figure-4. The map of Faisalabad (Punjab)

Meteorological Data
Table-5 Monthly minimum and maximum temperature (C)
during study period (Faisalabad)

Month
January
February
March
April
May
June
July
August
September
October
November
December

Minimum
Temperature C
2004-05
2005-06
5.7
5.81
7.6
8.1
13.5
13.8
20.8
20.8
28.5
26.8
28.5
28.3
27.6
27.8
27.5
27.6
24.6
23.9
19.2
19.5
12.3
11.3
6.5
6.2

Maximum
Temperature C
2004-05 2005-06
18.2
16.6
21.2
24.5
28.4
29.8
38
34.9
41.8
41.5
40.1
39
36.8
36.6
37.7
37.1
36.2
36.6
35.2
34.5
28.5
28
23.8
23

Table-6 Mean monthly rainfall (mm) during the study period (Faisalabad)
Year
Month

2004-2005

2005-2006

January
February

9.5
10.8

-1.0
-1.0

March

0.8

4.5

April

1.0

35.0

May

1.0

10.2

June

56.5

140.1

July

92.9

96.0

August

49.5

65.0

September

27.7

2.6

October

1.0

-1.0

November

1.5

-1.0

December

1.0

10.2

Year Total

249.2

363.6

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3.3 Molecular Characterization of forest trees

The second segment of the thesis work was carried out at the Centre for
Agricultural

Biochemistry

and

Bioctechnology,

Uinversity

of

Agriculture,

Faisalabad during 2005-2006.

3.3.1 Plant material

Three farm forestry trees were identified in each ecological zones of the
Punjab to examine the genetic diversity within the species. The objective was to
obtain clear knowledge of the selected tree species with respect to ecological
adaptation, farmers preference, selection and performance in each zone. The
plant species were;

Dalbergia sissoo Roxb.(Family:Papilionaceae,Genus Dalbergia Linn)

Locally known as Shishum or Tali; Two types of the trees were observed
at forestry farms i.e. trees with dark green (healthy) and trees having
yellowish green (diseased)

Acacia

nilotica

(L.)

Delile,

syn.

Acacia

Arabic.

Willd.

(Family

Mimosaceae, Genus: Acacia Mill.) Locally known as Kiker. Trees selected

were dark green and spreaded crown.

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Populus deltoides Bert, (Family Salicaceae, Genus: Populus Linn.)

. Locally known as poplar. Trees were apparently healthy and dark green
in colour.( Khan;D.A. Floricultures and Landscape Gardening)

3.3.2 Tissue Collection

Young leaves of the test trees species were collected, wrapped in moist
muslin cloth, stored in an auto refrigerator and brought to the CABB Laboratory
where they were stored in refrigerator at - 80 C until processed. Efforts were
made to process them for DNA extraction with out any delay.

Florescence
Dalbergia sissoo

Seed Pods of
Dalbergia sissoo

Dalbergia sissoo

Seed pods
Acacia nilotica

Leaves/Flowers-13Acacia nilotica

Leaves
Populus deltoides

Florescence
Populus deltoides

Populus deltoides

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-15-

3.4

DNA extraction

DNA was extracted from young leaves using the N-cetyl-N, N, Ntrimethylammonium bromide (CTAB) method described by (Doyle and Doyle
1990). DNA was extracted from younger leaves because good quality DNA can
be extracted as they have thin cell walls. Protocol for extraction using Qiagen
DNeasy kit is given below. In the present work both CTAB and Qiagen
DNeasy Kit were used for genomic DNA extraction according to the protocol
given below (David et al. 1996).

3.4.1 Protocol for DNA Extraction

100mg sample of leaf tissues was taken and frozen in liquid

nitrogen.

Frozen tissue was homogenized to a fine powder with a

mortar and pestle in liquid nitrogen.

The powder was immediately transferred in to microfuge tube

containing 400ml API buffer for cell lysis.

Added 4ul RNAse and vortex to mixed it up and remove

clumps and incubated it at 65C in water bath for 10 minutes.

Added 130ul AP2 buffer, mix and incubate for 5 minutes on

ice.

Cut blue tubes tips equal to the number of samples.

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Applied whole material with cut tips to Lilac QLA shredder

tubes and spun it for 2 minutes at maximum speed.

Transferred the material from collection tube to new

microfuge tube and discarded spin column and collection
tube.

Added 675ul AP3 buffer to clear lysate and miximum with tip,
flick and vortex.

Added 650ul of this mixture in DNeasy column, spin for 1

minute at 8000 rpm.

Added remaining material DNeasy column and spin again for

2 minutes at 8000 rpm and discard flow through at bottom
with tube.

Put DNeasy column in new collection tube and add 500ul AW

buffer into column, and spin for 1minute 8000 rpm discard
flow through but keep the tube.

Added 500ul AW buffer and spin for 2 minutes, at maximum

speed to dry column membrane.

Cut lids of microfuge tubes put DNeasy column in these

tubes and discard collection with contents.

Added 100ul preheated AE buffer directly in to column

membrane. Incubate at room temperature for 5 minutes spin
for 1 minute at 8000rpm.

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Transferred elute to other new microfuge or screw top tube

and label it. This would be first eluting.

Re-used microfuge tubes with same AE buffer to get 2nd

elute. Spec to estimate conc/analyse on gel.

3.4.2 Standardization of PCR conditions

An optimization of PCR conditions was conducted with respect to
concentration of genomic DNA, 10X PCR buffer, Mgcl2, dNTPs, primer and Taq
DNA polymerase because the reproducibility of RAPD technique is influenced by
these factors and type of thermocycler used (Scherwater and Ender, 1993).
Template DNA, Taq DNA polymerase and Mgcl2 with different concentrations
were used to standardize the PCR conditions in order to obtain bright and
reproducible RAPD banding pattern. Template DNA with five different
concentrations of (7, 10, 15, 20, 25 ng/ul) was tested and it was found that
concentration of 15ng/uL was optimum to produce more consistent and
reproducible banding pattern. Similarly Mgcl2 with eight different concentrations
(1.7, 2.0, 2.3, 2.5, 2.8, 3.0, 3.3, 3.5 mM) was used and 3mM was found optimum
for consistent results. In case of Taq DNA polymerase one unit concentration of
Taq was found optimum for better amplification of template DNA after studying
different concentrations (0.2, 0.5, 0.7, 1.0, 1.5 unit/25uL). Other PCR reaction
conditions were kept constant to achieve the consistent and reproducible RAPD
banding pattern in each replicate.

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-19-

. The optimized reaction conditions are given as follows.

Thermal Cycler

Eppendorf AG No. 533300839, Germany

DNA Template
Taq DNA polymerase

15 ng

1.0 U (MBI, Fermentas, Vilnius, Lithuania)

KCl

MgCl

50 mM
:

3 mM

dNTP

100 mM

10 per primers

0.2 mM

The total reaction was performed in a volume of 25 l.

PCR temperature Profile
Hot start

95 C for 5 minutes.

Denaturation

95 C for 1 minute.

Primer annealing

34 C for 1 minute.

Extension

72 C for 2 minutes.

Go to step 2, 40 times
Final Extension

72 C for 10 minutes.

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3.4.3 Estimation of DNA

Extracted DNA was estimated visually by comparing with different

concentrations of r DNA, (DNA with known concentration), results were
evaluated by Popgen software (Ver; 1.44). Different concentrations of r DNA
were made from 100ug/ul of rDNA.

1. 100g/l
2. 50g/l
3. 25g/l
4. 12.5g/l
5. 6.25g/l

1% agarose gel was used for quantification of genomic DNA, protocol for
making for gel (appendix-II)

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