DOMESTICATION OF ALLANBLACKIA SPECIES IN GHANA
REPORT FOR JANUARY – JUNE 2006
REPORT FROM
CSIR-FORIG / ICRAF, GHANA
UNIVERSITY BOX 63, KUMASI, GHANA, Tel. +233 51 60123, Fax +233 51 60121
PROJECT TEAM
OFORI, D. A., PEPRAH, T. AND COBBINAH, J. R.
June 2006
1
Introduction
The oil from the Allanblackia nuts is attractive to Unilever since it can be used for margarine
production with less chemical processing and refraction than palm oil. Unilever are therefore
exploring the possibility of building a sustainable production, a sizeable tonnage of
Allanblackia oil per annum, with fair returns to collectors and local processors. They are
committed to helping conserve natural forest areas where it occurs and stimulating a smallholder production supply chain.
Unilever have asked ICRAF to assist in the domestication process of the species. Of
particular concern is that material entering cultivation is of sufficient genetic diversity to
provide an adaptive capacity to potential changes in environment and user requirements. For
this to be sustainable, knowledge on the intra-specific genetic variation for development of
core collection (gene bank) is a prerequisite. One major bottleneck in the domestication
programme of Allanblackia is the development of suitable methods for propagation and
enhancement of phase change from juvenile to mature phase.
Unilever in collaboration with World Agroforestry Centre/ICRAF identified institutions
in Tanzania, Cameroon, and Ghana of which the Forestry Research Institute of Ghana is
included to assist in the domestication of the species.
The objectives of the project with FORIG / ICRAF, Ghana are;
1. To develop appropriate methods for seed germination of Allanblackia sp.
2. To develop the best method for vegetative propagation of Allanblackia sp.
3. To establish Allanblackia gene bank in Ghana.
Germplasm collection
Germplasm collection was undertaken between January and March 2006. Four zones
were covered to select new trees that could not be sampled in year 2005 and also to get a
top up to trees that few fruits were obtained in year 2005 (Table 1; Fig 1). The sampled
trees were marked and given identification numbers. Each tree was visited at least twice
for collection of fruits that fall on their own. Table 1 shows the four zones and the
2
number of trees from which fruits were collected. Leaf samples were also collected from
trees the new trees that were identified in 2006.
Table 1 Allanblackia germplasm collection zones and number of accessions collected
Zone ID
Name
Localities
Ecological zone
No. of
trees
SR
Samreboi
M. Amenfi, Sureso, W. Asikuma etc
Moist Evergreen
14
SW
Sefwi Wiawso
Wiawso, Sui Ano etc
Moist Evergreen
5
MK
Mankranso
Goaso (Ayumso), Mankranso
Moist
Semi- 10
deciduous N/W
AR
Atewa Range
Kadewaso, Kyebi, Atewa range
Moist
Semi- 2
deciduous S/E
TOTAL
Fig. 1. Areas in Ghana where Allanblackia germplasm was collected
38
3
Processing
All fruits collected were sent to FORIG’s seed processing shed. The following records
were taken before seeds were extracted from the fruits; Fruit size (weight, length and
circumference), fruit shape (straight, curved) grooves (shallow, deep, very deep) and
nipple size (Fig. 2).
After seed extraction, the number of seeds per fruit and weight per 100 seeds were
recorded (Fig. 3). A total of 16,757 seeds were obtained (Table 2). Some fruit and seed
characteristics are shown in Table 3.
Fig. 2 Taking records on fruits
Table 2
Fig. 3
Taking records on seeds
Number of seeds sowed per zone
Zone ID
Name
Ecological zone No. of seeds
SR
Samereboi
ME
6,271
SW
Sefwi Wiawso
ME
6,828
MK
Mankranso
MSNW
1,719
AR
Atewa Range
MSSE
1,939
TOTAL
16,757
4
Table 3
Fruit and seed characteristics of Allanblackia palviflora in Ghana
Characters
Length (cm)
Circumference (cm)
Fruit wt (kg)
No seed/fruit
Wt of seeds / fruit (g)
Wt of 100 seeds (g)
Mean
30.1 ± 0.37
33.1 ± 0.34
1.4 ± 0.06
26.5 ± 1.18
210 ± 14
730 ± 30
min
20
25.3
0.5
18
100
600
max
40
41
3.4
48
800
1200
Germination on nursery bed (seeds sowed in year 2005)
Monitoring of seed germination (total of 63,393 seeds) from 150 mother trees is in
progress. As at 30th June 2006 (approx. 16 months after sowing), germination had been
recorded in 97 accessions forming 64.7% of the total number of accessions tested. The
germination percentage range from 0.3 – 35.0 % (Fig. 4).
Fig. 4 Germination percentages of 97 accessions of Allanblakia palviflora in Ghana
Seed germination in greenhouse (seeds sowed in year 2005)
Monitoring of effect of pre-sowing treatment on germination in the green house that
maintains 50% of the incident sun light is in progress. Treatments evaluated were;
1. Sowing with testa removed
2. Sowing with testa on
3. Testa not removed + soaking for 24 hours before sowing
5
Germination percentages so far obtained show that removal of seed coat enhances
germination. For seeds with testa removed, 81% of the accessions have germinated with
germination percentage ranging between 4 and 30, while seeds with intact testa, only
19% of the accessions have germinated with germination percentage ranging between 1
and 2. For intact seeds plus 24hr soaking in water, only 3 accessions were used. One of
them has germinated with germination percentage of 1 (Table 4).
Table 4
Effect of testa removal and soaking of seeds in water for 24 hrs before
sowing on germination
Accessions
MN 3
B4
SA 47
MH 7
SA 42
NEN 4
B 17
ASN 10
KAP 4
K 14
DB 12
NZA 27
NZA 29
AKO 2
DM 1
AS 3
NZA 26
AS 5
GB 12
KAP 16
GB 11
Testa removed
% Germination
20
30
9
11
0
0
10
6
4
4
6
8
4
18
0
14
0
0
22
6
22
Testa intact
% Germination
1
0
0
0
0
0
0
0
0
0
2
1
0
0
0
0
0
0
0
2
0
Testa intact +1 day
soaking; % Germ
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
0
1
0
N/A
N/A
N/A
N/A = experiment not conducted
Pre-sowing treatments on germination - 2006
From the results of pre-sowing treatments obtained, efforts are being made to break
dormancy through mechanical, chemical and physiological methods (Fig. 5). A total of
29 treatments were used. These were;
Mechanical
Testa removed
6
Scarified seeds
Seed coat intact
GA3 Treatment 1000 ppm
Intact seed soaked in 1000 ppm solution for 24, 48, 72 and 96 hours
Scarified seeds soaked in 1000 ppm solution for 24, 48, 72 and 96 hours
Seeds with testa removed soaked in 1000 ppm for 24, 48, 72 and 96 hours
GA3 Treatment 500 ppm
Intact seed soaked in 500 ppm solution for 24, 48 and 72 hours
Scarified seeds soaked in 500 ppm solution for 24, 48 and 72 hours
Seeds with testa removed soaked in 500 ppm for 24, 48 and 72 hours
Sulphuric acid treatment
Intact seed put in 5%, 10%, 25%, 50% and 75% sulphuric acid for 1 minute
Samples of seeds from each treatment were taken and were sown in 4 different media.
These were saw dust, coarse sand, coarse sand + sawdust (1:1) and sawdust + coarse sand
+ sandy loam soil (1:1:1). Trials were set in germination bowls in green house and on
nursery beds. A total of 16,757 seeds from 38 trees were sowed (Table 2).
Fig. 5a Seeds with testa removed
Fig. 5b Scarified seeds
7
Fig. 5c Seeds with test removed and soaked
in GA3 solution
Fig. 5e
Fig. 5d Scarified seeds soaked in GA3
solution
Intact seeds
Within three months after sowing, germination was recorded in seeds with testa removed
and sowed in coarse sand.
Gene bank establishment
Two hundred and sixty (260) seedlings from 70 accessions have been planted in a 1.5 ha
plot at Pra Anum Research Station of Forestry Research Institute of Ghana. The station is
located within the Moist Semi-deciduous forest zone with an average annual rainfall of
1,500-1,800 mm. The design was replicates of 2-tree plots. See the attached design in
8
appendix 2. Figs 6 and 7 show carting of Allanblakia seedlings from trucks to the field
and planted Allanblackia in the gene bank respectively.
Fig. 6
Carting of Allanblackia seedlings to field
Fig. 7
Planting of Allanblackia seedlings
Vegetative propagation
Propagation unit
The improvement of the propagation unit was completed during this reporting period.
Three large non-mist propagators (Fig. 8a) were built together with two weaning planks
(Fig. 8b) following the improved design by ICRAF Cameroon.
Fig. 8a Non mist propagators
Fig 8b Weaning plank
9
Cuttings
Propagation by stem cuttings continued this year. The sixteen trees felled in year 2005
produced coppiced shoots (Fig. 9) for cutting propagation. Shoots have been harvested
for propagation by cuttings. The harvested shoots were kept in humid bags specially
designed for collection of shoots for propagation (Fig 10). One to two node cuttings
ranging between 5 and 7 cm with about 40 cm2 leaf area were prepared and the based
dipped in a rooting powder (Hormex, 0.8% IBA) before inserting into the rooting
medium in the propagator (Fig.11). Rooting success of the first trial using Hormex was
35%. Now that lots of shoots have been produced, a second experiment has been set up
using 7 treatments. These include;
1. Hormex; quick dip
2. Rootone F; quick dip
3. IBA 500 ppm; base of cuttings soaked for 30 minutes (Fig. 12)
4. IBA 1000 ppm; base of cuttings soaked for 30 minutes (Fig. 12)
5. GA3, 500 ppm; base of cuttings soaked for 30 minutes (Fig. 12)
6. GA3, 1000 ppm; base of cuttings soaked for 30 minutes (Fig 12)
7. Control; cuttings prepared and inserted into the rooting medium without any treatment
Assessment is made fortnightly and cuttings potted when the longest root is at least 1 cm
long (Fig. 13).
Fig. 9a Coppice shoots
Fig. 9b Collection of coppice shoots
10
Fig. 10 Shoots in collection bags
Fig. 12 Base of cuttings soaked in
rooting hormone solution
Fig. 11 Cuttings in a propagator
Fig. 13 Rooted cutting of Allanblackia
ready for potting
Air layering
The possibility of propagating Allanblackia by air layering began in year 2005. This was
done by removal of 2 cm width of the bark from small branches (about 1-3 cm diameter)
and a ball of rooting medium (soil + fibre from decomposed palm tree) tied round the
wounded portion. A total of 40 layers were made from eight trees. Monitoring continued
during the first half of the year. For most of them the wounds had been healed and were
re-opened. So far only 5% of the layers have produced roots (Fig 14).
11
14a
Fig. 14. Air layering of Allanblackia palviflora;
a)
Roots developing from the base of the wound
b) Potted layer of A. palviflora
14
b
Potting and seedling management
Seedlings were potted into potting pots when stems were at least 2 cm long. Cuttings
were also potted when the longest root was at least 1 cm long. The potting medium
initially used was humus soil but leaves were found yellowing. A mixture of humus soil
and soil collected under Allanblackia trees has improved growth and greening of the
leaves. Potted propagules were kept in the nursery for at least 6 months before
transplanting to the field.
Monitoring of growth of Allanblackia
An agroforestry plot consisting of Allanblackia and maize has been established at FORIG
campus, Kumasi (Fig. 15). The growth of the Allanblackia is being monitored to establish
the growth rate and the number of years required for the tree to flower and fruit.
12
Fig. 15 Agroforestry with Allanblackia and maize at FORIG campus, Kumasi
DNA analysis
Protocol for DNA extraction has successfully been developed (see Appendix 3). Rapid
freezing of leaves in liquid nitrogen just after collection from the trees improves the DNA
yield and quality. Electrophoreses of 10 µl of the DNA extract showed that the DNA was
pure and of high quality. RAPD analysis has begun with testing of 20 different primers.
DNA amplification failed in all reactions except with one DNA sample that worked with
only one primer. More primers need to be tested to select best primers for the species.
Fig 16 Electrophoresis of DNA extracts to check the presence and quality of DNA
Awareness creation
Both farmers and Research Scientists have developed interest in undertaking cocoa
agroforestry with Allablackia. Arrangements have been completed to establish a
demonstration farm with an Agroforester who is interested in cocoa agroforestry with
trees. Our plan is to supply him with seedlings after the establishment of the two gene
banks.
13
Discussion
The behaviour of Allanblackia was unknown at the beginning of the project. It has been
found to be a difficult species in terms of germination of seeds, vegetative propagation
and DNA extraction from the leaves. Our experience however shows that seed
germination takes a long time, starting at about 7 months after sowing but could be
reduced by removal of testa before sowing. Seeds with testa removed and sowed in bowls
in green house had higher germination than seeds sowed on nursery beds with intact
testa. Germination within three months after sowing has been recorded on seeds with
testa removed before sowing. Due to this, several treatments are being tested to break
seed dormancy. These include mechanical, chemical and physiological treatments. For
propagation by cuttings, improvement of the propagation unit has enhanced the rooting
percentage from 12% to 35%. Application of the various hormone treatments currently in
progress would certainly give better results. Success in air layering is being improved by
widening the length of the bark removed. The layers obtained could then be multiplied by
serial propagation.
Large variability in morphological characteristics such as fruit and seed morphology has
been observed. Similarly the germination percentages of the seeds differ markedly among
genotypes. The extent of these variations is such that it not only exists among trees from
different provenances but also occurs among trees from the same provenance. This
suggests that the observed variability may have little to do with environmental factors but
rather has a genetic basis that may be reflected in molecular analysis. Analysis of genetic
diversity at DNA level is in progress. Difficulty in obtaining good quality DNA for the
analysis has now been overcome. Good quality DNA has been obtained but the problem
now lies with getting good amplification products. More primers therefore need to be
tested for identification of good primers for Allanblackia.
When the molecular analysis becomes a reality, a combination of the morphological
traits, germination behaviour and molecular diversity could enhance the selection of
superior genotypes for propagation.
14
Planned activities for July to December 2006
1. Monitoring of seed germination will continue
2. Vegetative propagation using cuttings will continue
3. Air layering will continue.
4. Grafting studies will begin
5. The established of gene bank at Amantia (Pra Anum Fores Reserve) will be
expanded as more seedlings reach planting stage.
6. Testing of primers for good amplification products will continue
15
Appendix 1. Accessions and the number of seeds sowed in year 2006
Zone
Atewa
Atewa
Mankranso
Mankranso
Location
Kyebi Apapam
Kadewaso
Akraboakrom
Akraboakrom
Accessions
KAP 6
WSO 1
AK 2
AK 3
Number of seeds sown
Mankranso
Akraboakrom
AK I
100
Mankranso
Ayumso-Kwahu
KW 1
710
Mankranso
Mankranso Hiapae
MH 3
10
Mankranso
Mankranso
Mankranso
Mankranso
Mankranso Hiapae
M. Nyamebekyere
Nyame Bekyere
Nyame Bekyere
MH 4
MN 3
NB 1
NB 3
106
64
974
539
Mankranso
Mankranso
Mankranso
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Samereboi
Sefwi Wiawso
Sefwi Wiawso
Sefwi Wiawso
Sefwi Wiawso
Sefwi Wiawso
Otaakrom
Otaakrom
Otaakrom
Kamaso
Kamaso
Bisaso
Bisaso
Bisaso
Bisaso
Nkrankrom
Nkrankrom
Nkrankrom
Nkrankrom
Wassa Asikuma
Wassa Asikuma
Manso Amenfi
W. A. Mensakrom
W. A. Mensakrom
W. A. Mensakrom
W. A. Mensakrom
W. A. Mensakrom
W. A. Mensakrom
Sui Ano
Sui Ano
Sui Ano
Sui Ano
Sefwi Wiawso
OT 1
OT 2
OT 5
KAMAS0 4
KAMASO 1
LAHO 3
LAHO 4
LAHO1
LAHO2
NK 10
NK 6
NK 7
NK 9
WAA 1
WAA 3
WAM 2
WAMK 1
WAMK 2
WAMK 4
WAMK 5
WAMK 7
WAMK3
SA 2
SA 3
SA 34
SA 4?
WR 4
380
1559
250
325
42
12
100
300
637
300
600
1145
250
230
100
538
300
1162
250
300
566
525
600
375
210
990
78
250
300
1250
330
Appendix 2. The design of the gene bank at Amantia
Amantia
BLOCK 1
OT7
OT7
KAP1
KAP1
AK3
AK3
MH5
MH5
KA16
KA16
DM4
DM4
OT5
OT5
KAP14
KAP14
GB11
GB11
WSO1
WSO1
WAMK7
WAMK7
AP6
AP6
OT1
OT1
D5
D5
KW2
KW2
LAHO5
LAHO5
NK2
NK2
B1
B1
GB10
GB10
DB10
DB10
AS43
AS43
TKA2
TKA2
WH1
WH1
B22
B22
AK16
AK16
K13
K13
MH8
MH8
B19
B19
ASN11
ASN11
SA3
SA3
K2
K2
BLOCK 2
WAMK13 B10
WAMK13 B10
TKA3
TKA3
DB2
DB2
BMK7
BMK7
NSN4
NSN4
B4
B4
NZA27
NZA27
WAMK10 SA47
WAMK10 SA47
AKO2
AKO2
MN3
GB12
MN3
GB12
AK1
A3
AK1
A3
WANK15 B12
WAMK15 B12
WR12
K4
WR12
K4
NEN1
LAHO2
NEN1
LAHO2
OT6
KAP16
OT6
KAP16
B18
MN1
B18
MN1
K8
K8
MH7
MH7
AS3
AS3
AP4
AP4
NZA26
NZA26
B17
B17
K14
K14
DB12
DB12
Pine plantation
Muronoim
AK16
AK16
K13
K13
MH8
MH8
B19
B19
ASN11
ASN11
SA3
SA3
K2
K2
ASN10
ASN10
WAMK13 B10
WAMK13 B10
TKA3
MN3
TKA3
MN3
DB2
AK1
DB2
AK1
BMK7
WANK15
BMK7
WAMK15
NSN4
WR12
NSN4
WR12
B4
NEN1
B4
NEN1
NZA27 OT6
NZA27 OT6
WAMK10 SA47
WAMK10 SA47
AKO2
AKO2
GB12
GB12
A3
A3
B12
B12
K4
K4
LAHO2
LAHO2
KAP16
KAP16
B18
B18
K8
K8
MH7
MH7
AS3
AS3
AP4
AP4
NZA26
NZA26
B17
B17
K14
K14
MN1
MN1
ASN10
ASN10
KAP1
KAP1
AK3
AK3
MH5
MH5
KA16
KA16
DM4
DM4
OT5
OT5
DB12
DB12
GB11
GB11
WSO1
WSO1
WAMK7
WAMK7
AP6
AP6
OT1
OT1
D5
D5
KW2
KW2
KAP14
KAP14
NK2
NK2
B1
B1
GB10
GB10
DB10
DB10
AS43
AS43
TKA2
TKA2
WH1
WH1
LAHO5
LAHO5
OT7
OT7
B22
B22
Appendix 2. DNA extraction protocol
PREPARATION OF DNA EXTRACTION BUFFER AND DNA EXTRACTION
You need the following
5 M NaCl (solution prepared)
1 M Tris, (solution prepared) adjust pH to 8
0.5 M EDTA pH8 (solution prepared) adjust pH to 8
2 % PVP40
2% Mercaptoethanol
1% Sodium sulphite
20% SDS
For preparation of DNA extraction solution use the following quantities per the desired
volume:
Components
50 ml vol
100 ml vol
500 ml vol
1 litre vol.
5M NaCl
14 ml
28 ml
140 ml
280 ml
1M Tris, pH8
10 ml
20 ml
100 ml
200 ml
1 M EDTA, pH8
4 ml
8 ml
40 ml
80 ml
PVP40, 2%
1 g ml
2g
10 g
20 g
CTAB, 1%
0.5g
1.0g
5g
10g
Water approx
29?
58?
NOTE !!!: 1. CTAB is very toxic and must be opened only in the fume chamber
2. Quantity of water will not be the same as the volume written. Prepare
the buffer in a beaker of cylinder with the desired volume indicated so that
it will be easy to top up with water to the desired volume.
STEPS IN DNA EXTRACTION
Prepare the following:
1. Chloroform: isopropanol mix – mix 24 ml chloroform with 1 ml isopropanol in fume
chamber. Note that chloroform does not like light
2. Wash buffer:
Ethanol
= 76 ml
2.5 M sodium acetate = 8 ml
Water
=16 ml
Total vol
= 100 ml
You can prepare 50 ml vol by using half of the quantities
3. 1x TE check from the protocol, otherwise use sterile distilled water instead
1
DNA EXTRACTION PROTOCOL
1. Pre-heat DNA extraction buffer to 65 °C
2. Grind 0.04g dry leaf in liquid nitrogen (0.1g fresh leaf)
3. Add 600ul extraction buffer
4. Add 50 ul 20% SDS
5. Incubate at 65°C for 20 min, mix gently by inversion at 3 min intervals
6. Add 150 ul of 5M potassium acetate, mix thoroughly by gentle inversion of tube
7. Place on ice for 20 min
8. Centrifuge at 12,000 rpm for 10 min
9. Transfer solution into new tube without any debris
10. Add 500 ul chloroform: isopropanol mix (24:1), mix and put on shaker for 5 min (set
to 15 rpm)
11. Gently pipette supernatant solution into a new tube
12. Add 1 ml of cold isopropanol
13. Incubate on ice for 30 min
14. Centrifuge at 10,000 rpm for 10 min
15. Discard supernatant
16. Wash pellets with 500 ml wash buffer
17. Wash pellets with 70% ethanol and invert tube to dry
18. Dissolve pellets in 150 ul 1x TE (pH8), warm at 37 oC for 30 min
19. Place on shaker and leave over night
20. Centrifuge at 8,000 rpm for 5 min
21. Transfer solution into fresh tube
DNA quality and quantity
Check DNA by running 10 ul on 1% agaros gel