ALLANBLACKIA SEED GERMINATION, SESSIONS 2.2 and 2.

3 LECTURE NOTES FOR THE ALLANBLACKIA DOMESTICATION WORKSHOP

23RD TO 27TH OCTOBER 2006 SUMMARY OF ACTIVITIES by Lucy Mwaura

Learning Objectives Participants will learn the current methods for Allanblackia seed germination in theory and also observe and carry out practical demonstrations at the nursery. Instructional methods Theoretical presentation with group discussion (45 min) and nursery demonstrations (45 min) Instructional materials Training materials will be provided as lecture notes and power-point presentation given. Summary Methods for seed germination: theory (session 2.2) A review of approaches across all countries used to stimulate germination will be presented in this lecture note. Difficulties and advantages associated with different techniques in terms of cost, effectiveness, material availability and technical requirements will be discussed amongst the participants. A topic on relevance and application of local knowledge i.e. utility of harvesting rat caches, burying the fruit and collecting seeds that have been buried by animals in the forest will also be considered. Participants representing all countries will discuss any species-specific issues. The current lessons/state of knowledge on best methodologies for large scale deployment and key gaps in knowledge currently limiting practical deployment in the Allanblackia initiative will be discussed during this session. We will be able to debate on best practice based on our current knowledge to practically improve germination performance of Allanblackia seeds. Written protocols for germination will be discussed to see whether they fit the current best practices from various countries. Short introduction of seed germination practical will be given to the participants. Methods for seed germination practice (session 2.3) Participants will learn in the nursery current best practices of Allanblackia germination for large scale deployment. Participants from each country will be required to relate best germination practice as carried out in their countries. This demonstration will take place at ICRAF HQs nursery. Demonstration will include the best techniques to store and treat seed before germination, and how to set up appropriate nursery seedbeds for germination and how to manage emergent seedlings in beds before transplanting. Participants will have the opportunity to compare the different germination methods as used by different countries as well learn new techniques and improve their current ones. Seed viability assessment before and after sowing (notes will be provided) It is important to find out if seeds put in nursery for germination remain viable or not. AB takes many months to germination, therefore it may be difficult to tell if fresh seed is viable or not. Biochemical TTZ test is a quick method that can be used to assess seed viability before storage if seed has to be stored and after seeds take several months before germinating. A few dormant seeds can be tested for viability

while still in the nursery beds, seeds being removed from time to time from the nursery bed and dipped in 1% tetrazolium salt and stored in the dark for 48hrs. Viable seed will stain brilliant red, while pale pink indicating dead tissue. Continuous TTZ test provides guidance to whether the ungerminated seed is still viable or not. Further reading - FAO FORESTRY PAPER 20/2 1985. A guide to Forest Seed Handling. By William R.C for DFSC. John. M. Riley, growing rare fruits from seed, California rare fruits growers, Inc, 1997 - International Rules for Seed Testing, Rules (1996). (Volume24, Supplement, Rules 1996) Seed Science and Technology.

Allanblackia seed germination Allanblackia germination is challenging, the report is received from all participating countries in this initiative. The germination rate is slow and germination capacity quite low. Small-scale nursery-based research trials in Cameroon (on A. floribunda and A. gabonensis) and Ghana (on A. parviflora) and lab based research trials in Kenya (on A. stuhlmannii) indicate that seed is very slow to germinate. In a trial in Cameroon, first germinants emerged only six months after seeding, with significant germination only observed after 12 months (Fig. D1). It took 18 (A. floribunda) and 19 (A. gabonensis) months for germination levels to reach 50 %. Although initial germination was very slow in Cameroonian trials, after a period of almost two years (22 months) overall germination had reached quite high levels, being > 80 % for A. floribunda and > 60 % for A. gabonensis. Like wise, it took 9 months for germination levels of A stuhlmannii to reach 4% from a trial done in Kenya in 2004. This lecture note theferefore summarizes the pre-sowing methods that have been used by countries participating in this initiative to improve seed germination that has been found to be slow and low.

Seed germination from specific countries

1 CAMEROON Objectives The main objectives of this study were to initiate Allanblackia trees improvement in Cameroon based on the first three steps elaborated by Zobel and Talbert (1984), which are: Identification of methods for propagation (sexually for rootstock production, and rooting of leafy stem cuttings as well as grafting methods) The determination of extent and pattern of variability in each species fruit and nut traits of nutritive and commercial importances The location of suitable package (ideotypes) for fruit and nut traits for plus trees selection (clones to be propagated after progeny tests).

RESULTS SO FAR OBTAINED Identification of methods for A. floribunda propagation: activity 1. Germination. Experiments on dormancy-breaking of Allanblackia seeds using warm stratification and gibberellic acids are in progress in ICRAF AHT nursery in Yaound, Cameroon. Preliminary results from an experiment investigating the effects of a mixture of forest soil and sand (2:1) on Allanblackia seeds germination indicated that the germination rate (T 50) is 18 months for A. floribunda and 19 months for A. gabonensis (Figure 1) seeds.

A. floribunda (n = 102) 90 80 70 Germination percentage 60 50 40 30 20 10 0 6 12 18 Tim e/Months

A. gabonensis (n = 450)

19

20

Fig. 1. Germination curve s of Allanblackia se eds in ICRAF-AHT nurse ry, Yaound, Cameroon

These results indicate that 70% germination success can be obtained in Allanblackia seeds. This experiment is still in progress, and preliminary results so far obtained indicated that Allanblackia seeds have a slow germination rate, as germination percentages of more than 50 % occurred after at least 18 months. Following these results, experiments investigating the hastening of Allanblackia seeds germination are planned. However, a preliminary experiment set up in September 2004 showed that a germination rate of 48.33 % can be obtained from A. floribunda seeds previously immerged in 90% gibberellic acid (GA3) for 72 hours (n = 60). After a period of 22 months, the overall germination has reached quite high levels 2 TANZANIA

2.1

TFCG at Kwezitu

Establishment of the Allanblackia stuhlmannii is taking place under three different approaches; the first approach is where the seeds are sown on the seed beds following recommended nursery techniques for Allanblackia stuhlmannii and the second one is where farmers are sowing the whole fruit in one hole covered with various forest materials and soil collected under the Allanblackia tree from the forest in trying to mimic the actual forest condition. The idea behind this is that, when found in the forest, germination of the Allanblackia seeds was better than in the seed beds using modern technology. Third approach is where farmers are collecting newly emergent seed of the Allanblackia stuhlmannii from the forest and raise them in the polythene tubes( harvesting rat caches.) Normally, the Allanblackia

stuhlmannii seeds take long time from 4 to 8 month to germinate thus they thought that it is better to collect the seedling and raise them while waiting for the seeds on the seed beds to germinate. Even after 8 months since sowing dates, germination rate has observed to be very low (sporadic). From the indigenous knowledge point of the view, it is believed that the higher seed oil content has caused the slow germination rates. No one seed could germinate until these amounts of oil is reduced to certain level (Personal communication). 2.2 Amani Nature Reserve (ANR)

The seeds were collected from the nature reserve from the selected 70 mother trees. The fruits were collected from the ground and transported to a central place where extraction was carried out. The seeds were extracted immediately they arrived, cleaned with water and the floating ones discarded

then buried in a hole with river sand as a way of storing. When a considerable amount was collected and extracted the 1st stage treatment, which was cracking the seed coat, was conducted. The cracking was done on the distal end of the nut and the reburied again in another hole awaiting the GA3 treatment. This second reburying was carried out to avoid oxidation and drying out of the seeds. Seed extraction and treatment The extraction of seeds takes place immediately followed by cleaning, discarding the floating lot of empty and unripe, removing off the fruit fresh by rubbing off the outer covering (to avoid fermentation and absorption of much of GA3), cracking/making a cut on the seed coat and then treating They are then cracked by nicking the dorsal end and then soaked in 1000ppm GA3 for 48 hrs before sowing in the nursery beds.( see annex II for GA3 preparation) Sowing at the nursery The method used here is bury them in a trench with each seed on its place for a period of time like three month and by the time the first germinant is spotted they will be transferred into the poly-pots.( please refer to annex II for sowing protocol and bed preparation)) Under the domestication programme on AB, Moses, Mr Sawe and others in Tanzania have this year collect very large quantities of AB seed (primarily from Amani Nature Reserve [ANR] forest, East Usambara). This seed is currently being cleaned and treated, and is then being distributed to nurseries. Presently, two principal treatments are being applied to AB seed before planting cracking (that is, perforation of the testa), followed by soaking in a solution of gibberellic acid (GA3) for 48 hours.

GHANA

In Ghana, ICRAF is collaborating with Forestry Research Institute of Ghana to undertake gene bank establishment, genetic diversity analysis and

vegetative propagation to capture quick genetic gains. During the first quarter of the year 2005, activities were concentrated on fruit collection in seven selected zones in Ghana. Fruits were collected from 150 trees and a total of 63,393 were sown. 3.1 The Allanblackia nursery at FORIG

Improvement of the Allanblackia nursery is in progress. A one- thousand capacity water storage tank has been installed at the nursery and connected to a pump that draws water from a nearby dam. In order to provide adequate and uniform shade, work on provision shade netting is in progress. Sixty treated teak posts have been purchased and erected. Shade net is yet to be procured.

Fig 2. View of Allanblakia nursery, with shade provided by palm fronds being replaced with shade net. Permanent metal labels provided for each accession.

(Fig 2)

Germination of seeds No germination was obtained from the 150 progenies of 60,000 seeds sowed at main nursery, however cutting through the seeds showed massive development of the embryo. Seeds sown in coarse river sand in the green house have started germinating. For seeds with testa removed before sowing, germination ranges from 0 4% depending upon the genotype. No germination has been recorded on the other two treatments (i.e. intact seeds sowed without removing the testa and soaking of intact seeds for 24 hours before sowing) (Fig.2)

Fig. 3. Seeds sown in bowls in green house

3.2

Seed germination at International Tree Seed

Centre( ITSC) At Offinso central nursery, 600,000 seeds of (A. parviflora ) were seeded into nursery beds in early 2005,however, seed emergency was so low that the conversion rate of seed to potted seedlings was < 0.0% this was germination during practical deployment. 4 KENYA Second germination trial with treatments Germination reports that were carried out in 2004 had been disappointing since levels of germination were quite low with the highest germination being 2% without treatment, we did another germination test and applied the following treatment:

Stage 1 Random sample of 100 extracted seed picked from the bulk collection.

Stage II-Floatation method used to sieve off immature and empty seed from the sinkers.

Stage III-Sinkers soaked in 800C water for 72 hrs.

Stage IV- Dried for one week under shade in the open.

A slight opening done to the seed tested before sowing

Seeds sown in sterile sand incubated at 20-30 0C with 12 hrs light/12 hrs dark, 78% RH.

No germination was reported here and most of the seeds were rotten after a duration of eight months. Another seed collection was carried out in December 2005, this was not the peak season and so very few seeds were brought to Nairobi for this test. Following methods were applied, The treatments applied,

250ppm GA3 48hrs 500ppm GA3 48hrs 1000ppm GA3 48hrs Cold water treatment 48hrs KNO3 1% 48hrs KNO3 0.2% 48hrs KNO3 0.5% 48hrs Combined treatment KNO3 0.2% plus 250ppm Control

Each treatment carried 21 seeds. Chart below shows the rooting results for the 9 treatments after 8 months of incubation and monitoring. Seeds treated with KNO 3 rooted earlier and was reported with highest rooted seeds as compared to the treatment with GA3 and cold water. No rooting was reported with the control. There were variances in rooting within the three concentrations that were used with KNO3 and GA3 concentrations (refer to chart). The number of seeds used in this germination test was quite small and therefore we need to consider setting up another germination test using

at least 200 seeds for each treatment. This experimental set up has however provided a guide to seed dormancy breaking methods.

A stuhlmannii germination report

30

25

20

%germinated seeds 15

10

0 250ppm GA3 soak 48hrs 500ppm GA3 soak 48hrs 1000ppm GA3 soak 48hrs KNO3 0.2% KNO3 0.5% KNO3 1.0 % Cold water 250ppm 48hrs 48hrs 48hrs soak 48hrs GA3 +KNO3 0.2% 48hrs Treatments Control

Biochemical viability test The quick method to test if the seeds are still viable while still in soil is to use the biochemical test. Seeds that had previously been sown in sand for three months were dug up cut across and put in a solution of 1% tetrazolium salt and later put in the dark for 48hrs. The brilliant red staining pattern observed under the microscope show seed viable tissue. This observation was evident even after the seeds stayed in sand for 3 months, about 50% of the seeds were found to be still viable at the close of the experiment.( please refer to annex 1 for TTZ preparation) .

Lessons learnt Seed have physical and embryonic dormancies which inhibit seed germination for seed to germinate, this also makes the germination process slow and low Choice of medium used for germination has influence of seed germination ( sand has been used successfully as pre-germination media in Tanzania and Kenya) There is still no pre-sowing method that has been proved to break seed dormancy although several methods have been tried in different countries. Use of seed nicking and soaking in GA3 has been use on large scale in Tanzania but still no improvement in germination has been reported We therefore all have a role to play in devising appropriate procedures for seed handling and treating.

References. Atangana Alain.R, Zac Tchoundjeu and Ebenezar Asaah (May 2005), Progressive Report on Allanblackia Domestication in Cameroon. Dawson Ian (August 2006) Novella Project elements: a detailed review of domestication activities on Allanblackia, developing propagation techniques. Meshack C,(2004) Indigenous knowledge of Allanblackia stuhlmannii in the East Usambara mountain, Tanzania

Mwaura L (May 2004), Lab report on Allanblackia stuhlmannii fruit and seed handling and storage, ICRAF Nairobi Kenya.

Munjuga M, (September 2005), Tanzania trip report. Ofori .D.A et al, (July 2005), Domestication of Allanblackia in Ghana, ICRAF/FORIG-Ghana.

TFCG team, (2005) Progress report of establishment of Allanblakia stuhlmannii in Kwezitu village of Tanzania.

ANNEX 1: Tetrazolium test Salt Preparation Solution 1 Solution 2 Dissolve 9.078gms in KH2 PO4 in 1000mls Dissolve9.472gms in Na2HPO4 in 1000mls or dissolve 11.876gms

Na2HPO4 x 2H2O in 1000mls water. Mix two parts of solution 1 with three parts of solution 2 and check the PH which must be between 6.5 7.5.

Dissolve the correct amount of tetrazolium salt (either chloride or bromide) in this buffer to obtain the right concentration, e.g.1g salt per 100mls buffer gives a 1% solution. Objectives The objectives of these biochemical tests are: 1. To make a quick estimate of the viability of seed samples in general and those showing dormancy in particular. This mainly includes species of hardwoods and confers which germinate slowly by regular germination methods. 2. In the case of particular samples which at the end of a germination test reveal a high percentage of dormant seeds, to determine the viability of individual dormant seeds or the viability of a working sample. Principle In the topographical tetrazolium test a colourless solution of 2,3,5-triphenyl tetrazolium chloride or bromide is used as an indicator to reveal the processes, which take place within living cells .The indicator is imbibed by the seed. Within the tissues it interacts with the reduction processes of living cells and accepts hydrogen from the dehydrogenases. By hydrogenation of the 2,3,5-triphenyl tetrazolium chloride a red, stable and non-diffusible substance, triphenyl forazan, is produced in living cells. This makes it possible to distinguish the red-coloured living parts of the seeds from the colourless dead ones In addition to completely stained viable seeds and completely unstained nondiffusable seeds partially stained seeds may occur. Varying proportions of necrotic tissue are found in different zones of these partially stained seeds.

The position and size of the necrotic areas, and not necessarily the intensity of the colour, determine whether such seeds are classified as viable or nonviable. However, colour differences along with tissue soundness are to be considered decisive mainly to the extent that they permit recognition and location of sound, weak or dead tissue. Procedure Working sample A full test is carried out on four replicates of 100 seeds drawn at random from the pure seed fraction of a purity test. Test may be carried out also on individual seeds that are found dormant at the end of a germination test. The seeds are soaked in water for about 20hours, and then the seed coat is punctured to facilitate entry of the 1% aqueous solution tetrazolium (TZ) then they are immersed in the dark for 48 hours Seed preparation and treatment 1 2 The seeds are soaked in water for about 20hours. Bisect the seeds longitudinally through the embryo with a razor blade to facilitate entry of the 1% aqueous solution of tetrazolium (TZ). 3 Immerse seeds in the dark for 48 hours.

Evaluation of the results 1 2 Immerse seeds in plenty of running water to remove excess stain. Soak the seeds in lacto phenol solution for 1-2 hrs (200mls phenol, 400mls lactic acid, 200mls glycerine, 200mls water to make one litre) Phenol preparation (dissolve 0.5-1.0gms of phenol detached crystals in 100mls.use ethyl alcohol if the crystals do not dissolve).

Use low power binocular microscopy to evaluate the staining pattern. Viable tissues stain bright red. Pink and very dark red stains are indicative of dead tissue.

Classification Completely stained and viable seeds Completely unstained seeds that are non-viable Partially stained seeds Advantages and disadvantages Advantages Its a quick method for testing viability of a seedlot. Its useful for hardwood and conifer seeds that take longer time to germinate. Disadvantages There is danger of overstraining, and this may portray wrong evaluation. This biochemical test requires skilled staff to evaluate the results. The test is limited to certain species due to difficulties in staining. There is lack of uniform interpretation of staining and difficulty in interpreting the significance of different degrees of staining Recommendation The method is not practical due to many disadvantages and so is not recommended for routine seed quality testing.

ANNEX II Methods of Allanblackia seed treatments and germination on nursery beds Collection handling and processing On arrival to the site or nursery, store fruits under shade either in gunny bags or open containers for post-harvesting maturation until i.e. when they can be mashed between hands easily. Extract by smashing the fruit between hands and separate the seed from the pulp. Clean the seeds in running water to remove the pulp and other chuffs to avoid pathogenic infection. After cleaning the seeds, hold the seed with your left hand and nick the dorsal side with a penknife Since this method is tedious process, the seeds are first preserved in sawdust or sand to attain the required quantity for treatment The seeds are then soaked in GA3 at 1000ppm for 48hrs (to make 1000ppm of 1litre GA3, mix 1 gram of powered GA3 in one litre of water; use either tincture of alcohol or hot water to dissolve GA3). Medium used Standard soil mixture: Mix, 2 parts forest soil, 1 part compost and 1 part sand. Use of sand only In Cameroon 2 parts forest soil and one part sand is used as standard media Use raised beds or benches to prevent root injuries, if containers are used.

Trench Preparation. Dig a trench about 3 feet dip and fill it in with sand( this would depend on the number of seeds you need to pre-germinate Sowing seed Sow the seeds in the prepared seedbeds or poly-tubes (filled with appropriate substrate to 2-3 cm deep). Plant 1 seed per container if direct seeding is opted In central nursery, a trench one meter wide and six inches deep was dug and layer of sand was spread and seeds then placed on the sand and then covered with another layer of sand and mulch later placed on top

Alternatively, farmers in Tanzania bury the whole fruit for three months and then extract seeds to be later sown in poly-tubes. Placing the seed on the bed Place the proximal side of the seed facing down or slightly tilted. Put a slight layer of substrate on top. Press the soil down firmly to ensure good contact between soil and seed. Use raised beds where possible to avoid root injury while root pruning Water carefully with a watering can or a perforated tin. Cover the seedbeds/benches with a transparent polythene sheet or place containers in a glasshouse to hold the moisture and maintain the temperature above 250C. As soon as the seeds germinate, remove the cover. In its native range, cover with mulch after watering. Water the beds twice daily; making sure the water penetrates the media. Weed regularly. Transplanting and pricking out Seeds germinate (root first) after 8 months, they complete germination in eighteen months. If in a seedbed, prick out the germinated seedlings and transplant them into containers. Germination is reviewed by scooping of the media (substrate) with a stick. Remove all the seeds that have roots emerging. Transplant them carefully, using a small stick. Do not damage the roots. If you plant in containers, fill containers with soil mixture, make a hole in the middle of the container and place the roots straight down in the substrate. Water the seedbed and containers after transplanting. Weed regularly. Expected germination capacity would be 20% after complete germination. Harden-off of the seedlings 2 months before transplant to the field i.e. reduced watering and remove them from the shade completely. Transplant to the field after attaining three nodes.