African Journal of Biotechnology Vol. 10(32), pp. 5959-5966, 4 July, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Seed germination and in vitro regeneration of the
African medicinal and pesticidal plant, Bobgunnia
madagascariensis
Blackson L. K. Thokozani1, Donald Zulu2, Gudeta W. Sileshi3*, Zewge Teklehaimanot4,
Dominic S. B. Gondwe2, Viswambharan Sarasan5 and Philip Stevenson5
1
Mzuzu University, P/Bag 201, Luwinga, Mzuzu 2, Malawi.
Kasisi Agricultural Training Centre, Kasisi, P. O. Box 30652, Malawi.
3
World Agroforestry Centre (ICRAF), Southern Africa Programme, Chitedze Agricultural Research Station, P.O. Box
30798, Lilongwe, Malawi.
4
School of Environment and Natural Resources, Bangor University, Bangor, LL57 2UW, United Kingdom.
5
Royal Botanic Gardens, Kew, Surrey, TW9 3DS, United Kingdom.
2
Accepted 6 April, 2011
Propagation of the medicinal and pesticidal tree, Bobgunnia madagascarensis is difficult due to poor
and erratic germination of its seeds and slow growth of its seedlings. This study involved two separate
experiments. The first evaluated the effect of pre-sowing treatments and growing medium on ex vitro
seed germination and early seedling development. The second experiment involved in vitro
germination, shoot initiation and rooting of shoots. Pre-sowing seed treatments involved soaking seeds
in cold and hot water for 12 and 24 h and soaking in different concentrations (0, 100, 200, 400 and 800
mg/l) of gibberellic acid for 24 h. Soaking of seeds in cold or hot water for up to 24 h did not achieve
more than 45% germination, while seeds treated with gibberellic acid achieved <20% germination rates.
On the other hand, in vitro procedures achieved 30 to 70% germination of seeds. Seedling survival of
ex-vitro germinated seeds was higher (>76%) when seeds were sown in a growing medium without
compost compared with a medium with compost (<43%). All shoot-tips isolated from the in vitro
germinated seedlings on B5 media without plant growth regulators continued to grow as a single shoot,
while shoot-tips cultured on B5 supplemented with 0.1 mg/l of naphthaleneacetic acid (NAA) and
thidiazuron (TDZ) produced two shoots each after four weeks. It was concluded that B.
madagascariensis seeds had very low ex vitro germination percentages. Although, in vitro cultures
improved seed germination, axillary shoot multiplication and rooting were not satisfactory. Therefore,
further studies are needed to develop an optimal in vitro multiplication protocol for B.
madagascariensis.
Key words: Axillary shoot multiplication, gibberellic acid, in vitro regeneration, seed germination, Swartzia.
INTRODUCTION
Bobgunnia
madagascariensis,
formerly
Swartzia
*Corresponding author. E-mail: sgwelde@yahoo.com
sileshi@africa-online.net.
Tel:
+265-999-642-149
+265999642149. Fax: +265-1-707-319.
or
or
madagascariensis (Kirkbride and Wiersema, 1997), is a
leguminous tree widely distributed throughout the
savannahs and dry forests across Africa (Kirkbride and
Wiersema, 1997; Hostettmann et al., 2000; Schaller et
al., 2001; Smith and Allen, 2004). Different parts of the
tree are used in traditional medicine to treat diseases
such as malaria, fever, syphilis and leprosy
5960
Afr. J. Biotechnol.
(Cunningham, 1993; Schaller et al., 2001; Kirkbride and
Wiersema, 1997; Ouattaraa et al., 2006). In the
laboratory, extracts of the root bark had very high activity
against a chloroquine-resistant strain of the malaria
parasite Plasmodium falciparum (Ouattaraa et al., 2006).
The fruit pulp was shown to exhibit high trypanocidal
activity in Nigeria (Atawodi, 2005). Antifungal products
have also been isolated from the root bark (Hostettmann
and Marston, 2002; Hostettmann et al., 2000; Schaller et
al., 2001). The toxic properties of B. madagascariensis
have also been exploited in the control of insect pests of
medical, veterinary and agricultural importance (Schaller
et al., 2001; Sileshi et al., 2008). The powdered pods are
a potent insecticide against mosquito larva (Kirkbride and
Wiersema, 1997). The molluscicidal activity of saponins
from the aqueous extract of fruits (pods) has been
demonstrated (Borel and Hostettmann, 1987; Lwambo
and Moyo, 1991; Suter et al., 1986). In addition to its
medicinal and insecticidal properties, this tree has a very
strong, durable and termite resistant wood used in
building, making of local musical instruments, veneer,
curios and makes good firewood and charcoal (Kirkbride
and Wiersema, 1997; Vermeulen et al., 1996).
The commercial harvesting and sale of roots of this
species is widespread in Africa (Cunningham, 1993). This
may involve uprooting of the whole plant or removal of
the root, which may seriously damage the root system.
Unsustainable harvesting of the pods could also reduce
regeneration and thus, threaten local populations. This
calls for cultivation on the farm to increase availability
locally. Unfortunately, B. madagascariensis is a difficult
tree to propagate because it has low seed viability and
poor germination. Plants propagated from seed also
exhibit high degree of genetic variability (Berger and
Schaffner, 1995).
In vitro culture offers advantages over conventional
methods in the propagation of some crops and species of
conservation
importance.
Micropropagation
can
significantly improve regeneration rates in rare and
threatened species with very small quantities of seeds
and/or very poor seed germination (Sarasan et al., 2006).
However, development of good quality cultures for large
scale propagation for both commercial propagation and
conservation can be limited by the recalcitrant nature of
the species in many cases. Some leguminous tree
species are recalcitrant in culture (Jordan et al., 2001).
Berger and Schaffner (1995) made the first attempt to
develop
micropropagation
protocols
for
B.
madagascariensis and found that, the species show very
high genotypic variability in rooting ability and low shoot
induction on Gelerite-based media. Since then, no effort
has been made to propagate B. madagascariensis either
through in vitro or ex vitro methods. Therefore, the
specific objectives of this study were (1) to identify presowing seed treatments which can improve ex vitro
germination rates; (2) assess the effect of growing media
on seedling survival and growth; and (3) to undertake a
feasibility study using seeds to develop protocols for in
vitro propagation of B. madagascariensis.
MATERIALS AND METHODS
The study involved two separate experiments. The first experiment
involved assessment of seed pre-treatment and growing medium
on germination, seedling survival and growth at Chitedze
Agricultural Research Station in Malawi. The second experiment
involved in vitro culturing in the Conservation Biotechnology Unit
(CBU) at the Royal Botanic Gardens Kew, United Kingdom. The
seed used in both studies were from the same seed lots collected
from the natural stands of scattered trees of various age in eastern
Zambia.
Ex vitro germination and seedling survival
This experiment was conducted in January-April 2009 in a
greenhouse with average temperatures varying between 13 and
40°C. The pre-sowing seed treatments involved soaking seeds in
cold and hot water for 12 and 24 h and soaking in different
concentrations (0, 100, 200, 400 and 800 mg/l) of gibberellic acid
for 24 h. For each concentration, 200 seeds were immersed in 500
ml flasks containing 200 ml solution of a given gibberellic acid
concentration and shaken on a reciprocating shaker. After 24 h of
soaking, the solutions were drained off and seeds rinsed with
distilled water for one minute. Seeds soaked in 0 mg/l gibberellic
acid (soaking in distilled water) served as the control. The
hypothesis tested was that, there is a direct relationship between
gibberellic acid concentration and germination of B. madagascariensis
seeds.
Each treated seed was sown in black polythene tube filled with
either of the two growing medium: (1) compost manure + sand +
forest soil in a 1:1:1 mixture by volume, which is the recommended
potting mixture for growing tree seedlings in medium to light soils
(Jaenicke, 1999) and (2) sand + forest soil in a 1:1 mixture. The
compost manure was procured from the Four Seasons Nursery, a
commercial producer of ornamentals. The manure was produced
from a well composted mixture of maize stover, kitchen waste,
chicken and cattle manure. The forest soil was collected from a
mature miombo woodland (a deciduous forest type in southern
Africa where B. madagascarensis grows naturally) and was sieved
to remove unwanted materials.
The seed pre-treatment and growing media were combined in a
factorial experiment in a completely randomized design with four
replications (25 seeds per replicate). Watering was done when the
surface of the soil seemed to have started drying out. Data were
collected on germination, survival and height growth of the
seedlings. Germination was defined as emergence of the
cotyledons above the medium surface. The first germinating seeds
acted as the baseline for recording data. A value of "1" was
recorded if the seed had germinated and the value of "0" for
otherwise. Germinating seeds were counted and recorded daily
until no further germination was recorded for three consecutive
days. The germination test lasted for seven weeks. The total
numbers of the germinated seeds for each treatment were summed
up to determine the cumulative germination. 11 weeks after sowing,
the total number of seedlings that survived and their mean heights
were recorded for each treatment.
Thokozani et al.
5961
Table 1. Significance (P value) effect of pre-sowing seed treatment and growing medium on
germination of seeds, survival and height growth of B. madagascariensis seedlings.
Treatment
Germination
Survival
Height growth
Hot water
<0.001
0.876
0.524
Growing medium
<0.001
<0.001
0.003
Interaction
0.105
0.606
0.330
Cold water
<0.001
0.042
0.381
Growing medium
Interaction
0.551
0.009
0.167
0.002
0.005
0.081
Gibberellic acid concentration
0.144
0.218
0.259
Growing medium
0.824
0.019
0.135
Interaction
0.186
0.327
0.220
In vitro culture
The various preliminary in vitro culture tests were carried out
between March and August, 2008 in Conservation Biotechnology
Unit (CBU). Seeds were either scored along the middle with a
scalpel blade or left intact before being soaked in deionised water
with a drop of Tween 20 and placed on a shaker at 110 rpm for 24
h. Imbibed seeds were washed in deionised water to remove the
seed coat and were sterilized by soaking in 0.5% (w/v) of sodium
dichloroisocyanurate (SDICN) for an hour and 30 min. After
sterilisation, seeds were washed in deionised water again and
surfaces were dried on sterile filter paper. The sterilised seeds were
cultured on Gamborg’s B5 (Gamborg et al., 1968) and Murashige
and Skoog (MS) medium (Murashige and Skoog, 1962). All culture
media for seed germination and multiplication contained 3%
sucrose, while rooting media contained 2% sucrose unless
otherwise stated. Activated charcoal was added to the media at a
concentration of 1.5 g/l. All solid media were solidified with 0.8%
tissue culture agar (A-7002, Sigma Chemicals, UK) and pH was
adjusted to 5.8 before they were autoclaved at 121°C for 15 min.
For seed germination and multiplication, about 100 ml of media
were dispensed into honey jars (355 ml) with plastic screw-cap lids.
All aseptic culture procedures were done in a sterile laminar airflow
cabinet. The cultures were grown in a tissue culture growth room at
23±2°C and photosynthetic photon flux density (PFD: 50 µmol m–2
s–1) under 18/6 h (day/night) photoperiod.
For shoot multiplication, nine shoot-tips were isolated, while the
seedlings were still inside the jar by making a cut just below the
second node of the germinated seedlings. The rest of the seedling
(with the first node) was left in the medium for axillary shoot
proliferation. The shoot-tips were cultured on MS medium without
plant growth regulators (PGRs) (control) and MS medium
containing 1 mg/l of naphthaleneacetic acid (NAA) and 1 mg/l of
thidiazuron (TDZ). The in vitro germinated seedlings were
monitored for their ability to produce axillary shoots after providing
explants for sub-culturing. Six fresh explants harvested from the in
vitro germinated seedlings were cultured on B5 liquid media
supplemented with 0.1 mg/l of NAA and 0.1 mg/l of TDZ for axillary
multiplication.
To induce rooting, shoot-tips were cultured on Florialite® cubes
(Nisshinbo Industries Inc. Japan) in B5 liquid media supplemented
with 1 mg/l of NAA and 1 mg/l of indole butyric acid (IBA). These
were subjected to an improved air circulation with vented Magenta®
B-cap (Sigma Chemicals, UK). The liquid medium used for this trial
was devoid of sucrose.
Data analysis
Angular transformations of the seed germination and seedling
survival data were subjected to analysis of variance ( = 0.05).
Since seed germination was recorded as binary process (1 =
germinated and 0 = not germinated) at a particular time, it was
possible to model the probability distribution of germination in each
treatment over the entire period of observation. The probability of
germination was estimated using categorical models and the
cumulative probabilities were plotted against time.
RESULTS
Ex-vitro seed germination, seedling survival and
growth
Seed germination percentages significantly (P < 0.01)
varied with the length of soaking in hot water and growing
medium, but not with their interaction effect (Table 1).
Germination percentages also significantly (P < 0.01)
varied with the length of soaking in cold water and not
with the growing media and the interaction effect of the
length of soaking in cold water and growing medium
(Table 1). On the other hand, the concentration of
gibberellic acid, growing medium and their interactions
did not significantly (P > 0.05) influence germination
percentage (Table 1). Over the 50 day period, the highest
(45%) cumulative germination was recorded in the seeds
soaked in hot water for 24 h (Figure 1a). Higher and more
uniform germination was recorded in the seeds soaked in
hot water for 24 h and sown in the growing medium
without compost manure (Figure 1a). On the other hand,
lower and erratic germination was recorded in the seeds
soaked in cold water for 24 h and sown in either growing
5962
Afr. J. Biotechnol.
A
B
Figure 1. Cumulative distribution of germination of B. madagascariensis seeds as affected by soaking in
water and growing medium.
A
B
Figure 2. Cumulative distribution of germination of B. madagascariensis seeds as affected by soaking
in various concentrations of gibberellic acid and growing medium.
medium (Figure 1a, b). Seeds soaked in 400 mg/l of
gibberellic acid for 24 h had higher cumulative
germination compared with those soaked in 400 mg/l of
gibberellic acid when sown in growing medium without
compost manure (Figure 2a). When sown in a growing
medium with compost manure, cumulative germination
Thokozani et al.
5963
Table 2. The effect of pre-sowing seed treatment and growing medium on the survival rate (%) of B. madagascariensis seedlings.
Pre-treatment
Level
Germination (%)
Without
With
compost
compost
18
18
Survival (%)
Without
With
compost
compost
88.9
40.1
Height (cm)
Without
With
compost
compost
79.2±2.5
57.3±17.8
Control
0
Cold water
12
24
19
12
29
13
52.6
58.3
55.2
84.6
74.8±4.7
66.9±6.1
61.1±3.5
58.1±6.6
Hot water
12
24
22
45
14
22
76.2
80.0
35.7
31.7
69.5±4.1
73.8±3.0
56.4±4.5
60.0±6.9
Gibberellic acid
100
200
400
800
11
11
12
4
10
9
14
10
54.5
90.9
66.7
50.0
50.0
55.6
35.4
20.8
62.3±6.8
61.2±6.9
66.1±6.9
55.0±7.5
43.±7.5
42.2±9.7
45.8±9.0
55.0±1.0
was lower in seeds soaked in gibberellic acid compared
with the control (Figure 2b).
Seedling survival ranged from 21 to 92%, the highest
(91%) being in seeds soaked in 200 mg/l of gibberellic
acid and sown in media without compost (Table 2).
Seedling survival did not significantly vary (P > 0.05) with
the duration of soaking in hot water or various concentration of gibberellic acid. However, it significantly varied
(P < 0.01) with the growing medium in the case of hot
water treatment (Table 1). The growing medium without
compost manure after hot water treatment had higher
survival (76 to 80%) compared with the one with compost
manure (32 to 36%) irrespective of the duration of
soaking (Table 2).
Seedling height significantly differed (P < 0.01) only
with growing medium following either cold or hot water
treatment (Table 1). Untreated seeds (control) sown in a
medium without compost manure on average grew taller
(79.2 cm) than seeds sown in medium with compost
(57.3 cm). Similarly, seeds soaked in hot water and sown
in the medium without compost grew taller than those
sown in medium with compost (Table 2).
In vitro culture
Seeds pre-treated by scoring and soaking gave high germination percentage (70%) compared with those soaked
without scoring and cultured on Gamborg’s B5 (35%
germination) and MS media (30% germination). Explants
tested for shoot multiplication on both MS media without
PGRs and MS media supplemented with 1 mg/l of both
NAA and TDZ neither elongated nor multiplied (Figure
3a). All six in vitro germinated seedlings left in B5 media
without PGRs for axillary shoot proliferation produced a
single shoot, after removing the shoot-tip in seven weeks
(Figure 3c). All the six explants tested on B5 media
supplemented with 0.1 mg/l of NAA and TDZ produced
two shoots each after four weeks (Figure 3d). Explants
cultured on Florialite® cubes in B5 liquid media supplemented with 1 mg/l of NAA and IBA with an improved air
circulation failed to produce roots after four weeks.
DISCUSSION
This study and the literature (Mbuya et al., 1994)
indicated that, B. madagascariensis seeds had very low
ex vitro germination percentages. Efforts in the nursery at
Kasisi Agricultural Training Centre (KATC) in Zambia to
germinate it achieved less than 30% germination
(Lesseps, pers. comm.). Seeds of B. madagascariensis
could not be uniformly germinated by soaking in water.
Seed dormancy caused by the hard seed coat or due to a
deficit of endogenous gibberellins may cause low or
erratic germination at times taking up to six months, while
seeds are still germinating (Masamba, 1994). Treating
seeds with gibberellic acid has been reported to
overcome dormancy and ensure uniform germination (AlAbsi, 2010; Çetinba and Koyuncu, 2006). However, the
various gibberellic acid concentrations did not signifycantly improve germination percentage over the control,
which also achieved less than 20% germination. On the
other hand, in vitro procedures achieved over 70%
germination. This is probably because of the controlled
culture conditions provided under aseptic conditions for
seed germination.
Amendment of the growing medium with compost
5964
Afr. J. Biotechnol.
A
B
C
D
Figure 3. (A), Shoot-tips cultured on MS medium supplemented with 1 mg/l of NAA and TDZ; (B), MS medium without PGRs
showing no multiplication after 4 weeks; (C), axillary shoot proliferation from seedling without shoot tips on B5 without PGRs after
7 weeks; (D) those cultured on B5 supplemented with 0.1 mg/l of NAA and TDZ after 4 weeks.
Thokozani et al.
manure appeared to depress germination, seedling
survival and height growth, contrary to earlier reports that
compost suppresses disease incidence and increase
survival of plants (Hoitink and Boeh, 1999; Mazzola,
2007; Raaijmakers et al., 2008; Termorshuizen et al.,
2006). In traditional tree nurseries, growing media are
amended with compost manure to improve soil organic
matter content, increase soil water holding capacity and
suppress diseases (Hoitink and Boehm, 1999; Mazzola,
2007; Raaijmakers et al., 2008; Termorshuizen et al.,
2006). The lower germination percentage in the growing
medium with compost manure could be attributed to
factors such as poor water drainage and nitrogen supply.
Brady and Weil (2002) noted that, addition of manure to
growing medium increases nitrogen content.
Seeds and vegetative parts of some recalcitrant
species may pose challenges at all stages of in vitro
culture. The seed coat was proved to be the limiting
factor in this case to achieve good germination. By
scoring the seeds with a sharp blade germination
percentage were improved. The poor shoot induction on
B5 media is consistent with the findings of Berger and
Schaffner (1995). However, the species multiplied well on
MS and WP media (Berger and Schaffner, 1995). A half
strength MS media containing 26.8 µM (5 mg/l) of NAA
was used to effectively induce rooting (Berger and
Schaffner, 1995). Further studies are needed to find the
optimal culture medium and other growing conditions
required to obtain maximum shoot multiplication from
juvenile material. Achieving high percentage of rooting
from these shoots and the successful transplantation of
plantlets to the ex vitro conditions are further challenges
when considering developing an efficient system of in
vitro propagation. Initial trials done at CBU showed that,
combining rooting and weaning using a photoautotrophic
system of growing can be achieved (data not included).
Growing cultures in a sucrose-free medium with either
forced or diffusive ventilations systems simple
photoautotrophic systems can be developed. Plants
produced this way in culture will have functional stomata
and well developed cuticle once they leave the culture
environment to the greenhouse (Sarasan et al., 2006).
Large scale propagation of desired genotypes of B.
madagascariensis is required for the sustainable utilisation of this important medicinal and pesticidal species.
This approach can overcome the reported genetic
variability exhibited especially by plants propagated from
seed (Berger and Schaffer, 1995). Based on the trials
conducted in this study, it is clear that B.
madagascariensis seeds have very low ex vitro germination percentage. However, just developing in vitro raised
seedlings may not solve the propagation problem to meet
the demands in the market place. While in vitro cultures
can improve germination, axillary multiplication and
rooting efforts for rapid multiplication of this species
5965
were not satisfactory in this study. By using genetically
diverse seed stocks, media and culture conditions can be
improved. Therefore, further studies are needed to
develop an efficient micropropagation system to produce
quality propagules.
ACKNOWLEDGEMENTS
We thank Dr. Smart Lungu for the seed collection. This
work was financed by the European Union through the
Southern African Development Community (SADC)
Secretariat’s Implementation and Coordination of
Agricultural Research and Training (ICART) project and
the African, Caribbean and Pacific (ACP) Science and
Technology programme. The contents of this document
are the sole responsibility of the authors and can under
no circumstances be regarded as reflecting the position
of the SADC Secretariat or the European Union.
REFERENCES
Atawodi SE (2005). Comparative in vitro trypanocidal activities of
petroleum ether, chloroform, methanol and aqueous extracts of some
Nigerian savannah plants. Afr. J. Biotechnol., 4: 177-182.
Al-Absi KM (2010) The effects of different pre-sowing seed treatments
on breaking the dormancy of mahaleb cherries, Prunus mahaleb L.
seeds. Seed Sci. Tech., 38: 332-340.
Berger K, Schaffner W (1995). In vitro propagation of the leguminous
tree Swartzia madagascariensis. Plant Cell Tissue Organ., 40: 289291.
Borel C, Hostettmann K (1987). Molluscicidal saponins from Swartzia
madagascariensis Desvaux. Helv. Chim. Acta, 70: 571-576.
Brady NC, Weil RR (2002). The nature and properties of soils, 13th edn.
Pearson Education, New Jersey.
Çetinba M, Koyuncu F (2006). Improving germination of Prunus avium
L. seeds by gibberellic acid, potassium nitrate and thiourea.
Hortscience, 33: 119–123.
Cunningham AB (1993). African medicinal plants: setting priorities at the
interface between conservation and primary health care. People and
Plants working paper 1, Paris, UNESCO.
Gamborg OL, Miller RA, Ojima K (1968). Nutrient requirements of
suspension cultures of soybean root cells. Exp. Cell Res., 50: 151158.
Hoitink HAJ, Boehm MJ (1999). Biocontrol within the context of soil
microbial communities: a substrate-dependent phenomenon. Ann.
Rev. Phytopathol., 37: 427–446.
Hostettmann K, Schaller F (2000). United States patent No 5,929,124.
Hostettmann K, Marston A (2002). Twenty years of research into
medicinal plants: Results and perspectives. Phytochem. Rev., 1:
275–285.
Hostettmann K, Marston A, Ndjoko K, Wolfender J-L (2000). The
potential of African plants as a source of drugs. Curr. Org. Chem., 4:
973-1010.
Jaenicke H (1999). Good tree nursery practices. Practical guidelines for
research nurseries. World Agroforestry Centre (ICRAF), Nairobi,
Kenya. 90 pp.
Jordan M, Larrain M, Tapia A, Roveraro C (2001). In vitro regeneration
of from seedling explants. Plant Cell Tissue Organ., 66: 89-95.
Kirkbride JH Jr., Wiersema JH (1997). Bobgunnia, a new African genus
of tribe Swartzieae (Fabaceae, Faboideae). Brittonia, 49: 1-23.
Lwambo NJ, Moyo HG (1991). The molluscicidal activity of seed pods of
5966
Afr. J. Biotechnol.
Swartzia madagascariensis on Marisa cornuarietis. East Afr. Med. J.,
68: 827-30.
Masamba C (1994). Presowing seed treatments on four African Acacia
species: appropriate technology for use in forestry for rural
development. Forest Ecol. Manage., 64: 105-109.
Mazzola M (2007). Manipulation of rhizosphere microbial communities
to induce suppressive soils. J. Nematol., 39: 213–220.
Mbuya LP, Msanga CK, Ruff, CK, Birnie A, Tengas B (1994). Useful
trees and shrubs for Tanzania: identification, propagation and
management for agricultural and pastoral communities. Regional Soil
Conservation Unit (RSCU), Swedish International Development
Authority (SIDA).
Murashige T, Skoog F (1962). A revised medium for rapid growth and
bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.
Ouattaraa Y, Sanonb S, Traoréc Y, Mahioud V, Azase N, Sawadogoa L
(2006). Antimalarial activity of Bobgunnia madagascariensis Desv.
(leguminosae), Combretum glutinosum Guill. & Perr. (Combretaceae)
and Tinospora bakis Miers. (Menispermaceae), Burkina Faso
medicinal plants. Afr. J. Trad. Compl. Alt. Med., 3: 75-81
Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, MoënneLoccoz Y (2008). The rhizosphere: a playground and battlefield for
soilborne pathogens and beneficial microorganisms. Plant Soil DOI
10.1007/s11104-008-9568-6
Sarasan V, Cripps R, Ramsey MM, Atherton C, McMichen M,
Prendergast G, Rowntree JK (2006). Conservation in vitro of
threatened plants; progress in the past decade. In Vitro Cell Dev.
Biol. Plant, 42: 2006-214.
Schaller F, Rahalison L, Islam N, Potterat O, Hostettman K, StoeckliEvans H, Mavi S (2001). A new potent antifungal ‘Quinone methide’
diterpene
with
a
cassane
skeleton
from
Bobgunnia
madagascariensis. Helv. Chim. Acta, 84: 222-229.
Sileshi G, Kuntashula E, Matakala P, Nkunika PO (2008). Farmers’
perceptions of pests and pest management practices in agroforestry
in Malawi, Mozambique and Zambia. Agrofor. Syst., 72: 87-101.
Smith P, Allen Q (2004). Field guide to the trees and shrubs of the
Miombo woodlands. Royal Botanic Gardens, Kew, pp. 176.
Suter R, Tanner M, Borel C, Hostettmann K, Freyvogel TA (1986).
Laboratory and field trials at Ifakara (Kilombero District, Tanzania) on
the plant molluscicide Swartzia madagascariensis. Acta Trop., 43:
69-83.
Termorshuizen AJ, van Rijn E, van der Gaag DJ, Alabouvette C, Chen
Y, Lagerlöf J, Malandrakis AA, Paplomatas EJ, Rämert B, Ryckeboer
J, Steinberg C, Zmora-Nahum S (2006). Suppressiveness of 18
composts against 7 soilborne plant pathogens. Soil Biol. Biochem.,
38: 2461–2477.
Vermeulen SJ, Campbell BM, Matzke GE (1996). The consumption of
wood by rural households in Gokwe communal area, Zimbabwe.
Hum. Ecol., 24: 479-491.