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DOMESTICATION OF ALLANBLACKIASPECIES 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

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 small-holder 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 number of trees from which fruits were collected. Leaf samples were also collected from trees the new trees that were identified in 2006.

Table 1Allanblackia 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-deciduous N/W / 10
AR / AtewaRange / Kadewaso, Kyebi, Atewa range / Moist Semi-deciduous S/E / 2
TOTAL / 38

Fig. 1. Areas in Ghana where Allanblackia germplasm was collected

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 Fig. 3Taking records on seeds

Table 2 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 / AtewaRange / MSSE / 1,939
TOTAL / 16,757

Table 3Fruit and seed characteristics of Allanblackia palviflora in Ghana

Characters / Mean / min / max
Length (cm) / 30.1 ± 0.37 / 20 / 40
Circumference (cm) / 33.1 ± 0.34 / 25.3 / 41
Fruit wt (kg) / 1.4 ± 0.06 / 0.5 / 3.4
No seed/fruit / 26.5 ± 1.18 / 18 / 48
Wt of seeds / fruit (g) / 210 ± 14 / 100 / 800
Wt of 100 seeds (g) / 730 ± 30 / 600 / 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. 4Germination 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 lightis 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

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 4Effect of testa removal and soaking of seeds in water for 24 hrs before sowing on germination

Accessions / Testa removed
% Germination / Testa intact
% Germination / Testa intact +1 day soaking; % Germ
MN 3 / 20 / 1 / N/A
B 4 / 30 / 0 / N/A
SA 47 / 9 / 0 / N/A
MH 7 / 11 / 0 / N/A
SA 42 / 0 / 0 / N/A
NEN 4 / 0 / 0 / N/A
B 17 / 10 / 0 / N/A
ASN 10 / 6 / 0 / N/A
KAP 4 / 4 / 0 / N/A
K 14 / 4 / 0 / N/A
DB 12 / 6 / 2 / N/A
NZA 27 / 8 / 1 / N/A
NZA 29 / 4 / 0 / N/A
AKO 2 / 18 / 0 / N/A
DM 1 / 0 / 0 / N/A
AS 3 / 14 / 0 / 0
NZA 26 / 0 / 0 / 1
AS 5 / 0 / 0 / 0
GB 12 / 22 / 0 / N/A
KAP 16 / 6 / 2 / N/A
GB 11 / 22 / 0 / 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

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 (Table2).

Fig. 5a Seeds with testa removedFig. 5b Scarified seeds

Fig. 5cSeeds with test removed and soakedFig. 5dScarified seeds soaked in GA3 in GA3 solution solution

Fig. 5eIntact 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 appendix2. Figs 6 and 7 show carting of Allanblakia seedlings from trucks to the field and planted Allanblackia in the gene bank respectively.

Fig. 6Carting of Allanblackiaseedlings to fieldFig. 7Planting ofAllanblackia 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 propagatorsFig 8bWeaning plank

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 mediumin 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

Fig. 10Shoots in collection bagsFig. 11Cuttings in a propagator

Fig. 12 Base of cuttings soaked in Fig. 13 Rooted cutting of Allanblackia

rooting hormone solutionready 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).

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 plotconsisting of Allanblackiaand 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.

Fig. 15Agroforestry 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 16Electrophoresis 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.

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 layersobtained 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.

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 seedlingsreach planting stage.
  6. Testing of primers for good amplification products will continue

Appendix 1. Accessions and the number of seeds sowed in year 2006

Zone / Location / Accessions / Number of seeds sown
Atewa / Kyebi Apapam / KAP 6 / 380
Atewa / Kadewaso / WSO 1 / 1559
Mankranso / Akraboakrom / AK 2 / 250
Mankranso / Akraboakrom / AK 3 / 325
Mankranso / Akraboakrom / AK I / 100
Mankranso / Ayumso-Kwahu / KW 1 / 710
Mankranso / Mankranso Hiapae / MH 3 / 10
Mankranso / Mankranso Hiapae / MH 4 / 106
Mankranso / M. Nyamebekyere / MN 3 / 64
Mankranso / Nyame Bekyere / NB 1 / 974
Mankranso / Nyame Bekyere / NB 3 / 539
Mankranso / Otaakrom / OT 1 / 42
Mankranso / Otaakrom / OT 2 / 12
Mankranso / Otaakrom / OT 5 / 100
Samereboi / Kamaso / KAMAS0 4 / 300
Samereboi / Kamaso / KAMASO 1 / 637
Samereboi / Bisaso / LAHO 3 / 300
Samereboi / Bisaso / LAHO 4 / 600
Samereboi / Bisaso / LAHO1 / 1145
Samereboi / Bisaso / LAHO2 / 250
Samereboi / Nkrankrom / NK 10 / 230
Samereboi / Nkrankrom / NK 6 / 100
Samereboi / Nkrankrom / NK 7 / 538
Samereboi / Nkrankrom / NK 9 / 300
Samereboi / Wassa Asikuma / WAA 1 / 1162
Samereboi / Wassa Asikuma / WAA 3 / 250
Samereboi / Manso Amenfi / WAM 2 / 300
Samereboi / W. A. Mensakrom / WAMK 1 / 566
Samereboi / W. A. Mensakrom / WAMK 2 / 525
Samereboi / W. A. Mensakrom / WAMK 4 / 600
Samereboi / W. A. Mensakrom / WAMK 5 / 375
Samereboi / W. A. Mensakrom / WAMK 7 / 210
Samereboi / W. A. Mensakrom / WAMK3 / 990
Sefwi Wiawso / Sui Ano / SA 2 / 78
Sefwi Wiawso / Sui Ano / SA 3 / 250
Sefwi Wiawso / Sui Ano / SA 34 / 300
Sefwi Wiawso / Sui Ano / SA 4? / 1250
Sefwi Wiawso / Sefwi Wiawso / WR 4 / 330

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Appendix 2. The design of the gene bank at Amantia

Amantia

Muronoim

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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
  1. Wash buffer: Ethanol = 76 ml
  2. 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

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