NASECO (1996) LTD

RESEARCH PROJECT

ON NURSERY MANAGEMENT,

CULTIVATION AND EXTRACTION

OF

ARTEMISIA ANNUA

Technical Paper No. 1

Nursery Methods and Management

NASECO 1996 ltd

P. O. Box 497, Hoima
Uganda

With assistance from:

ASPS-DANIDA
Enterprise Development Fund December 2006

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Table of Contents:

GENERAL

1. The introduction
2. Conditions for successful introduction of Artemisia annua in Uganda
3. General conclusion of the research project on Artemisia.
4. Vote of thanks
5. Authorisation
6. Nursery management report
7. Cultivation methods
8. Adaptive research
9. Technical feasibility of extraction methods

TECHNICAL PAPER NO. 1: NURSERY METHODS AND MANAGEMENT

6.1.0 Abstract
6.2.0 Introduction:
6.3.0 Study Materials and Methodology:
6.3.1 Materials
6.3.2 Methodology
6.3.2.1 Seeding
6.3.2.2 Cuttings process
6.3.3 Irrigation/water application procedures
6.3.3.1 Manipulation of the Greenhouse Environment:
6.3.4 Statistical analysis
6.4 Results:
6.5.0 Discussion of result:
6.6.0 Conclusion:
6.7.0 Annex

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 1. INTRODUCTION

Due to increasing resistance of malaria parasites to existing synthetic treatments, the World
Health Organisation (WHO) is encouraging malaria affected countries to adopt
Artemisinin-based Combination Therapies (ACTs) as the first line treatment against
malaria.

Artemisia annua is a plant originating from Asia, where it holds an important place in
traditional healing systems. The discovery of its usefulness in the treatment of malaria is
not recent, but it is only in recent years that WHO and African governments have started to
promote ACTs as the main first line treatment against malaria.

In these recent years we have witnessed a surge in the interest in cultivation of Artemisia
annua in East Africa, where the climat and soil conditions exist to make cultivation a viable
undertaking. However, artemisin, the active substance extracted
from dried leaves of Artemisia annua, is unstable in the plant at different stages of maturity
and processing. Several attempts to grow and dry Artemisia and then send it for extraction
to Europe and Asia have failed because of this. Hence the need for local adaptive research
for cultivation and extraction techniques.

2. CONDITIONS FOR SUCCESSFUL INTRODUCTION

OF ARTEMISIA ANNUA IN UGANDA

In order to be viable, a programme for cultivation of Artemisia annua in Uganda needs to

succesfully adress following issues:

- Appropriate cultivation techniques, targeting optimal yields of artemisin,

should be developed both for medium and large scale commercial farms as
for smallholders around nucleus farms.

- Appropriate post-harvest techniques (drying, cutting and storage) should be

developed that allow for a continuous supply of high quality planting
materials for extraction.

- Appropriate extraction techniques should be developed to turn the plant

material in a stable, marketable product that can be used as a base material
for medicine production.

- A clear and straightforward link to the market for artemisia-based products

should be established.

- Last but not least, farmgate prices should be at a level where the cultivation
of Artemisia annua is a viable and profitable undertaking for farmers.

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 3. GENERAL CONCLUSION OF THE RESEARCH
PROJECT ON ARTEMISIA.

The project has enabled management of Naseco to have a clear picture on the pro and
contra Artemisia growing in Nalweyo and surroundings. The economics as a grower are
positive, provided the crop grows to expectation and long term contracts are covering the
risks and assure a steady income. The alternative means processing by extraction to a semi
finished product (eg high concentrated miscelia and/or artemisine Cristals), this again
requires heavier investments, assurance of year round leave and in addition to long term
supply contracts.

A very positive outcome is the fact that extraction using alternative solvents led to higher
recovery rates. This knowledge should be a guide to existing extractors to further
experiment on the solvents, solvent recovery rates, crude washing and purifying of the
crude extracts.

During the experimental phase experience was obtained into the nursery management and
growing of Artemisia.

Successfull production in Nalweyo has been limited by the stress factor of the plants,
assumed to be linked to high day night temperature and water stress (none or too much)
during the growing cycle. Naseco believes that profitable production can only be done in
area’s higher then 1500 meter with a good rainfall distribution during the growing season.
Production in lower area’s (1100-1200m) require a fully established irrigation on
production fields, which, looking into the direct investments and operational costs will not
be possible if production contracts are not assured on longer term.

The production of seedlings/cuttings is a possible activity we looked in and Naseco is still

willing to produce seedlings or cuttings against a well defined contract. All investments
have been done in order to do full production of seedlings/cuttings. The management
capacity is in place including committed supervisors and trained casual workers. As a
negative point, the farm location is out of the production area, transport logistic and costs
become a negative factor. Here again, fixed contracts are required and for cuttings a mother
garden needs to be established well in advance. Looking into the costs of the cuttings,
Naseco will only be able to sell at 15 Ush break even, not counting some overhead costs
and margin. The final price of 20 Ush/cutting, makes this far above the current 10-15 Ush
offered by farmers/firms.

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 4. VOTE OF THANKS

Naseco was not going to be able to carry out this research on it’s own and is very grateful
for the financial support received from the ASPS – Danida program.

We also thank the input of our collaborators for the adaptive research, including:
- Ssezibwa Estates – Mukono
- Mubuku Irrigation Scheme - Kasese
- Botanical gardens – Fort Portal

Mr. Latigo Patrick is highly acknowledged for his consultancy on the extraction methods.
Despite problems with some lab equipments, power cuts, difficulties to get the right
solvents amongst others, a clear report was established.

Mr. Walter De Boeck’s efforts into project planning, initial contacts and revising of the
study have been highly appreciated.

We appreciate the efforts of East African Botanicals and the Botanical Gardens for the
provision of planting materials, specific varieties used in the adaptive research, in addition
to a lot of practical experience.

The efforts of Chemiphar (U) Ltd to invest into a Thin Layer Chromatography (TLC) are a
real commitment towards investment into the agri business and direct support to small
starting enterprises. By this technology in Uganda, the artemisine quantification cost has
reduced at least by 5 times, enabling individual farmers to determine their harvesting dates
and analyse leave samples before commercialisation. We thanks Chemiphar for the good
services and fast delivery of analysis results.

Discussions with many stakeholders have led to a clear picture on the sectorial
developments, opportunities and constraints.

Mr. Simon Okiror, being appointed on a short term contract, as biometrician enabled the
smooth implementation of the trials: from design, establishment, follow up, data collection,
data analysis until draft report.

5. AUTHORISATION
Naseco authorises anybody to use the outcome of this study for the purpose of improving
production processes, improved agronomics and the production for the benefit of small
holders.

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 NURSERY METHODS AND MANAGEMENT

FOR ARTEMISIA ANNUA

IN UGANDA

Prepared By:
Simon Okiror (Research Assistant)

December 2006

6. NURSERY METHODS AND MANAGEMENT

6.1.0 Abstract:

Artemisia annua plantlets growth depends on the conditions of growing. The studies on
nursery methods at the Experiment Station in Nalweyo showed that the average weekly
growth rate of plantlets under the greenhouse nursery method is 5.08% higher compared to
open area nursery method. The 24 hour air and soil temperatures in the greenhouse undergo
greater fluctuations than in the open, creating microclimate with a definite effect on
Artemisia annua growth. Under this condition, the seedlings in the greenhouse take 5
weeks to attain the recommended transplanting average height of 7cm; 2 weeks earlier than
those in open area nursery method.
Two nursery methods (greenhouse and open area) where compared with a goal of
establishing plantlet prices, both when seeding and use of cutting is done in the nurseries
when raising plantlets respectively. The results showed that seeding in both the greenhouse
and open area produce cheaper plantlets.

6.2.0 Introduction:
Artemisia annua is a plant originating from Asia, where it holds and important place in
traditional healing systems (Ferreira and Janick 1996). The discovery of its usefulness in
the treatment of malaria is not recent, but its only recent years where WHO and African
governments have started to promote ACTs as the main first line treatment against malaria.
Artemisia annua is an annual shrub from which it is extracted artemisinin – an important
raw material - for anti malarial pharmaceuticals. Due to increasing resistance of malaria
parasite to existing synthetic treatment, the World Heath Organisation (WHO) is

4
encouraging the malaria affected countries to adopt artemisinin-based combination
therapies (ACTs) as the first line treatment against malaria (Pan American Health
Organisation-PAHO-, 2006). In these recent years, a surge has been witnessed in the
interest in cultivation of Artemisia in East Africa, where the climate and soil conditions
exist to make the cultivation a viable undertaking.
Artemisia annua seed needs to be germinated and established in a nursery prior to
transplanting in the fields. This as can be seen is a delicate and labour intensive process.
However, little literature is available about cultivation methods of the crop. Initial trials
undertaken by Nalweyo Seed Company - Naseco 1996 Ltd - indicated that growing
plantlets from seeds in polyethylene containers is too costly for large scale production. It
was therefore suggested that a better method be growing plantlets from seed mixed with
sand on a prepared soil bed in a greenhouse equipped with sprinkler irrigation system.
Based on the above facts, a study on nursery methods was carried out with the following
objectives
a) To compare greenhouse and open area nursery methods in raising the Artemisia
annua plantlets under the seeding and cutting processes.
b) To asses the costs of each plantlet grown under each method and process.

6.3.0 Study Materials and Methodology:

6.3.1 Materials
The material used in this study included; note book, pen and pencil, ruler, 100 cm2 frames,
four varieties of Artemisia annua seeds.

6.3.2 Methodology
Proceedings of raising the plantlets under the greenhouse nursery method were done in a
fully equipped greenhouse as opposed to those in the open area (shade net and palm leaves
nurseries); with the requirements of the agronomic measures, which assured their normal
development. Prior to seedbed preparations, the nutrient status of the soil was considered
for analysis (refer to annex i).

6.3.2.1 Seeding
With the use of the hand hoe to break the soil and a rake to remove stones, plant residue
and to level the soil, seedbeds measuring 10m by 10m and raised at 25cm above the ground
level were established: one in the greenhouse and the other in the open area. The
vermiculite was incorporated in both beds to help in retaining the moisture content in the
soil. The seedbeds were divided into four sections to accommodate the four varieties of
Artemisia Annua; Brazil, East African Botanical (EAB), Mediplant and Anamed.
Beds were brought to field capacity before each variety of seed was sprinkled, per section,
at a rate of about 1.46gm/m2. A polythene paper with perforations was spread over the
seedbed in the greenhouse and palm leaves for the other in open area during the first 3 days.
The covering was removed off the seedbed in the greenhouse whereas in the open area,
palm leaves were raised to form shading at a height of 1.2m after 3 days.

5
 6.3.2.2 Cuttings process
Cuttings in good health condition were identified from the mother garden and were cut at
an average length of 7cm. The prepared cuttings were immersed in the rooting hormone
solution (Hormoril) and were inserted firmly into the seedling tray holes fitted with loam
soil.

6.3.3 Irrigation/water application procedures

Water was regularly applied to the seedbeds and trays twice every single day to ensure
adequate soil moisture content. In the greenhouse method, the water was applied through
the sprinkler irrigation system to both the seed bed and cuttings while in the open area
seedbed; a spraying pump was used during the first 2½ weeks to ensure no run off, of
seeds. To the cuttings in the open area, water was provided to the rooting stems by a
watering can.

6.3.3.1 Manipulation of the Greenhouse Environment:

The greenhouse was maintained open during the day and closed during the night. However,
its manipulation depended more on the climatic conditions. Caution was always taken
during raining moments, where the greenhouse was closed.

6.3.4 Statistical analysis

Seedlings height (PH) where taken after every seven days, both in the greenhouse and in
the open area nursery seedbeds. Seedlings were randomly considered for measurement
during data collection. The number of days to determine time taken for the cuttings to
develop roots was also noted.
Records of cost expenditure under each nursery method were taken. The production costs
whose records where consider include; fertilizers, seeds, fuel, costs of land, labor,
machinery, management and irrigation.

6.4. Results:
The outcome of the seedling growth rates was characterised by the results provided on table
1. There is a clear marked difference between the growth of seedlings in the greenhouse
and those raised in open area nursery bed method.

Means
PH (cm) PH (cm) PH (cm) PH (cm) PH (cm) PH (cm) PH (cm)
Nursery Method 14 days 21 days 28 days 35 days 42 days 49 days 56 days
Irrigated 1.32 2.14 3.22 3.88 6.04 7.84 10.28
Greenhouse
Manual 1.29 2.11 3.16 3.84 6.21 7.80 10.40
Shadenet 1.25 1.84 2.56 3.00 4.31 6.03 8.01
Open area
Palm leaves 1.27 1.89 2.61 3.01 4.34 6.07 7.91

Table 1. Mean values for plant heights (PH) Assessed at different periods (7,
14,21,28,35,42,49,56 Days of the Nursery Growing)
The growth dynamics for seedlings as can be observed on table 1 and in fig. 1, show faster
vegetative growth in the greenhouse compared to the open area nursery method. The East
African Botanical Growers’ Production Manual April 2005 recommends that, plantlets are
ready for transplanting at a height of 7 – 10 cm. On simultaneous planting, the lineal
6
 equations shown in figure 2 below indicates that greenhouse plantlets attain an average
transplanting height of 7 cm in 5 weeks (37.8 days) compared to open area plantlets taking
7 Weeks (48.7 days).

Open a r ea gr een h ou se
12.00
12.00
Gr een H ou se open a r ea

10.00 10.00 Lin ea r (gr een h ou se)

y = 0.1996x - 0.5248
Lin ea r (open a r ea )
8.00
8.00

plantlets height (cm)

Plantlet height
6.00
(cm) y = 0.1488x - 0.2402
6.00

4.00

4.00
2.00

0.00 2.00
0 7 14 21 27 35 42 49
Days (from germination)
0.00
0 10 20 30 40 50 60
Plantlets growth (days )

Figure 2. Linear equations for Artemisia

Figure 1. Graphical view of values for
annua progressive growth under the two
mean plant heights assessed at different nursery methods.
periods (days)

Based on this result, it can be deduced that seedling in the greenhouse take less time to
attain the required height for transplanting as compared to those in open area seedbed.
However, plants from rooted cuttings even proved to take shorter time (21 days) compared
to seedlings.
Despite the fact that seedling growth is fast in the greenhouse than in open area (refer to
figure 1), the vigour is even more pronounced in the greenhouse on application of
equivalent doses of fertilizer (0.6125g/l, per squared meter of NPK).
Application of fertiliser on 21 days in both nursery methods resulted to a remarkable rise on
seedling growth noted in space of 7 days. However, seedlings were seen burned after the
application of the fertilizer and afterwards, were also seen weak during transplant
establishment.
During this study, the advantage established in seeding process was the ease to control
spread of diseases and less labour involved during establishment of nurseries. However, the
disadvantage is that, seeds are accessed at a high cost and are hard to spread uniformly on
the bed. On the other hand, transportation of the seedlings is quite difficult during
transplant as they are easily stressed during the process.
Cuttings however, provide the advantage of giving the pure variety of the crop required,
easy to establish and to transport. The disadvantage is the ease to pass disease and also is
labour intensive.

The structure of the prime costs of raising seedlings under greenhouse methods is not the
same under natural economic conditions to that in open area nursery method (refer to annex
ii).
According to the calculations made with respected to the total number of plantlets raised
under the nursery methods, a plantlet in average costs 21 /= when cuttings are used as
opposed to 8.5 /= when seeding process is done.

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In seeding condition however, irrigated and manually watered seedlings in the greenhouse
and; shade net together with palm leaves nurseries in the open area show no difference in
cost. Nevertheless, the cost per seedling under the greenhouse is slightly low compared to
open area. This cost variation is accredited to the high population of seedlings obtained in
greenhouse than in open area (germination percentages). Cuttings when also irrigated in
both the greenhouse and shade net show a slight variation in pricing per plant (refer to
annex ii).

6.5.0 Discussion of result:

Artemisia growth depends to the highest degree on the conditions of growing. Though
climatic data was not taken during this study in both nursery methods, the 24 hour air and
soil temperatures in the greenhouse undergo greater fluctuations than in the open because
they are directly dependent on sunlight intensity, restricted air circulation and polyethylene
high infra-red rays’ permeability.

These peculiarities in the greenhouse microclimate have a definite effect on Artemisia

annua growth. The study conducted with four varieties of Brazil, East African Botanical,
Mediplant and Anamed showed that at simultaneous planting (27th/Feb/2006), the
greenhouse plantlets develop at an accelerated rate and plantlets mature two weeks earlier
than those in the open area nursery method.
There is considerable variation in plantlets costs under the seeding and the cutting process
in the two nursery methods adopted in this study. The cost of a rooted cutting is high
compared to that of a seedling. This variation as seen is based on the unique character of
each operation under the nursery method used. The sources of difference are found in some
key categories such as seedling trays, cutting costs in the mother garden and supervision.

6.6.0 Conclusion:

Artemisia annua growth rate is higher in the green house than in open. This is attributed to
the microclimate in the greenhouse which is seen to provide a definitive effect. The period
taken by the plantlet to reach transplanting height is shorter as compared if grown on open
area seedbed. It is assumed that the effect is higher at higher altitudes, eg Fort Portal.

The choice of cutting or direct seeding will mainly depend on the availability and cost of
seeds. The cost of seeding is 3 times lower then cutting.

The level of investment has quasi no influence on the price, provided the investments are
spread over a number of years, with guaranteed market. Additional investments, eg
greenhouse provide increased production / area. Investment in irrigation improves water
management and assures equal water distribution thus leading to a higher success rate.

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 Annex I ; Laboratory soil analysis results detail

Texture
Lab
Details PH O.M N Av.P K Na Ca Mg (percentage) Zn Cu
No:
Percentage mg/kg C.moles/kg Sand Clay Silt mg/kg
Sample B (Green
1038
House) 8.1 5.29 0.09 14.12 2.03 0.08 29.2 12.3 56.0 8.0 36.0 7.64 3.06
1039 Sample A (Garden) 7.3 8.4 0.24 11.2 2.93 0.08 23.1 3.49 58.0 14.0 28.0 16.5 1.87

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 Annex ii : General analysis of plantlet costs under the nursery methods used in the
study.

SEEDING CUTTINGS
COSTING FOR Greenhouse Open Area Irrigated No irrig
100M2 Out
Irrigated Manual Shadenet Palmleave Greenhouse shadenet Out palm
Production cycles 1.5 1.5 1 1 3 2.5 2.5
Duration/cycle (days) 37 37 48 48 17 22 22
Total occupation
(days) 55.5 55.5 48 48 51 55 55

Investments
Greenhouse 291,666 291,666 0 0 291,666 0 0
Irrigation system 62,500 0 62,500 0 62,500 62,500 0
Water pump 62,500 0 62,500 0 62,500 62,500 0
Shadenet 0 0 62,500 0 0 62,500 0
Trays 0 0 0 0 250,000 250,000 250,000

TOTALS Ush/season 416,666 291,666 187,500 0 666,666 437,500 250,000

Running costs per

cycle
Seeds (F2) 70,000 70,000 35,000 35,000 0 0 0
Mother garden 0 0 0 0 65,000 65,000 65,000

Casual labour
- seedbed prep 15,000 15,000 15,000 15,000 7,500 7,500 7,500
- seeding 10,000 10,000 10,000 10,000 0 0 0
- tray preparation 0 0 0 0 90,000 90,000 90,000
- shade palmleaves 0 0 0 50,000 0 0 50,000
- cutting 0 0 0 0 210,000 210,000 210,000
- watering 0 65,000 65,000 0 0 65,000
- weeding 20,000 20,000 20,000 20,000 0 0 0
- uprooting and
packing 100,000 100,000 100,000 100,000 30,000 30,000 30,000

Fertiliser (NPK 17-17-

17) 20,000 20,000 20,000 20,000 0 0 0
Foliar Feed 35,000 35,000 35,000 35,000 35,000 35,000 35,000
Supervision (8000
Ush/day) 444,000 444,000 384,000 384,000 408,000 440,000 440,000
Overhead cost 150,000 150,000 150,000 150,000 150,000 150,000 150,000
Fuel Water pump 30,000 0 30,000 0 30,000 30,000 0

Total Running Costs 894,000 929,000 799,000 884,000 1,025,500 1,057,500 1,142,500

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Plants/100m2/cycle 225,000 225,000 225,000 225,000 100,000 100,000 100,000
Germination/rooting
% 95% 90% 90% 80% 85% 80% 80%
Seasonal production 320,625 303,750 202,500 180,000 255,000 200,000 200,000

Cost/cycle 894,000 929,000 799,000 884,000 1,025,500 1,057,500 1,142,500

Cost Total 1,341,000 1,393,500 799,000 884,000 3,076,500 2,643,750 2,856,250
Seasonal depreciation 416,666 291,666 187,500 0 666,666 437,500 250,000

TOTALS 1,757,666 1,685,166 986,500 884,000 3,743,166 3,081,250 3,106,250

Price/plantlet 5.5 5.5 4.9 4.9 14.7 15.4 15.5

Margin 25% 25% 25% 25% 25% 25% 25%

Sales price 6.9 6.9 6.1 6.1 18.3 19.3 19.4

Calculation notes
Investment
- The greenhouse, shade net and the irrigation system are depreciated in 6 season’s period (3
years). The cost to establish: greenhouse (7m × 30m, but calculated over 100m2) is and the
shade net (10 × 10m) is 3,000,000Ush and 750,000Ush respectively.
- The irrigation system (200m) is depreciated over a period of 12 season- 6 years and costs
750,000Ush
Running cost
- All casual labour cost calculations are best on an average wage of 3000 Ush/person/day
Cutting costs
- Establishment of the mother garden is 750,000 Ush/ha with plant population of 10,000plant.
It’s considered that each plant makes up to 150 cuttings. Thus the price per cutting is 0.5
Ushs.
- 500 trays are calculated to fit 100m2 area with 25% space for walking during supervision.
Each tray has 200 holes to contain plants.

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