Plant Cell Culture & Micropropagation ISSN:1808-9909
In vitro culture of Annona emarginata: A rootstock for  
commercial annonaceae species 
Cultivo in vitro de Annona emarginata: Porta-enxerto para espécies  
de anónaceas comerciais 
Rodrigo Therezan de Freitas1*, Renato Paiva1, Nádia Alves Campos5,  
Luciano Coutinho Silva2, Rony Swennen3,4,5, Bart Panis3
ABSTRACT
Annona emarginata is a native fruit tree in the Brazilian Cerrado which, unlike the commercial species, does not present a high fruit 
quality. On the other hand, it stands out based on its rootstock value. However, there are some problems that hinder the large-
scale production of Annona emarginata seedlings for use as a rootstock. In order to overcome these difficulties, micropropagation 
has become a viable alternative for the rapid and efficient propagation of Annona spp, but Annona emarginata micropropagation 
has not yet been reported.  Therefore, the aim of this study was to initiate Annona emarginata in vitro growth. For axillary shoot 
proliferation, in vitro nodal segments of 0.5 cm were transferred to MS or WPM supplemented with BA at different concentrations. 
Nodal segments were also cultured on WPM medium with ranging concentrations of different plant growth regulators, aiming 
for either shoot elongation or rooting. The results showed that the use of 1 µM BA in WPM medium is recommended for in vitro 
multiplication of Annona emarginata. This is based on the low adventitious shoot formation, combined with a higher number of 
buds and leaves. The use of GA3 at any concentration tested induced the formation of malformed plants. Root formation could not 
be stimulated, regardless the duration of auxin treatment.
Keywords: Araticum-mirim, microprogation, plant growth regulators. 
RESUMO
Annona emarginata é uma frutífera presente no Cerrado brasileiro que diferentemente das espécies comercias não apresenta alta 
qualidade de frutos, porém esta espécie destaca-se baseado unicamente em seu valor como porta-enxerto. Entretanto, existem 
alguns problemas que impedem a produção em larga escala de mudas de Annona emarginata para utilização como porta-enxerto. 
Para superar estas dificuldades a micropropagação tornou-se uma alternativa viável para a propagação rápida e eficiente de Annona 
spp. Porém a micropropagação de Annona emarginata ainda não foi relatado. Objetivou-se com o presente estudo o cultivo in vitro 
de Annona emarginata. Para o aumento de brotações in vitro, segmentos nodais de 0,5 cm foram transferidos para meio MS ou 
WPM, suplementado ou não com BAP em diferentes concentrações. Segmentos nodais também foram cultivados em meio WPM 
com diferentes concentrações de diferentes reguladores de crescimento, visando o alongamento ou enraizamento. Os resultados 
mostraram que o uso de 1 uM de BAP em meio WPM é recomendado para a multiplicação in vitro de Annona emarginata, baseado na 
baixa formação de gemas adventícias combinado com um maior número de brotos e folhas. O uso de GA3 em qualquer concentração 
testada induziu a formação de plantas malformadas e a formação de raízes não pode ser estimulada, independentemente do tipo 
ou da duração do tratamento com auxina testada. 
Termos para indexação: Araticum-mirim, microprogação, regulador de crescimento. 
INTRODUCTION the hybrid atemoya (Annona cherimola Mill. x Annona 
Annona ssp. represents a group of important fruit squamosa L.), the sugar apple or sweetsop (Annona 
trees found in the Brazilian Cerrado and the Amazonian squamosa L.), known as pinha or fruta do conde and the 
rainforest (Egydio and Santos, 2011). Five Annonaceae soursop (Annona muricata L.), known as graviola (Orsi 
species are of major commercial importance (Pareek et et al., 2012), which are mainly used for pulp or juice 
al., 2011), such as cherimoya (Annona cherimola Mill.), production in Brazil. 
Recebido em 24 de agosto de 2015 e aprovado em 5 de maio de 2016 
1Universidade Federal de Lavras/UFLA, Departamento de Biologia, Lavras, MG, Brazil 
2Universidade Federal da Paraíba/UFPB, Departamento de Biologia Celular e Molecular, João Pessoa, PB, Brazil 
3Bioversity International, KU Leuven, Willem De Croylaan, Leuven, Belgium 
4International Institute of Tropical Agriculture, c/o AVRDC, The World Vegetable Center, Arusha, Tanzania
5Laboratory of Tropical Crop Improvement, KU Leuven, Willem De Croylaan, Leuven, Belgium
*Corresponding author: rodrigotfreitas@hotmail.com 
Plant Cell Cult. Micropropag., Lavras, 12(1)1-6, 2016
2  –  FREITAS, R. T. de et al.  
Due to difficulties inherent in conventional (Murashige and Skoog, 1962), supplemented with 5 µM 
propagation through seeds (Padilla and Encina 2003; 6-benzylaminopurine (BA), 30g L-1 sucrose and solidified 
Santana et al., 2011), the propagation of commercial with 0.65% agar. The pH was adjusted to 5.8 ± 0.1 before 
Annona species happens through grafting or budding autoclaving. All cultures were maintained in a growth room 
(Costa, Bueno and Ferreira, 2011; Nagori and Purohit, at 25 ± 2 °C with a 16-hour photoperiod and 56 µmol m-2 
2004; Rasai, George and Kantharajah, 1995). Due to s-1 PAR (Photosynthetic Activity Radiation). 
problems of incompatibility between the scion and the 
rootstock (Schaffer, Davies and Crane, 2006) and the Shoot Induction
presence of diseases like root rot infection (Neto et al. For axillary shoot proliferation, in vitro nodal 
2006), it is important to choose a suitable rootstock. segments of 0.5 cm with a single bud, were transferred to MS 
A suitable rootstock for other Annonaceae species or Woody Plant Medium (WPM) (Lloyd and McCown, 1980) 
is Annona emarginata, locally known as Araticum-mirim supplemented or not with BA at different concentrations 
(Lorenzi, 2009). When grafted on this species, the graft (0, 1, 3 or 5 µM). The media were supplemented with 30g 
shows 90% survival (Baron et al. 2011). This rootstock L-1 sucrose and solidified with 3g L-1 Phytagel® and the pH 
also displays a high resistance to soil fungi and it is less was adjusted to 5.8 ± 0.1. Each tube contained one explant 
attractive to stem borers (Neto et al. 2006). However, and was considered as one replicate, with each treatment 
there are some problems that hinder the large-scale consisting of 15 replicates. After 45 days of culture, the 
production of Annona emarginata seedlings for use as number of buds and leaves of each explant was counted, 
a rootstock, such as the low and uneven germination and the height of the main shoot was measured. 
percentage and the rapid loss of viability after seed drying 
(Costa, Bueno and Ferreira, 2011). Shoot elongation 
Micropropagation is a viable alternative for the rapid Shoots of 1 cm length derived from the axillary 
and efficient propagation of several species. Although several proliferation with four buds were transferred to WPM 
Annonaceae species have already been micropropagated supplemented with 1µM BA, in combination with different 
(Encina et al. 2014), few protocols proved to be successful for concentrations (0, 1, 5 and 10 µM) of gibberellic acid-3 
the micropropagation process. Most of the species studied, (GA3), 30g L-1 sucrose, solidified with 3g L-1 Phytagel® and 
show problems with rooting and acclimatization (Lemos and pH adjusted to 5.8 ± 0.1. The explants were maintained in 
Blake, 1996; Oliveira et al. 2010). a dark room at 25 ± 2 ºC for 14 days, and then transferred 
Annona emarginata micropropagation has not been to a culture room in light at 25 ± 2 °C with a 16-hour 
reported yet. Therefore, the aim of this study was to develop photoperiod and 56 µmol m-2 s-1 PAR, or were cultured 
a micropropagation protocol for Annona emarginata. throughout the whole experimental period in a light. After 
45 days, the number of buds and leaves of each explant 
MATERIAL AND METHODS was counted and the height was measured. Treatments 
Plant material and culture initiation consisted of 12 replicates with one explant per tube. 
Nodal segments of 0.5 cm were excised from Rooting 
three-year-old Annona emarginata plants, maintained For root induction, shoots of 1-2 cm length with four 
in a greenhouse at the Plant Tissue Culture Laboratory buds previously maintained in WPM + 1 µM BA for 60 days, 
of the Federal University of Lavras (UFLA), Brazil. The were transferred to a WPM basal medium without plant 
explants were immersed in 70% ethyl alcohol (v/v) for growth regulators (control), or supplemented with different 
30 seconds, followed by sodium hypochlorite treatment auxins: Indole-3-acetic acid (IAA), indole-3-butyric acid 
(1% active chloride) during 15 minutes. They were (IBA) or 1-naphthaleneacetic acid (NAA) at 1, 10 and 100 
then washed three times in sterile distilled water and µM. All media were supplemented with 30g L-1 sucrose and 
inoculated into test tubes containing 20 mL MS medium 3g L-1 Phytagel® with pH adjusted to 5.8 ± 0.1. Treatments 
Plant Cell Cult. Micropropag., Lavras, 12(1)1-6, 2016
In vitro culture of annona emarginata: a rootstock for commercial annonaceae species  –  3
consisted of 24 replicates, with one explant per tube. this increase also takes into account the formation 
Shoots were maintained in a medium containing auxin for of  “clumps”. Clumps is a swelling at the base of the 
seven or 30 days and then, transferred to a growth regulator explant, but which doesn’t looks such as callus with 
free medium. After 45 days of culture, the number of roots undifferentiated and loose cells (Figure 2). This is followed 
was counted and the presence of callus was noted.  by the appearance of small buds that can sometimes 
Statistical analysis develop into new plants. It was, however, observed that 
most of the clumps or adventitious buds gave rise to 
Statistical analyses were performed using the malformed leaves or buds. These malformations observed 
SISVAR program (Ferreira, 2014). All the experiments are undesirable in clonal multiplication cycles, since they 
were set up in a completely randomized design. The data may cause somaclonal variation. 
were analyzed through analysis of variance (ANOVA) and, Clumps were observed at a rate over 80% when MS 
when significant (P<0.05) by F test, they were submitted was used, or over 66% in WPM medium when supplemented 
to polynomial regression. with any concentration of BA.  In contrast, in the absence of 
BA in either MS or WPM medium, no clumps were visible.
RESULTS AND DISCUSSION Such increase in the number of buds, following an 
It was possible to observe that the WPM medium increased concentration of BA, was also described by Lemos 
is always superior to MS after 45 days of culture regarding and Blake (1996) when BA was used to induce multiplication 
the number of buds produced, provided a source of in Annona muricata. However, these authors did not describe 
cytokines is supplemented (Figure 1A). In general, the the formation of clumps and malformed leaves. 
number of leaves increased in both media in the presence Shoot elongation reached the highest values at 5 
of BA and the highest number of leaves was found in WPM µM GA3, irrespective of light conditions (Figure 3).
medium, regardless of the BA concentration (Figure 1B). The results obtained in the present study differ 
Shoot height was not statistically different when using MS from other authors that also tested the effect of GA3 in 
or WPM medium (P>0.05). the culture medium of Annonaceae. Nagori and Purohit 
Cytokines have been routinely used in tissue (2004) reported that, when initiated on MS medium 
culture due their action in promoting the unfolding of containing BA, even after repeated subcultures, or when 
a complex gene expression program that results in a treated with GA3 in combination with BA, explants of 
variety of traits, including shoot formation during plant Annona squamosa did not undergo any significant growth 
propagation (Howell, Lall and Che, 2003). stimulation. Lemos and Blake (1996) also reported that 
The highest number of buds (9) was obtained the addition of GA3 to the basic medium did not stimulate 
with the highest concentration of BA tested. However, any significant growth in Annona muricata. 
Figure 1 – Number of buds (A) and leaves (B) after 45 days in MS and WPM media supplemented with different 
concentrations of BA. 
Plant Cell Cult. Micropropag., Lavras, 12(1)1-6, 2016
4  –  FREITAS, R. T. de et al.  
on root formation, compared to the control (p > 0.05). 
Irrespective of the exposure time to auxins, less than 10% 
of the explants showed root formation when IAA or IBA 
were used. NAA did not result in any root induction in any 
of the concentrations or exposure time tested. 
Figure 2 – Clump formation at the base of a shoot 
(indicated by an arrow) in the presence of 5 µM BA after 
30 days on WPM medium. bar = 0.5 cm. 
Figure 4 – Annona emarginata control supplemented with 
only 1µM BA, compared to plants supplemented with 1µM 
BA plus 5 µM GA3 under dark conditions with irregular shapes 
Figure 3 – Shoot height in WPM medium with 1 µM BA and color of leaves after 45 days of culture.  bar = 0.5 cm.
and different concentration of GA3 after 45 days of culture.
Callus formation occurred after 30 days of 
Although the use of 5 µM GA3 increased elongation, culture, despite the auxin treatment (Figure 5A). The 
the addition of GA3 induced malformed plants with presence of 100 µM NAA, IBA or IAA for 30 consecutive 
irregular shapes and color of leaves, regardless of light days promoted 50%, 25% and 75% callus formation, 
conditions (Figure 4). respectively. When the same concentrations were 
Classically, GA3 is used for elongation of in vitro applied for seven consecutive days, less callus (16%, 16% 
plants.  This compound stimulates an increase in internode and 8.33%, respectively) was observed. The induction 
length. However, in this study, the addition of GA3 showed of such callus was also reported by Nagori and Purohit 
a negative effect. Thus, further studies related to the use of (2004) and by Zobayed, Armstrong and Armstrong, 
GA3 or their combination with other plant growth regulators (2002) in Annona squamosa. Moreover, a prolonged 
could significantly contribute to determine an appropriate use of auxin, especially at high concentrations (100 
concentration for Annona emarginata. µM), adversely affected the development of Annona 
The addition of NAA, IAA or IBA at any of the emarginata shoots, showing leaf yellowing and bud 
concentrations tested did not show any statistical influence necrosis in all treatments (Figure 5B).  
Plant Cell Cult. Micropropag., Lavras, 12(1)1-6, 2016
In vitro culture of annona emarginata: a rootstock for commercial annonaceae species  –  5
Figure 5 – (A) Callus formation at the base of a shoot (indicated by an arrow) in the presence of 100 µM NAA after 
45 days of culture. (B) Leaf abscission and bud necrosis (indicated by an arrow) in the presence of 10 µM NAA after 
30 days of culture. bar = 0.5 cm.
Lemos and Blake (1994) and Oliveira et al. (2007) based on the low rate of adventitious shoot formation, 
reported that leaf abscission in Annonaceae in vitro cultures combined with a higher number of buds and leaves. 
are a serious problem. This can be due to the induction and Independent of light conditions, GA3 induced the 
accumulation of ethylene in the tissue culture container, formation of malformed plants with irregular shapes and 
when auxins are applied for a prolonged period of time lighter color of leaves, at any of the concentrations, when 
(De Klerk, 2002). An increase in ethylene production can combined with 1 µM BA. 
be due to a decrease in cytokinin concentration in mature In the present study, root formation could not be 
organs (Oliveira et al. 2007) or due to the use of exogenous stimulated, regardless of the duration of auxin treatment, 
auxins acting as an antagonist of cytokinins (Shimizu-Sato, type or concentration of auxin. Further experiments are 
Tanaka and Mori, 2009). Ethylene production may also be thus needed to optimize root formation.
stimulated by mechanical cutting of the explant (Khalafalla 
and Hattori, 2000). ACKNOWLEDGEMENTS
Leaf abscission was also observed by Farooq, The authors would like to thank CNPq, CAPES and 
Farooq and Rao (2002) with Annona squamosa, when FAPEMIG for the financial support and the Laboratory for 
cultured on medium enriched with IBA. Zobayed, Tropical Crop Improvement (KU Leuven) for guidance of 
Armstrong and Armstrong (2002) also describe that the the practical work. Bart Panis also gratefully acknowledge 
addition of NAA to the medium increased leaf abscission the support from the CGIAR Research Programmes (CRP) 
in A. squamosa and A. muricata. Roots, Tubers and Bananas CRP and The Genebank CRP.
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