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Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a

temporary immersion system

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DOI: 10.1007/s11627-014-9602-8

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In Vitro Cell.Dev.Biol.—Plant (2014) 50:576–581
DOI 10.1007/s11627-014-9602-8
PLANT TISSUE CULTURE
Improved propagation of vanilla (Vanilla planifolia Jacks. ex
Andrews) using a temporary immersion system
A. Ramos-Castellá & L. G. Iglesias-Andreu &
J. Bello-Bello & H. Lee-Espinosa
Received: 8 August 2013 / Accepted: 17 February 2014 / Published online: 16 April 2014 / Editor: J. Forster
# The Society for In Vitro Biology 2014
Abstract Here, we evaluated the efficiency of shoot multi-
plication of Vanilla planifolia Jacks. ex Andrews using solid
medium, partial immersion, and a temporary immersion sys-
tem (TIS) to improve micropropagation in this species.
Clusters of shoots were cultivated in vitro using Murashige
and Skoog (MS) medium supplemented with 9.55 μM
benzyladenine (BA) and 100 mL L−1 coconut water. For the
TIS, a RITA® system was used and three immersion frequen-
cies were evaluated (every 4, 8, and 12 h) with an immersion
time of 2 min. After 30-d culture, the TIS produced the
maximum multiplication rate (14.27 shoots per explant) when
using an immersion frequency of 2 min every 4 h, followed by
the partial immersion system (8.64 shoots per explant), and
solid medium (5.80 shoots per explant). Next, the effect of the
volume of culture medium per explant was also evaluated for
TIS. The most suitable volume of culture medium for shoot
formation was 25 mL per explant, which increased the rate of
multiplication to 17.54 shoots per explant. Root initiation was
90% successful in TIS using half-strength MS medium sup-
plemented with 0.44 μM naphthaleneacetic acid (NAA) and
an immersion frequency of 2 min every 4 h. With this system,
the shoot multiplication rate increased threefold compared to
A. Ramos-Castellá : L. G. Iglesias-Andreu (*)
Instituto de Biotecnología y Ecología Aplicada (INBIOTECA),
Universidad Veracruzana, Campus para la Cultura, las Artes y el
Deporte, Av. de las Culturas Veracruzanas No. 101, Colonia
Emiliano Zapata, 91090 Xalapa, Veracruz, México
e-mail: xliglesias@gmail.com
J. Bello-Bello
Colegio de Postgraduados-Campus Córdoba, Carretera Federal
Córdoba-Veracruz km 348, Congregación Manuel León,
94946 Municipio de Amatlán de los Reyes, Veracruz, México
H. Lee-Espinosa
Facultad de Ciencias Biológicas y Agropecuarias de Córdoba, Ver.,
Km. 1, Universidad Veracruzana, 94950 Peñuela-Amatlán de los
Reyes, Veracruz, México
that obtained with solid medium. In addition, this system
produced good results for the transplantation and acclimation
(90% of survival) of in vitro-derived plants. These results offer
new options for large-scale micropropagation of vanilla.
Keywords Immersion frequency . Partial immersion .
RITA® . Solid medium . Volume of culture medium
Introduction
Vanillin is one of the most highly appreciated flavorings in the
food, pharmaceutical, and fragrance industries (Bory et al.
2008; Greule et al. 2010) and is extracted from the pods of
Vanilla planifolia, an orchid native to the tropical forests of
southeastern Mexico (Soto Arenas and Cribb 2010; Salazar-
Rojas et al. 2012). This species is currently listed as threatened
owing to the overexploitation that has decimated the wild
populations and reduced genetic diversity (Soto Arenas
1999; SEMARNAT 2002).
In Mexico, vanilla is commercially propagated exclusively
through stem cuttings. However, because of poor quality
control, this method has contributed to the spread of pests
and diseases such as red bug (Tenthecoris confusus), root and
stem rot (Fusarium oxysporum), and anthracnosis
(Colletotrichum gloeosporioides; Hernández 2011); all of
which are responsible for significant financial losses in vanilla
production (Pinaria et al. 2010). Sexual propagation, on the
other hand, has the limitation of low or null germination from
seeds (Torres-González et al. 2011). Therefore, the best option
for propagation of this species is to use micropropagation
techniques.
There are several micropropagation protocols for
V. planifolia, whether by direct organogenesis (Geetha and
Sudheer 2000; Giridhar and Ravishankar 2004; Kalimuthu
et al. 2006; Lee-Espinosa et al. 2008; Minno and Nirmal
 IMPROVED PROPAGATION OF VANILLA
2009; Sreedhar 2009; Zerihun et al. 2009) or indirect organo-
genesis (Janarthanam and Seshadri 2008; Tan et al. 2011).
Most of these protocols used solid medium of a solid medium,
and their efficiency is rather low considering that the multi-
plication rate for most protocols is more than 60 d. Also the
double-phase medium has been used during the multiplication
stage (George and Ravishankar 1997) and liquid medium with
agitation in combination with a solid medium (Lee-Espinosa
et al. 2008) has been applied. Upon comparing different
systems, Sreedhar (2009) did not find significant differences
for vanilla shoot multiplication rates among treatments using
solid medium, complete immersion, and a temporary immer-
sion system (TIS) using Growtek™.
TIS, among other advantages, allows for the semi-
automation of the culture process, facilitates medium renewal,
and also combines aeration and explant immersion with pro-
grammed duration and frequency of immersion, thus reducing
hyperhydricity of the plant and increasing survival during the
acclimatization stage (Etienne and Berthouly 2002; Levin and
Tanny 2004). The RITA® (Récipient à Immersion Temporaire
Automatique) system has been used in the micropropagation
of several species including Malus sp. (Zhu et al. 2005),
Eucalyptus spp. (McAlister et al. 2005), Saccharum spp.
(Mordocco et al. 2009), Fragaria ananassa, Rubus sp.,
Vaccinium sp. (Debnath 2011), Camptotheca acuminata
(Sankar-Thomas and Lieberei 2011), and Quercus robur
(Mallón et al. 2012), among others. The objective of this study
was to establish an efficient protocol for the large-scale
micropropagation of V. planifolia using a TIS, based on
RITA®.
Materials and Methods
Plant material and culture media. Young stem cuttings of
vanilla (V. planifolia) were collected in Papantla, Veracruz,
Mexico. For the in vitro establishment of axillary buds, the
disinfection method by Lee-Espinosa et al. (2008) was follow-
ed. The buds were cultivated in Murashige and Skoog (MS)
medium (Murashige and Skoog 1962) supplemented with
9.55 μM benzyladenine (BA), 35 mg L−1 cysteine hydrochlo-
ride, 30 g L−1 sucrose, 2 g L−1 activated charcoal, and
2.2 g L−1 Gelrite™ (Sigma-Aldrich, St. Louis, MO). After
three subcultures of 30 d each, the cultivated explants were
used for different treatments.
For the multiplication phase cluster shoots below 10 mm in
height were used, cultivated in MS medium supplement-
ed with 9.55 μM BA, 150 mg L −1 ascorbic acid,
35 mg L−1 cysteine hydrochloride, 30 g L−1 sucrose,
100 mL L−1 coconut water, and 2.2 g L−1 Gelrite™ (Sigma-
Aldrich) was used as a gelling agent (where solid medium was
577
used). The pH was adjusted to 5.8 before autoclaving at
1.2 kg cm−2 for 15 min at 120°C.
All cultures were kept at a temperature of 25±2°C, with a
16/8-h light/dark photoperiod, under a light intensity of 40–
50 μmol m−2 s−1 using white-light lamps.
Evaluation of the different culture systems. The explants
(clusters shoots 10×10 mm) were cultivated in different cul-
ture systems for a period of 30 d: solid culture medium, liquid
culture medium with partial immersion (~5 mm of the shoot
base was submerged in liquid medium), and temporary im-
mersion using the automated system, RITA® (VITROPIC,
Saint-Mathieu-de-Tréviers, France). A total of 20 explants
were used per treatment, two explants/Magenta® GA-7 vessels
of 77×77×97 mm (Sigma-Aldrich) for solid culture medium
and partial immersion, with 20 mL of multiplication medium
per vessel. For the TIS, 54 explants were used (six explants/
per 1-L RITA® container, 150×130 mm) with 200 mL of
liquid multiplication medium per container (i.e., 33.3 mL/
explant), with 2 min per immersion every 4 h.
Evaluation of the different immersion frequency. For the TIS,
three types of immersions frequency (2 min per immersion)
were evaluated: every 4, 8, and 12 h were applied, and in all
assays, six explants/per RITA® container were assessed after
30 d. Three containers per immersion frequency were used.
The effect of culture medium volume per explant. To evaluate
the effect of the volume of culture medium per explant for the
TIS, three treatments were tested: 50.0, 33.3, and 25.0 mL per
explant (four, six, and eight explants/per RITA® container,
respectively). In all cases, the same multiplication medium
and an immersion frequency of 2 min every 4 h were used.
Three containers per treatment were used.
For all treatments, after 30-d culture, the number of shoots
per explant was counted and the length, diameter, and number
of leaves on the shoots obtained were measured for each
treatment.
Rooting and acclimatization. Shoots longer than 2 cm were
transferred to RITA® containers with half-strength MS medi-
um supplemented with 0.44 μM NAA and 15 g L−1 sucrose.
The explants were immersed for 2 min every 4 h. Nine
containers were used (ten shoots/per RITA®). Percent rooting
was determined after 45-d culture. Rooted seedlings that were
5- to 10-cm long were transplanted in 50×30×7 cm trays with
a 1:1 mixture of peat moss (Premier, Rivière-du-Loup,
Canada) and agrolita ® (Agrolita, Tlalnepantla de Baz,
Mexico) as substrate and transferred to a greenhouse. Plants
were watered three times a wk and foliar fertilizer
(Nitrofoska® foliar PS, COMPO, Zapopan, Mexico) was ap-
plied weekly. Once the plants had grown to a height greater
than 12 cm, they were transferred to individual pots (20×
578 RAMOS-CASTELLÁ ET AL.

5 cm) containing a mixture (1:1:1) of compost, agrolita®, and
peat moss and watered twice weekly.
Statistical analyses. For all trials, the experimental design was
completely randomized with three replicates. The data for the
number of shoots obtained were subjected to analysis of
variance (ANOVA) using the statistical program SPSS
(Version 11.5 for Windows Inc., Chicago, IL). To detect
differences among treatments, Tukey’s test was used
(P<.05). The variables, length, diameter, and number of
leaves, were analyzed using generalized linear model (GLM)
with the program R 2.15.2. To determine differences between
the levels of treatments, the contrast routine was used.
Results and Discussion
Evaluation of the different culture systems. To evaluate the
three culture systems, solid media, partial immersion with
liquid culture, and the automated temporary immersion sys-
tem (TIS) RITA®, four vegetative growth parameters of
V. planifolia were measured after 30 d (Table 1). The results
revealed significant differences among the three culture sys-
tems evaluated, with the best multiplication rate achieved with
TIS (14.27±0.89 shoots per explant), followed by the partial
immersion system (8.64±0.51 shoots per explant), and solid
medium (5.80±0.69 shoots per explant; Fig. 1a–c). The shoot
length also differed significantly between TIS (13.50 ±
0.88 mm) and solid medium (10.47±1.01 mm), but no signif-
icant differences were observed for TIS and the partial immer-
sion system (12.67±1.2). Furthermore, there were no differ-
ences detected in the number of leaves or shoot diameter
among treatments. Therefore, the greatest impact of the three
culture systems was a substantial increase in the multiplication
rate of V. planifolia using a liquid medium system, compared
with that of the solid medium. This increase was the greatest
when the TIS was used.
Sreedhar (2009) found no differences in the multiplication
rate for V. planifolia among solid medium, complete immer-
sion, and TIS with Growtek™ system. This author obtained
2.46 shoots/explant and doubled the shoot elongation and the
biomass with the TIS over 5 wk. This may be because the
author used explants that had been cultured for 10 yr, and the
shoots’ tips were not removed to allow for the formation of
new shoots.
Our results also concur with those obtained in other spe-
cies. Hempfling and Preil (2005) found that the shoot multi-
plication rate in Phalaenopsis spp. cultivated using a tempo-
rary immersion bioreactor (TIB) increased around fivefold
over that obtained using solid medium. Yan et al.
(2010) obtained a greater shoot multiplication in monk
fruit (Siraitia grosvenorii) using the Plantima® system
than when using solid or liquid media. Similarly, for
banana (Musa spp.), Farahani and Majd (2012) achieved
double the shoot formation rate relative to that obtained using
solid or liquid media. Arencibia et al. (2013) also doubled
the shoot multiplication rate in raspberry (Rubus spp.)
using a TIB.
Determination of immersion frequency. We also evaluated the
effect of different immersion frequencies (2-min immersions
every 4, 8, and 12 h) using the RITA® system. The only
differences observed were in shoot length and vigor, and there
were no significant differences in the number of new shoots
formed, shoot diameter, or the number of leaves (Table 1).

Table 1. Effect of the culture system, immersion frequency, and volume of culture medium per explant on multiplication rate, shoot height, diameter,
and number of leaves in V. planifolia

Micropropagation system Multiplication ratez X ± S.E. Shoot lengthy X ± S.E. (mm) Diametery X ± S.E. (mm) No. of leavesy X ± S.E.

Solid culture medium 5.80±0.69 c 10.47±1.01 b 5.07±0.51 a 1.20±0.10 a

Partial immersion 8.64±0.51 b 12.67±1.20 ab 4.80±0.29 a 1.73±0.17 a
Temporary immersion 14.27±0.89 a 13.50±0.88 a 4.83±0.23 a 1.67±0.16 a
Immersion frequencies in RITA® with 2-min immersion
Frequency every 4 h 14.27±0.89 a 13.50±0.88 a 4.83±0.23 a 1.67±0.16 a
Frequency every 8 h 14.00±0.61 a 13.53±1.46 a 4.53±0.30 a 2.40±0.15 a
Frequency every 12 h 14.90±0.59 a 8.67±0.78 b 4.47±0.32 a 1.80±0. 14 a
Volume of culture medium per explant (mL)
25.0 17.54±1.14 a 13.30±1.01 a 4.40±0.31 a 1.80±0.13 a
33.3 14.27±0.89 ab 13.50±0.88 a 4.83±0.23 a 1.67±0.16 a
50.0 13.45±1.08 b 13.10±0.57a 4.70±0.40 a 1.90±0.28 a

For multiplication rate, means with different letters per column represent significant differences between treatments (Tukey P=.05). For shoot height,
diameter, and number of leaves, means with different letters per column represent significant differences between treatments detected using the contrast
routine.
 IMPROVED PROPAGATION OF VANILLA 579

Figure 1. Micropropagation of
Vanilla planifolia Jacks. ex
Andrews multiple shoots
proliferating after 30-d culture. a,
Solid culture medium. b, Partial
immersion in liquid medium. c,
Temporary immersion with 2-min
immersions every 4 h. d,
Immersion frequency every 4 h. e,
Immersion frequency every 8 h. f,
Immersion frequency every 12 h.

Shorter shoots were observed using the 12-h immersion fre- Volume of culture medium per explant. Our experiments also
quency (8.7±0.78 mm), compared to 4- and 8-h (~13.5 mm determined that a smaller volume (25 mL) of culture medium
for both frequencies). Differences were observed in the ap- per explant significantly increased the multiplication rate, to
pearance of the leaves of shoots formed in the different fre- 17.54±1.14 shoots per explant in comparison with volumes of
quencies tested. All shoots of the 4-h frequency were greener 33.3 and 50 mL, which produced 14.27±0.89 and 13.45±1.08
and regular leaf shape and size (Fig. 1d). In the 8-h shoots per explant, respectively. There were, however, no
frequency, callus formation in leaves was observed in
approx. 40% of the shoots formed (Fig. 1e). While for
the 12-h frequency, all the leaves were whitish in color
and thickened by callus tissue (Fig. 1f). We conclude
the optimal results were achieved using a system with 2-min
immersions every 4 h for the micropropagation of
V. planifolia. As the immersion frequency decreased, the
plants obtained were less vigorous, suggesting that
V. planifolia is a nutrient-demanding species.
In contrast to our results, Sreedhar (2009) obtained a max-
imum of 2.42 vanilla shoots per explant with 30-min immer-
sions every 8 h using the Growtek™ system. Furthermore,
hyperhydricity was observed in the plantlets generated with
45-min immersions every 8 h. Our results showed that vanilla
does not require prolonged immersion times, but responds
best to shorter times (2 min) and more frequent immersions
(every 4 h). This may prevent hyperhydricity and callus
growth on plantlets and result in production of vigorous
plants. In the micropropagation of banana (Musa spp.) using
a TIB, Roels et al. (2005) found no significant differences in
shoot production per explant with 4-min immersions every 3,
5, and 7 h, but in the latter treatment, shoots were longer. In
habanero chili (Capsicum chinense), Bello-Bello et al. (2010)
increased the multiplication rate using the modular temporary Figure 2. In vitro rooting and acclimatization of V. planifolia. a, Rooting
immersion bioreactor (BioMINT™) with 2-min immersions in RITA® after 45-d culture with 2-min immersions every 4 h. b, Plantlets
every 8 h. in the process of acclimatization in the greenhouse after 30-d acclimation.
580 RAMOS-CASTELLÁ ET AL.
significant differences in the length, diameter, or number of
leaves produced per shoot among the medium volumes eval-
uated (Table 1).
In banana (Musa spp.), Roels et al. (2005) found 30 mL of
medium per explant was the best volume of medium for the
maximum multiplication rate. Jin et al. (2013) in grape root-
stock (Vitis vinifera) using the air lift bioreactor doubled the
plantlet biomass using 40, 30, and 76 mL of medium per
explant compared to 25 mL of medium per explant.
According to Lorenzo et al. (1998), larger culture medium
volumes produced lower multiplication rates in sugarcane
(Saccharum spp.). A possible explanation is that explants
excrete some compounds that promote shoot formation,
which would be diluted when large volumes of culture medi-
um are used.
Rooting and acclimatization. After 45-d culture, rooting of
shoots under the RITA® system was 90%. Once rooted, the
shoots were transferred to the greenhouse for acclimatization.
Plant survival was 90% after 30-d growth under our green-
house conditions (Fig. 2a–b). These results concur with those
obtained by Yan et al. (2010) for S. grosvenorii, who obtained
100% rooting using the Plantima® system with an immersion
frequency of 4 min every 4 h. In Phalaenopsis, Hempfling and
Preil (2005) achieved 88.7% rooting of shoots in the TIB
system with an immersion frequency of 10 min every 6 h.
The propagation protocol for V. planifolia in a double-phas e
medium proposed by George and Ravishankar (1997) and the
culture in solid medium followed by 14 d in liquid medium
with agitation (Lee-Espinosa et al. 2008) produced, at 45 d, a
total of seven and 18.5 shoots per explant, respectively. Over a
similar time period, Giridhar et al. (2001) obtained a mean of
5.6 shoots per explant with solid medium. Later, these authors
(Giridhar and Ravishankar 2004) described a protocol that
enabled them to obtain 17 shoots per explant over a period
of 105 d, carrying out three subcultures in the multiplication
stage. Kalimuthu et al. (2006) obtained 9.43 shoots per ex-
plant over 55 d in solid medium. Gonzalez-Arnao et al. (2009)
reported a multiplication rate of 15 shoots per explant in a
period of 4 mo, while Zerihun et al. (2009) obtained 4.17
shoots per explant by 45 d using a solid medium.
In comparison to other micropropagation protocols report-
ed for V. planifolia, the protocol presented here is considerably
more efficient as it allowed for substantial increases in the
number of shoots formed in a shorter period of time (30 d),
with the advantages of avoiding delays in the multiplication
process and not requiring subcultures that may increase pro-
duction costs.
Acknowledgments Authors would like to thank the “Programa de
Mejoramiento del Profesorado (PROMEP)” for the financial support
provided for the project “Biotechnological Basis for the Genetic
Improvement of Vanilla planifolia” within the “Conservation, Manage-
ment and Plant Breeding” network.
References
Arencibia AD, Vergara C, Quiroz K, Carrasco B, García-Gonzales R
(2013) Establishment of photomixotrophic cultures for raspberry
micropropagation in Temporary Immersion Bioreactors (TIBs). Sci
Hortic 160:49–53
Bello-Bello JJ, Canto-Flick A, Balam-Uc E, Gómez-Uc E, Robert ML,
Iglesias-Andreu LG, Santana-Buzzy N (2010) Improvement of
in vitro proliferation and elongation of habanero pepper shoots
(Capsicum chinense Jacq.) by temporary immersion. HortSci 45:
1093–1098
Bory S, Lubinsky P, Risterucci AM, Noyer JL, Grisoni M, Duval MF,
Besse P (2008) Patterns of introduction and diversification of Vanilla
planifolia (Orchidaceae) in Reunion Island (Indian Ocean). Am J
Bot 95:805–815
Debnath S (2011) Bioreactors and molecular analysis in berry crop
micropropagation. A review. Can J Plant Sci 91:147–157
Etienne H, Berthouly M (2002) Temporary immersion systems in plant
micropropagation. Plant Cell Tiss Organ Cult 69:215–231
Farahani F, Majd A (2012) Comparison of liquid culture methods and
effect of temporary immersion bioreactor on growth and multipli-
cation of banana (Musa cv. Dwarf Cavendish). Afr J Biotechnol 11:
8302–8308
Geetha S, Sudheer A (2000) In vitro propagation of Vanilla planifolia, a
tropical orchid. Curr Sci 79:885–889
George PS, Ravishankar GA (1997) In vitro multiplication of Vanilla
planifolia using axillary bud explants. Plant Cell Rep 16:490–494
Giridhar P, Obul B, Ravishankar GA (2001) Silver nitrate influences
in vitro shoot multiplication and root formation in Vanilla planifolia
Andr. Curr Sci 81:1166–1170
Giridhar P, Ravishankar GA (2004) Efficient micropropagation of Vanilla
planifolia Andr. under influence of thidiazuron, zeatin and coconut
milk. Indian J Biotechnol 3:113–118
Gonzalez-Arnao MT, Lazaro-Vallejo CE, Engelmann F, Gamez-Pastrana
R, Martínez-Ocampo YM, Pastelin-Solano MC, Díaz-Ramos C
(2009) Multiplication and cryopreservation of vanilla (Vanilla
planifolia Andrews). In Vitro Cell Dev Biol–Plant 45:574–582
Greule M, Tumino L, Kronewald T, Hener U, Schleucher J, Mosandl A,
Keppler F (2010) Improved rapid authentication of vanillin using
δ13C and δ2 H values. Eur Food Res Technol 231:933–941
Hempfling T, Preil W (2005) Application of a temporary immersion
system in mass propagation on Phalaenopsis. In: Hvoslef-Eide
AK, Preil W (eds) Liquid culture systems for in vitro plant propa-
gation. Springer, Dordrecht Netherlands, pp 231–242
Hernández J (2011) Paquete tecnológico Vainilla (Vanilla planifolia
Jackson). Establecimiento y mantenimiento. Programa Estratégico
para el desarrollo Rural Sustentable de la Región Sur-Sureste:
Trópico Húmedo. INIFAP. Tlapacoyan (in Spanish)
Janarthanam B, Sheshadri S (2008) Plantlet regeneration from leaf de-
rived callus of Vanilla planifolia Andrews. In Vitro Cell Dev Biol–
Plant 44:84–89
Jin MY, Piao XC, Xiu JR, Park SY, Lian ML (2013) Micropropagation
using a bioreactor system and subsequent acclimatization of grape
rootstock ′5BB′. Sci Hortic 164:35–40
Kalimuthu K, Senthilkumar R, Murugalatha N (2006) Regeneration and
mass multiplication of Vanilla planifolia Andrews. A tropical orchid.
Curr Sci 9:1401–1403
Lee-Espinosa HE, Murguía-González J, García-Rosas B, Córdova-
Contreras AL, Laguna-Cerda A, Mijangos-Cortés JO, Barahona-
Pérez LF, Iglesias-Andreu LG, Santana-Buzzy N (2008) In vitro
 IMPROVED PROPAGATION OF VANILLA
clonal propagation of vanilla (Vanilla planifolia ‘Andrews’). HortSci
43:454–458
Levin R, Tanny G (2004) Bioreactors as a low cost option for tissue
culture. In: Low cost options for tissue culture technology in devel-
oping countries. Conference proceedings. International Atomic
Energy Agency, Vienna Austria, pp 47–54
Lorenzo JC, González BL, Escalona M, Teisson C, Espinosa P, Borroto C
(1998) Sugarcane shoot formation in an improved temporary im-
mersion system. Plant Cell Tiss Organ Cult 54:197–200
Mallón R, Covelo P, Vieitez A (2012) Improving secondary embryogen-
esis in Quercus robur: application of temporary immersion of mass
propagation. Trees 26:731–741
McAlister B, Finnie J, Watt M, Blakeway F (2005) Use of the temporary
immersion bioreactor system (RITA) for production of commercial
Eucalyptus clones in Mondi Forests (SA). Plant Cell Tiss Organ Cult
81:347–358
Minno D, Nirmal K (2009) Micropropagation and in vitro conservation of
vanilla (Vanilla planifolia Andrews). In: Mohan J, Praveen K (eds)
Protocols for in vitro cultures and secondary metabolite analysis of
aromatic and medicinal plants, Methods in Molecular Biology.
Humana, New York, pp 129–138
Mordocco AM, Brumbley JA, Lakshmanan P (2009) Development of a
temporary immersion system (RITA®) for mass production of sug-
arcane (Saccharum spp. interspecific hybrids). In Vitro Cell Dev
Biol–Plant 45:450–457
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Pinaria AG, Liew EC, Burgess LW (2010) Fusarium species associated
with vanilla stem rot in Indonesia. Australas Plant Path 39:176–183
SEMARNAT (Secretaría de Medio Ambiente y Recursos Naturales)
(2002) Norma Oficial Mexicana (NOM-059-ECOL-2001) de
Protección especial de especies nativas de México de Flora y
Fauna silvestres. Diario Oficial de la Federación, marzo 6, pp 2–
56. (in Spanish)
Roels S, Escalona M, Cejas I, Noceda C, Rodriguez R, Canal MJ,
Sandoval J, Debergh P (2005) Optimization of plantain (Musa
View publication stats
581
AAB) micropropagation by temporary immersion system. Plant
Cell Tiss Organ Cult 82:57–66
Salazar-Rojas VM, Herrera-Cabrera BE, Delgado-Alvarado A, Soto-
Hernández M, Castillo-González F, Cobos-Peralta M (2012)
Chemotypical variation in Vanilla planifolia Jack. (Orchidaceae)
from the Puebla-Veracruz Totonacapan region. Genet Resour Crop
Evol 59:875–887
Sankar-Thomas YD, Lieberei R (2011) Camptothecin accumulation in
various organ cultures of Camptotheca acuminata Decne grown in
different culture systems. Plant Cell Tiss Organ Cult 106:445–454
Soto Arenas MA (1999) Filogeografía y recursos genéticos de las
vainillas de México. Instituto Chinoin AC. SNIB-CONABIO
proyecto No. J101. México DF (in Spanish)
Soto Arenas MA, Cribb P (2010) A new infrageneric classification and
synopsis of the genus Vanilla Plum. ex Mill. (Orchidaceae:
Vanillinae). Lakesteriana 9:355–398
Sreedhar RV (2009) Novel approaches for molecular analyses,
micropropagation and curing of vanilla (Vanilla planifolia). PhD
thesis. University of Mysore, New Delhi, India
Tan BC, Chin CF, Alderson P (2011) Optimisation of plantlet regenera-
tion from leaf and nodal derived callus of Vanilla planifolia
Andrews. Plant Cell Tiss Organ Cult 105:457–463
Torres-González MJ, Aguirre-Medina JF, Iracheta-Donjuan L (2011)
Germinación de semillas y obtención de plántulas de Vanilla
planifolia Andrews en condiciones in vitro. Agroproductividad 4:
3–8 (in Spanish)
Yan H, Liang C, Li Y (2010) Improved growth and quality of Siraitia
grosvenorii plantlets using a temporary immersion system. Plant
Cell Tiss Organ Cult 103:131–135
Zerihun A, Ayelign M, Alemayehu T, Wondyfraw T (2009) Efficient
in vitro multiplication protocol for Vanilla planifolia using nodal
explants in Ethiopia. Afr J Biotechnol 8:6817–6821
Zhu LH, Li XY, Welander M (2005) Optimization of growing
conditions for the apple rootstock M26 grown in RITA
containers using temporary immersion principle. Plant Cell Tiss
Organ Cult 81:313–318