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Progress in developing in vitro systemsfor shea tree (Vitellaria paradoxa C.F.Gaertn.) propagationPeter N. Lovett a & Nazmul Haq aa Environment Research Group, Faculty of Engineeringand Environment, University of Southampton, Highfield ,Southampton , SO17 1BJ , UKPublished online: 13 Feb 2013.
To cite this article: Peter N. Lovett & Nazmul Haq (2013) Progress in developing in vitro systemsfor shea tree (Vitellaria paradoxa C.F. Gaertn.) propagation, Forests, Trees and Livelihoods, 22:1,60-69, DOI: 10.1080/14728028.2013.765092
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Progress in developing in vitro systems for shea tree(Vitellaria paradoxa C.F. Gaertn.) propagation
Peter N. Lovett1 and Nazmul Haq*
Environment Research Group, Faculty of Engineering and Environment,University of Southampton, Highfield, Southampton SO17 1BJ, UK
Recent demand growth for shea butter from the international, multi-billion dollar,cosmetic and confectionary sectors, coupled with widespread occurrence of sheaparklands across Sahel–Savannah landscapes, provides an unprecedented opportunityto mitigate rural communities’ vulnerability to food insecurity while adapting to localand global climate variability. The key outcome of this study is to establish a startingpoint from which culture protocols can be developed for shoot and root regeneration ofexplants taken from mature materials. Once possible, superior genotypes withagronomic values could be selected and multiplied for production of high-qualityplanting materials for African farmers. Axillary shoot proliferation was induced inapical shoots of Vitellaria paradoxa seedlings when cultured on growth media withMurashige and Skoog macro- and micronutrients reduced to half-strength in thepresence of a combination of the plant growth regulators, 6-benzyladenine (BA) anda-napthaleneacetic acid. Shoot regeneration was maximal when the cytokinin/auxinratio was between 5:1 and 50:1 with BA in between 8.9 and 22.2mM. Adventitiousroots were stimulated when cultured on rooting media with Murashige and Skoogmacro- and micronutrients reduced to quarter-strength in the presence of the plantgrowth regulator, indolebutyric acid between 4.9 and 14.8mM.
Keywords: Vitellaria paradoxa; shoot proliferation; adventitious roots; cytokinin/auxin ratio
Abbreviations: BA, 6-benzyladenine; IBA, indolebutyric acid; MS, Murashige andSkoog medium; NAA, a-napthaleneacetic acid
Introduction
The shea tree (Vitellaria paradoxa C.F. Gaertn., syn. Butyrospermum paradoxum, Family:
Sapotaceae) is indigenous to the semi-arid zone of sub-Saharan West Africa and commonly
seen at high densities on inter-cropped farms in the parkland landscapes of the Sahel–
Savannah (Lovett & Haq 2000a; Elias 2012). It is a highly valued tree species with its most
notable product being the oil, known as shea butter (French: beurre de karite) obtained from
the seeds traditionally harvested by women in this zone. The income from shea has been
proven to be a significant contribution to rural livelihoods across the Sahel–Savannah
(Pouliot 2012). The wooded parklands, of which shea trees form a high-percentage biomass,
also protect against environmental degradation prevalent in this area and contain substantial
carbon stores with vast potential for future sequestration of climate change mitigation
(Luedeling & Neufeldt 2012). On the world market, shea butter is highly valued for use in
luxury cosmetic (moisturising creams, sun lotions and soaps) or pharmaceutical products
(cholesterol-lowering and anti-arthritic remedies). The main demand for shea, however, is for
the production of edible stearin, an exotic speciality fat utilised in the formulation of cocoa
q 2013 Taylor & Francis
*Corresponding author. Email: [email protected]
Forests, Trees and Livelihoods, 2013
Vol. 22, No. 1, 60–69, http://dx.doi.org/10.1080/14728028.2013.765092
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butter improvers for chocolate confectionary (Loh Moong Ming 2008; Talbot & Slager 2008).
The other edible by-product – shea olein – is often used in vegetable spreads, and the residue
remaining after oil extraction is used as biofuel (Hall et al. 1996). With rapid growth in the
shea industry over the past decade, estimates suggest that sheanut volumes (Lovett 2010)
multiplied by recent tonnage prices, even prior to butter extraction, can earn West African
Sahel–Savannah rural communities in the region of US$ 150 million.
Current populations of shea trees result from natural regeneration, protected and wild
managed during cycles of cultivation. Because of cross-pollination, these wild-managed
stands consist of heterozygous individuals and produce an unreliable crop in terms of
quantity and quality. The growing importance of this tree species, cutting for fuelwood,
the threat of over-harvesting (Elias 2012) and the opportunity to produce superior true-to-
type varieties in order to capture superior genetic traits (Sanou et al. 2006), has prompted
research into the clonal propagation of this tree (Ræbild et al. 2011). However, vegetative
methods, such as grafting, budding, cuttings and air-layering, have only produced limited
success (Picasso 1984; Frimpong et al. 1987; Grolleau 1989; Lovett et al. 1996; Sanou
et al. 2004; Yeboah et al. 2010).
Micro-propagation of woody species has become a widely used technique not only for the
rapid regeneration of superior germplasm, but also to reduce the post-propagation juvenile
period and for conservation purposes (Ahuja 1992; Pijut et al. 2012). Furthermore, in vitro
propagation techniques have been successful for other members of Sapotaceae, such as
Argania spinosa, which have previously been difficult to propagate (Nouaim & Chaussod
1994). Fotso et al. (2008) were also successful in inducing embryogenic callus and somatic
embryo formation in leaf fragments of bothBaillonella toxisperma andV. paradoxa, but were
unable to induce fully formed shoots or roots. Adu-Gyamfi et al. (2012) successfully
germinated somatic embryos from embryogenic callus of immature cotyledon explants, which
subsequently developed shoots, and were rooted and acclimatised. Ræbild et al. (2011)
highlighted micro-propagation as a ‘gap in research’ for this species and because no reports
exist for in vitro propagation from shoot tips; this study is therefore offered to demonstrate
whether in vitropropagation or conservation of selected varieties is possible for shea trees. This
paper reports successful shoot and root inductions during in vitro culture of apical shoot tips
from seedlings of V. paradoxa, thereby providing additional evidence that these techniques
potentially offer a valuable means for the multiplication and conservation of this species.
Materials and methods
Mature seeds collected from trees across northern Ghana were surface planted, hilum side
down, in trays containing a 5-cm layer of a 50:50 mix of sharp sand and Levington’s No. 1
compost. Seeds were maintained in Southampton University glasshouses at 15–27 ^ 28C
(night/day) and irrigated every 3 days. On germination (when the pseudo-radicle
emerged), the seeds were transferred to 17.5-cm-diameter pots containing the same soil
mixture. True shoots were observed after a further 4–6 weeks and used as explants.
Shoot tips of approximately 10–15 mm long were washed three times in distilled water
before being surface sterilised by washing in 70% (v/v) ethanol for 1.5 min followed by
immersion in 8% (v/v) Domestos (Lever Brothers, Port Sunlight, Merseyside, United
Kingdom) for 25 min and then washed four times in sterile distilled water. The tips were
then trimmed by removing 2–3 mm of stem and all remaining leaves and petioles before
being aseptically placed randomly into shoot regeneration media. Cultures were
maintained for 42 days in a culture room at 26 ^ 18C under a photoperiod of 16 h daylight
provided by cool white fluorescent at 18.5mE m22 s21.
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Shoot regeneration media consisted of 30 g l21 sucrose (Fisons, Ipswich, Suffolk,
United Kingdom) and 8 g l21 Gum Agar (Sigma, Gillingham, Dorset, United Kingdom)
with MS (Murashige & Skoog 1962) macro- and micronutrients at full or half-strength
(50%, w/v). Combination of the plant growth regulators, 6-benzyladenine (BA) (0–
22.2mM) and a-napthaleneacetic acid (NAA) (0–5.4mM) (Sigma), was added to
stimulate shoot initiation from lateral buds.
Regenerated shoots from lateral bud breaks were transferred to rooting media
consisting of 30 g l21 sucrose (Fisons) and 8 g l21 Gum Agar (Sigma) with MS macro- and
micronutrients at half- (50%, w/v) or quarter-strength (25%, w/v). The plant growth
regulators, NAA or indolebutyric acid (IBA) (0 and 16mM) (Sigma), were added to
stimulate adventitious root indication. Cultures were maintained for 70 days in a growth
room under conditions described earlier. All media were adjusted to pH 5.7 with 0.1 M
NaOH and autoclaved at 1208C for 15 min.
Data collection and statistical analysis
Callus size and growth were rated using the following scale. Callus width (scaled 0–6,
respectively): none, ,1, 1–5, 6–10, 10–15, 15–20 and .20 mm. Growth (scaled 0–6
respectively): necrotic, alive but no growth signs, only leaf bud growth, leaf growth/stem
elongation (,10 mm), leaf growth/stem elongation (10–30 mm), leaf growth/stem
elongation (30–50 mm) and leaf growth/stem elongation (.50 mm). One-way ANOVA
was used to compare the number of axillary shoots induced, growth and callus size after
42 days in culture period. For shoot proliferation experiments 20 explants were used per
treatment, and for rooting experiments 24 regenerated shoots were used per treatment
(seedlings randomly selected). Post-hoc pairwise multiple comparisons were performed to
determine treatments that were significantly different at the 95% or 99% levels using the
least significant difference test (SPSS for Windows ver. 7.5).
Results
Survival and growth of explants
V. paradoxa explants cultured on shoot regeneration media containing MS macro- and
micronutrients at full or half-strength showed no difference in the mean number of axillary
shoots induced (identical range of treatments for plant growth regulator combinations).
However, survival, induction of callus and shoots from calli and growth were observed to
be significantly lower in shoot regeneration media containing MS macro- and
micronutrients at full strength (Table 1), and were therefore considered unsuitable for
the maintenance of V. paradoxa explant cultures.
Shoot proliferation
Further proliferation of axillary shoots occurred when shoot regeneration media,
containing MS macro- and micronutrients at half-strength (1/2 MS), was supplemented
with various combinations of the plant growth regulators, BA and the NAA. Table 2 shows
the results for average growth, callus formation and the number of axillary shoots from
axillary buds per explant for each treatment. The maximum number of shoots produced
from one explant was 16 after 56 days of culture on media containing 1/2 MS and either
BA 4.4mM and NAA 10.7mM or BA 15.5mM and NAA 0.5mM. Adventitious shoots
from the base of calli were also occasionally found to be induced when V. paradoxa
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Table 1. Effect of MS concentration on survival, growth, callus induction and shoot regeneration ofV. paradoxa explants after 42 days in culture.
MSnutrients(w/v) (%) Survivala (%)
Cultures exhibitingcallusb (%)
Meancallus sizeb
Meangrowthb
Meanshootsb
100 27.4 30.4 0.4 ^ 0.3c 2.4 ^ 0.5d 0.2 ^ 0.350 72.0 91.1 1.7 ^ 0.2c 3.0 ^ 0.2d 0.2 ^ 0.2
a As a percentage of original number of explants after 42 days in culture.b As a percentage or mean of all surviving explants (^95% confidence limits).c Significantly different, p , 0.01.d Significantly different, p , 0.05.
Table 2. Effect of BA and NAA concentrations on growth, callus formation and axillary shootproliferation of V. paradoxa explants after 42 days culture on half-strength MS media.
Plant growthregulator (mM)
BA NAAMean growtha
(scale 0–6)Mean callus sizea
(scale 0–6) Mean new shoots per explanta
0 0.0 2.4 ^ 0.4 0.6 ^ 0.4 0.3 ^ 0.70.3 – – –0.5 2.8 ^ 0.5 0.8 ^ 0.7 0.0 n/a2.7 – – –5.4 2.8 ^ 0.5 1.8 ^ 0.6 0.3 ^ 0.7
2.2 0.0 3.3 ^ 1.4 1.5 ^ 2.2 0.3 ^ 1.40.3 – – –0.5 – – –2.7 – – –5.4 4.5 ^ 1.2 2.3 ^ 5.0 0.0 n/a
4.4 0.0 3.0 ^ 1.0 1.3 ^ 0.8 0.0 n/a0.3 – – –0.5 2.4 ^ 1.0 1.4 ^ 1.0 0.3 ^ 0.52.7 – – –5.4 3.1 ^ 0.5 2.1 ^ 1.0 0.8 ^ 1.8
8.9 0.0 3.2 ^ 0.9 1.5 ^ 0.6 0.4 ^ 0.60.3 3.3 ^ 0.5 1.7 ^ 0.8 1.7 ^ 0.80.5 2.6 ^ 1.1 1.4 ^ 0.8 0.1 ^ 0.32.7 3.1 ^ 1.5 2.0 ^ 0.9 0.8 ^ 1.65.4 3.1 ^ 0.4 1.9 ^ 2.6 0.0 n/a
15.5 0.0 2.8 ^ 2.3 1.4 ^ 2.0 0.0 n/a0.3 3.3 ^ 1.1 1.4 ^ 0.7 0.8 ^ 1.20.5 3.4 ^ 0.6 1.5 ^ 0.4 1.3 ^ 1.22.7 3.0 ^ 1.2 1.8 ^ 0.7 1.7 ^ 2.95.4 – – –
22.2 0.0 3.0 ^ 0.6 1.5 ^ 0.6 0.7 ^ 0.80.3 3.8 ^ 0.6 2.1 ^ 0.9 0.6 ^ 1.10.5 3.2 ^ 0.8 2.0 ^ 0.5 1.4 ^ 1.02.7 3.6 ^ 1.6 1.8 ^ 0.5 2.5 ^ 2.85.4 3.3 ^ 0.8 3.1 ^ 0.8 0.3 ^ 0.9
Note: – Indicates no explants set at this concentration. n/a: non-availability.a As mean of all surviving explants ^95% confidence limits.
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explants were cultured on media containing 1/2 MS and either BA 4.4mM and NAA
10.7mM or BA 15.5mM and NAA 0.5mM (Figure 1).
Results showed high variance and few significant differences between treatments. This
indicates high variability in the explant’s response to different combinations of plant
growth regulators, but this may also be due to heterozygosity of the original plant materials
(Lovett & Haq 2000b; Fontaine et al. 2004). A comparison of means, however, calculated
using cytokinin/auxin ratios (irrespective of concentration) indicated a significant shoot
induction response of treatments containing BA/NAA at a ratio of between 5:1 and 50:1,
as shown by the peak of axillary shoot formation in Figure 2. The presence of an outlier
with low shoot induction response (BA/NAA ¼ 16.5) suggests the occurrence of two
peaks in activity; however, this observation needs further verification because of a
statistically low sample size.
Root induction
Adventitious roots were induced when regenerated axillary shoots of V. paradoxa were
cultured for 70 days on root regeneration media containing MS macro- and micronutrients
at quarter-strength. Eight per cent of cultures (n ¼ 100) formed one root per axillary shoot
on media with 4.9mM IBA, and 8% of the cultures formed three roots per axillary shoot on
media containing 14.8mM IBA (Figure 3). No signs of rooting were observed when shoots
were cultured on media containing MS macro- and micronutrients at half-strength. These
results demonstrate a low success rate, but it should be noted that 76% of cultures were still
healthy on completion of the experiment. Similar observations have been reported during
attempts to root V. paradoxa cuttings using poly-propagators in northern Ghana and
Uganda and under mist in heated European glasshouses. Under these conditions, the
formation of hormone-induced adventitious roots usually occurred between 90 and
120 days after cutting, although the minimum recorded time was 67 days (Lovett et al.
1996; Lovett 2000; Ouna 2001; Yeboah et al. 2011).
Discussion
Reduced survival, growth and callus formation of explants in shoot regeneration media
containing MS macro- and micronutrients at full strength confirm other studies on woody
plant species where macro- and micronutrients at full strength can have inhibitory effects.
This inhibition on in vitro growth has often been overcome by diluting the medium strength
or by decreasing the total ionic strength through a reduction in the concentration of macro-
elements as with other studies on in vitro propagation of V. paradoxa (Fotso et al. 2008).
Numerous morphogenetic responses have been recorded for other Sapotaceae in the
presence of different plant growth regulators, for example with Pouteria lucuma (Jordan &
Oyanedel 1992). Likewise, this study has shown that explants from V. paradoxa seedlings
also demonstrate a variable response to different concentrations and combinations of the
plant growth regulators BA and NAA. However, a large variation in response was also
noted between accessions cultured on the same media. Because the seedlings were
obtained from randomly sampled wild-managed populations, the main source of variation
is probably genotypic diversity. Comparable genetic variability, in terms of
responsiveness to in vitro culture, has been clearly demonstrated for A. spinosa using
the material collected from wild populations (Nouaim et al. 2002).
It has frequently been observed that plants exhibit growth and shoot proliferation in
response to a high cytokinin/auxin ratio, while root induction usually occurs in the
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presence of high auxin/cytokinin levels, for example this was well noted for in vitro
regeneration of wild chervil (Anthriscus sylvestris) (Hendrawati et al. 2012). It is,
therefore, possible that the observed results could be explained if the shoot proliferation
Figure 1. Axillary shoot proliferation in explants of V. paradoxa cultured on half-strength MSmedia, BA 15.5mM and NAA 0.5mM.
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response were induced by a narrow range of cytokinin/auxin ratios. If this ‘ratio effect’
were genuine, only minimal changes to the BA/NAA ratio could produce large changes in
the numbers of shoots proliferated. Conversely, small genotypic differences could induce
Figure 2. Graph showing model of in vitro shoot induction, with cytokinin/auxin ratio, for explantsof V. paradoxa.
Figure 3. Adventitious roots induced from V. paradoxa explants after 70 days culture on quarter-strength MS media with 14.8mM IBA.
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large shifts to the shoot proliferation response for a particular cytokinin/auxin ratio. In
support of our claim, it appears that a growing number of studies are reporting the effect of
cytokinin/auxin ratios and genotype during in vitro propagation of tree species, for
example this was well studied during in vitro studies of the temperate tree species Arbutus
unedo (Gomes et al. 2010). For tropical tree species, Leakey and Newton (1994) gave the
optimal cytokinin/auxin ratio for inducing root proliferation in African Mahogany as 100–
200:1 at the sub-culture and 25:1 at the second sub-culture. With regard to root
regeneration, Villarreal and Rojas (1996) showed maximal root induction of Mimosa
tenuiflora when the auxin/cytokinin ratio was greater than 10:1. Ogunsola and Ilori (2008)
demonstrated that the variation of this ratio has effects on in vitro regeneration of another
Sapotaceae species from West Africa, Synsepalum dulcificum, as did Anis et al. (2010) for
Balanites aegyptiaca. In addition, Adu-Gyamfi et al. (2012) suggest that the relationship
between auxin concentrations and induction of embryogenic calli or somatic embryos of
V. paradoxa is non-linear and follows a quadratic pattern. This apparent effect of
cytokinin/auxin ratios and genotype on in vitro shoot or root induction may, therefore, be
widespread and important for other woody tropical plant species from the African Sahel–
Savannah in addition to V. paradoxa.
This paper has aimed to describe shoot and root induction during in vitro culture of
shea (V. paradoxa), a widespread, yet difficult to propagate, tree species (Yeboah et al.
2011). The study also indicates that adventitious shoot formation in vitro, originating from
the apical shoots of seedlings, may prove a useful means of multiplication because young
trees of this species normally exhibit only apical growth, with axillary branches forming
after 5–6 years, as noted in early scientific research on this species (Delome 1947). This is
a limiting factor for other forms of clonal propagation of shea trees, and thereby suggests
that micro-propagation techniques offer solutions to multiplication of this economically
important oleaginous African tree crop. It is recommended that further research be
undertaken to standardise in vitro propagation techniques for this species. One outcome of
this study is that a starting point has been established from which culture media and plant
growth regulators, and protocols can now be developed for the shoot and root regeneration
of explants taken from mature materials. Once this is possible, superior genotypes with
known agronomic values can be selected and multiplied for the production of high-quality
planting materials for farmers. Given the recalcitrant nature of V. paradoxa seeds, these
results also demonstrate that in vitro techniques can be an important tool for the
conservation of this species.
Acknowledgements
We are grateful to shea farmers of northern Ghana, to the staff of the Cocoa Research Institute ofGhana (CRIG) and to Leverhulme Trust funding for making this study possible.
Note
1. Email: [email protected]
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