American Journal of Plant Sciences, 2013, 4, 1685-1692
http://dx.doi.org/10.4236/ajps.2013.49205 Published Online September 2013 (http://www.scirp.org/journal/ajps)
A Simple and Efficient Protocol for the Mass Propagation
of Vanilla planifolia
Ab Rahman Zuraida1*, Kamarulzaman Hassan Fatin Liyana Izzati1, Othman Ayu Nazreena1,
Wan Sem bok W an Z ali ha2, Che Mohd Zain Che Radziah3, Zainal Zamri3, Subramaniam Sreeramanan4
1Biotechnology Research Centre, MARDI Headquarters, MARDI HQ, Persiaran MARDI-UPM, Serdang Selangor, Malaysia;
2Department of Agrotechnology, Faculty of Agrotechnology and Food Science, Universiti Malaysia Terengganu, Kuala Terengganu,
Malaysia; 3Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia; 4School of Biological Sciences,
Universiti Sains Malaysia (USM), Minden Heights, Malaysia.
Received June 12th, 2013; revised July 13th, 2013; accepted July 31st, 2013
Copyright © 2013 Ab Rahman Zuraida et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The present study describes a direct shoot regeneration-based micropropagation procedure for Vanilla planifolia. Two
types of explant (i.e. shoot apex and stem nodal segment) were screened for their shoot induction potential following a
three-month treatment with 6-benzylaminopurine (BAP) and
 -Naphthaleneacetic acid (NAA). Results indicated that
the shoot apices were poor candidates for shoot induction whereas the stem nodal segments showed potential for shoot
initiation at a rate of up to 6 shoots/explant. Stem nodal segments were the most responsive as shoots formed (55 shoots)
directly following treatments with 1 mg/L BAP at half strength MS medium after the third subculture. In addition, more
shoots were produced on solid medium treatments compared to the liquid medium treatments in two strengths of me-
diums tested. Regenerated plantlets derived from the 1 mg/L of BAP treatment were induced to root following a one
month culture in growth regulator-free MS medium. There was 90% survival rate of the rooted plantlets after acclima-
tization in the greenhouse. The findings in the present study would be helpful for large-scale mass propagation of Va-
nilla planifolia using this simple and efficient protocol.
Keywords: Vanilla planifolia; Mass Propagation; Growth Regulator; In Vitro; BAP; NAA; IBA
1. Introduction
Vanilla planifolia, a species of vanilla orchid, is grown
for its fruit which yields the vanilla flavour used in foods
and beverages [1]. Vanilla is a flavouring and aromatiz-
ing vanillin produced naturally, and is considered to be
better than its synthetic substitute. The seeds of the va-
nilla pod are used to flavour ice creams, liquor, soft
drinks and candies. Vanilla is also used in the production
of pharmaceuticals, cosmetics, tobacco and handicrafts.
According to the International Trade Centre, only 30% of
the vanilla world market comprises pure vanilla extract
while the remaining consists of synthetic vanilla extract,
which is produced from chemical components. Currently,
the pure vanilla extract world market is dominated by
Madagascar and Indonesia. During the past decade, the
increase in health awareness and preference for natural
products have strengthened the demand of vanilla beans
[2].
Generally, Vanilla is propagated by the conventional
method, i.e. using stem-cuttings. However, this method
of propagation is inefficient, time-consuming and un-
economical [3-5]. In order to meet the demand for pro-
pagules, rapid micropropagation of this plant is essential.
Micropropagation is now the basis of a large commercial
plant propagation industry involving hundreds of labora-
tories around the world. The technology is used to meet
market demand for vanilla, and to overcome difficulties
in alternative methods of propagation. In vitro multipli-
cation of V. planifolia has been reported using the culture
of callus masses [6,7], protocorms, root tips [8] and stem
nodes [9]. Even though few reports are available on in
vitro propagation of vanilla, most of the protocols are
complicated and utilise coconut water [3,10,11]. How-
ever, as coconut water is currently in demand in the mar-
ket, it has become too expensive to use in the propaga-
tion of vanilla in Malaysia. Moreover, there are very li-
*Corresponding author.

A Simple and Efficient Protocol for the Mass Propagation of Vanilla planifolia 1686
mited reports on in vitro propagation protocols of V.
planifolia in Malaysia. Thus, the aim of this research is
to establish a simple and efficient micropropagation pro-
tocol with further development (third subculture) for
plant tissue culture of vanilla for large scale production.
2. Materials and Methods
2.1. Plant Material and Aseptic Culture
The plant, V. planifolia, was grown in a pot for 6 months,
and maintained in a glass house for a few weeks prior to
explant excision and establishment in vitro. Shoot apices
and stem nodal segments were used to initiate shoot cul-
ture. These explants were cut into pieces of about 2 - 3
cm and kept in a conical flask. They were then washed in
a detergent (Teepol) solution for 20 min. before being
rinsed in distilled water. Next, the explants were trans-
ferred to a laminar air flow chamber, and sterilized with
20% of Clorox® containing several drops of Tween-20
for 5 - 20 min., followed by 5% of Clorox® for another
20 min. The sterilized explants were inoculated onto MS
mediums for different treatments.
2.2. Establishment and Micropropagation of
Shoot Cultures
All sterilized segments of the explants were cultured in-
dividually under aseptic conditions on basal MS medium
containing 0.1 g arginine, 0.1 asparagine, 0.1 g glutamine,
3.0% sucrose, 0.3% gelrite agar for gelling and supple-
mented with five different concentrations of BAP (0, 1.0,
2.0, 3.0, 5.0 or 10.0 mg/L) for shoots initiation. Medium
sterilization was performed by autoclaving at 121˚C for
20 min. pH was adjusted to 5.7 - 5.8 before adding agar.
Explants were inoculated in a 150 ml flask containing 40
mL of the desired medium. The cultures were incubated
in a plant growth room maintained at a temperature of
25˚C ± 1˚C and with 16 h photoperiod by cool-white
fluorescent lamps (1000 - 2000 lux). Observations were
recorded at weekly intervals. Results were expressed as
the number of shoots per explant and length of the shoots
after 45 days of culture.
The increments in shoot multiplication for mass pro-
pagation in separate experiments were compared. Shoot
initials derived from stem nodal segments were cultured
on half or full strength solid MS mediums supplemented
with different concentrations of BAP (1.0, 3.0 or 5.0 mg/l)
alone or combined with NAA (0.5 or 1.0 mg/l) for shoot
proliferation. The treatments were conducted in two me-
diums, viz. liquid and solid. All the explants were sub-
cultured onto the same fresh medium at 1 month intervals.
Results were expressed as percentage of proliferation and
number of shoots produced after 30 days of first subcul-
ture and third subculture (90 days after first subculture).
The experiment was carried out within 3 - 4 months of
culture.
2.3. Rooting of Shoots and Transplantation of
Plantlets
Shoots (>3.0 cm long) from the best treatment (1 mg/L
BAP and half strength MS medium) were cultured on
MS medium supplemented with either IBA (0.5 and 1.0
mg/L), or NAA (0.5 and 1.0 mg/L) or a combination of
both. The number of shoots producing roots, as well as
the number and length of the induced roots were re-
corded after 30 days. In vitro raised plantlets were re-
moved from the culture medium and roots were washed
under running tap water to remove the agar. Regenerated
shoots with well-developed roots were transferred to
plastic pots containing hardening medium and a top soil-
compost mixture (2:1). They were maintained at about
70% relative humidity in the greenhouse. The plantlets
were hardened for 60 days so that they would be well
established. Observations were recorded with respect to
the survival rate of plantlets rooted in the acclimatization
condition.
2.4. Statistic Analysis
All statistical analyses were performed using SPSS soft-
ware. The experiment followed a completely randomized
design, using 15 flasks for each treatment. Each flask
contained two samples and all experiments were repeated
thrice. The means and standard errors (indicated as ±
values) were calculated for the treatment responses.
3. Results and Discussion
3.1. Shoot Initiation and Micropropagation
A simple and effective protocol has been developed for
the in vitro micropropagation of Vanilla planifolia. Dif-
ferent types of explants (shoot apex and stem nodal seg-
ment) were cultured on MS media supplemented with
different concentrations of BAP to evaluate their effect
on shoot production at the initiation stage. Explants
grown on BAP-containing media resulted in varying suc-
cess in shoot initiation depending on the type of explants
cultured. The findings in this study indicated that ex-
plants from stem nodal segments performed better in pro-
ducing multiple shoots compared to those of the shoot
apices (Table 1 and Figures 1(a)-(c). Similar results
were reported by Malek et al. [12] in their studies on
pointed gourd. They observed that the nodal segments
showed a higher percentage of shoot induction (98.00%),
shoot number per explant (5.50) and shoot length per ex-
plant (6.55 cm) compared to shoot tip explants. Similarly,
according to Hutchinson [13], nodal segments of apple
roved to be good explants for micropropagation. These p

A Simple and Efficient Protocol for the Mass Propagation of Vanilla planifolia 1687
Table 1. Effects of type of explants and BAP on the numbe r of shoots/explants and shoot length after a 40-day culture.
Type of explants BAP (mg/L) Number of shoots per explant Shoot length (cm)
0 2.46 ± 0.12 2.2 ± 0.05
1.0 6.06 ± 0.19 4.5 ± 0.14
2.0 3.45 ± 0.13 3.4 ± 0.45
3.0 3.31 ± 0.15 3.5 ± 0.23
5.0 2.56 ± 0.09 2.8 ± 0.12
Stem nodal segment
10.0 1.45 ± 0.05 1.9 ± 0.11
1.0 1.0 ± 0.02 3.1 ± 0.23
2.0 1.56 ± 0.13 4.1 ± 0.34
3.0 2.10 ± 0.34 3.3 ± 0.23
5.0 1.60 ± 0.45 2.6 ± 0.14
Shoot apex
10.0 0.89 ± 0.05 0.9 ± 0.07
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 1. Micropropagation of Vanilla planifolia on MS medium. Shoot formation from shoot apices (a)-(b), and stem nodal
segments (c). Propagation of shoots (d)-(e). Shoots cultured in liquid medium (f). Root initiation and elongation on basal me-
ium (g)-(h). In vitro plant acclimatization, robust growth in the greenhouse (i). d

A Simple and Efficient Protocol for the Mass Propagation of Vanilla planifolia 1688
results also were in agreement with the findings of
Zaman et al. [14], which demonstrated the effect of BAP
on shoot elongation in nodal segment culture of Verbena
spp. It was also observed that MS mediums without any
hormone were ineffective in shoot regeneration for all
the explants.
The stem nodal segment of V. planifolia that was cul-
tured on a medium containing 1 mg/L BAP showed the
highest number of shoots per explant (6.06 shoots), and
maximum shoot length (4.5 cm). Treatments containing
BAP with more than 1 mg/L showed a smaller number of
shoots as well as shorter shoot length. The results clearly
indicated that shoot proliferation was higher at lower
concentrations of BAP, and gradually decreased when
BAP concentration was increased. According to Soledad
Diaz et al. [15], the suitability of growth regulators de-
pends upon the types of plant tissues or explants used. In
this study, the results were in agreement with those ob-
tained by Tan et al. [11], i.e. the highest number of
shoots per explant (5.03 shoots) with a mean length of
3.9 cm was observed from the medium supplemented
with 1.0 mg/l BAP. According to Firoozabady and Gut-
terson [16], the addition of BAP in MS medium was es-
sential for the regeneration plantlets from shoot apices.
This was reiterated by Kalimuthu et al. [3] who stated
that a combination of coconut water (150 ml/L) and BAP
(1 mg/L) was the most effective in shoot proliferation of
vanilla from shoot tip and nodal explants.
Shoots from the stem nodal segments of vanilla were
cultured on solid or in liquid (0.7% agar) MS medium
containing different concentrations of growth regulators
(BAP and NAA) and varying strengths of MS medium
for a 4-week period to compare the effects of these me-
diums on shoot proliferation. All the treatments showed a
proliferation of shoots ranging from 60% - 100% (Table
2). Generally, the medium that was supplemented only
with 1 mg/L BAP showed the best results for shoot pro-
liferation (100%) when cultured on solid or in liquid me-
diums, either at full or half strength. However, when
BAP treatments were combined with NAA, the result
was a decrease in shoot proliferation in all treatments ex-
cept the treatment with MS liquid medium at half
strength. The shoots that were cultured on MS medium
containing a higher concentration of BAP (3 - 5 mg/L)
showed a low level of development. Although multiple
shoots were produced, the plantlets were stunted after 4
weeks of culture.
In many plants, the steps for micropropagation proto-
col require a series of growth regulators. A balance be-
tween two groups of growth regulators, viz. auxins and
cytokinins, constitutes the most effective group of plant
hormones regulating cell division and elongation as well
as determining morphogenesis [15]. The use of BAP and
NAA in tissue culture media, which are associated with
in vitro morphogenetic events and cytokinins, is the most
promising in shoot formation. As reported in previous
studies, 97% of the explants produced a mean of 9.6
shoots with a mean shoot length of 4.70 cm when cul-
tured in a medium containing coconut water with 1.0
mg/l BAP [11]. Findings by Zuraida et al. [17] showed
that sub-culture of microshoots in a medium with 1 mg/L
BAP produced the highest proliferation of pineapple
plantlets. Furthermore, Alderete et al. [18] reported that
the nodal segments of Mecardonia tenella cultured in a
medium with 1 mg/L BAP showed the best multiplica-
tion rates, with values around 29 shoots per explant. The
positive effect of BAP and NAA in promoting the prolif-
eration of shoots was also detected by Boulay [19], who
indicated that the best shoot proliferation of Sequoia
sempervirens occurred on MS medium with 0.5 - 1.0
mg/L BAP and 0.02 mg/L NAA. As Abebe et al., [20]
had done with vanilla planifolia using a combination of 1
mg/L BAP and 1.5 mg/L KIN, Boulay observed up to 4
shoots from the nodal culture. The number of shoots ge-
nerated from vanilla explants cultured on the same me-
diums (as listed in Table 2) was recorded after the first
and third subculture. The in vitro plantlets required sub-
culture every eight weeks to remain healthy. The result
indicated that the number of multiple shoots was small
during initial or first subculture, but increased after the
third subculture. After 30 days of first subculture and 90
days of third subculture, the explants that were cultured
on a solid medium produced more shoots compared to
those in the liquid medium (Figures 1(d)-(f)). It was ob-
served that the physical state of the solid medium af-
fected shoot multiplication. A higher multiplication rate
was observed in those treated on solid medium, whereas
those continuously cultured in liquid medium resulted in
stunted growth and the leaves became whitish and light
green (Figure 1(f)). These results are in disagreement
with the findings of Tan et al. [11] for vanilla where the
liquid MS medium gave higher multiplication rate com-
pared to the semi-solid medium. Zuraida et al. [17] found
that with the pineapple plant, shoot multiplication in-
creased up to nine-fold in liquid medium when com-
pared to the cultures maintained on solid medium after
the third subculture. According to Hung et al. [21], a li-
quid medium based culture is a more economical method
for mass propagation in a number of plant species.
The explants, when continuously cultured on solid MS
medium containing 1 mg/L BAP with half salt strength
and full salt strength, showed a higher number of shoots,
producing 17 and 11 shoots respectively after the first
sub-culture. A medium containing 1 mg/L BAP concen-
tration with half salt strength evoked the best response
after the third subculture (55 shoots) (Table 2). Incorpo-
ration of more than 1 mg/L BAP in the third subculture
showed improved shoot multiplication but the shoots

A Simple and Efficient Protocol for the Mass Propagation of Vanilla planifolia 1689
Table 2. Effects of plant growth regulator (BAP and NAA), and strength MS medium on shoot proliferation and number of
shoots after being subcultured in solid or liquid MS me dium.
Plant growth regulator (mg/L)
MS Solid
BAP NAA
Percentage of shoot proliferation
(3 weeks)
Number of shoots:
after first sub-culture
Number of shoots:
after third sub-culture
0 - 75 ± 8 5 ± 1.5 8 ± 2.3
1.0 - 100 ± 5 11 ± 2.4 33 ± 4.8
3.0 - 90 ± 5 8 ± 1.5 21 ± 3.5
5.0 - 70 ± 7 7 ± 0.9 17 ± 2.3
1.0 0.5 85 ± 8 12 ± 3.1 21 ± 4.7
3.0 0.5 80 ± 5 8 ± 1.4 17 ± 2.8
5.0 0.5 70 ± 8 8 ± 4.6 16 ± 3.5
1.0 1.0 85 ± 3 14 ± 1.3 17 ± 2.7
3.0 1.0 70 ± 6 8 ± 2.4 17 ± 3.7
Full
salt strength
5.0 1.0 70 ± 7 9 ± 1.9 15 ± 1.2
0 - 75 ± 9 7 ± 1.3 18 ± 1.7
1.0 - 100 ± 4 17 ± 2.4 55 ± 4.5
3.0 - 90 ± 3 13 ± 1.9 31 ± 3.9
5.0 - 70 ± 8 11 ± 3.4 28 ± 2.5
1.0 0.5 85 ± 9 13 ± 2.1 32 ± 6.9
3.0 0.5 70 ± 4 9 ± 2.3 14 ± 3.1
5.0 0.5 65 ± 3 7 ± 1.5 12 ± 1.2
1.0 1.0 85 ± 6 8 ± 1.4 23 ± 2.4
3.0 1.0 70 ± 7 6 ± 1.2 15 ± 2.9
Half
salt strength
5.0 1.0 60 ± 8 6 ± 1.2 12 ± 3.4
MS Liquid
0 - 80 ± 8 5 ± 0.7 7 ± 2.0
1.0 - 100 ± 5 9 ± 1.5 29 ± 3.5
3.0 - 95 ± 9 8 ± 1.2 16 ± 2.1
5.0 - 80 ± 11 3 ± 0.6 11 ± 1.2
1.0 0.5 85 ± 14 6 ± 0.5 18 ± 2.3
3.0 0.5 70 ± 12 7 ± 0.5 17 ± 1.3
5.0 0.5 70 ± 4 7 ± 1.2 13 ± 3.4
1.0 1.0 85 ± 5 8 ± 1.5 13 ± 2.5
3.0 1.0 75 ± 7 5 ± 0.6 14 ± 2.4
Full
salt strength
5.0 1.0 60 ± 8 5 ± 0.9 14 ± 3.1
0 - 100 ± 8 4 ± 0.8 8 ± 1.2
1.0 - 100 ± 9 9 ± 1.4 18 ± 3.5
3.0 - 100 ± 6 8 ± 1.6 17 ± 2.5
5.0 - 95 ± 11 6 ± 2.3 18 ± 3.2
1.0 0.5 95 ± 6 6 ± 2.1 16 ± 2.3
3.0 0.5 95 ± 8 4 ± 0.8 12 ± 2.6
5.0 0.5 80 ± 9 5 ± 0.6 13 ± 1.8
1.0 1.0 100 ± 6 5 ± 0.7 13 ± 1.1
3.0 1.0 100 ± 3 3 ± 1.1 7 ± 2.3
Half
salt strength
5.0 1.0 80 ± 5 2 ± 0.5 6 ± 1.0

A Simple and Efficient Protocol for the Mass Propagation of Vanilla planifolia 1690
remained stunted. A number of regenerated plantlets that
were continuously cultured in liquid medium containing
1 mg/L BAP with half and full strength MS medium pro-
duced 29 shoots and and 18 shoots, respectively for the
third subculture. There was rapid shoot multiplication
when they were cultured on solid medium containing 1
mg/L BAP with half salt strength. Generally, a combina-
tion of cytokinin (BAP) and auxin (NAA) in the medium
would enhance the multiplication of shoots in the culture.
In this study, however, BAP alone proved to be better
than its combination with NAA. Firoozabady and Gut-
terson [16] stated that the addition of BAP in MS me-
dium was essential for the regeneration plantlets from
shoot apices. Ammirato [22] reported that the addition of
cytokinin at moderate concentrations enhanced shoot de-
velopment of Dioscorea spp. Abebe et al. [20], in their
culture of vanilla, observed clearly separated and fully
grown multiple shoots when 1 mg/L BAP alone was used,
whereas no shoot induction was observed in hormone-
free mediums and mediums supplemented with 2 or 3
mg/L of BAP combined with different levels of NAA
(0.5, 0.75, 1.0 and 1.5 mg/L). This observation is in
agreement with the present work.
Based on Table 2, a higher number of shoots were
produced in both strengths of MS mediums tested for the
third subculture. However, in overall treatments at full
strength medium, the number of shoots obtained was less
compared to those in half strength medium. This finding
was supported by Rafia et al. [23] who stated that the
half strength MS with 15 μM BAP developed the highest
number of shoot buds (30 ± 0.6) and was superior to
full-strength MS with the same level of BAP. As Malek
et al. [12] had done with in vitro grown plantlets which
had shoot tip explants cultured in half strength MS media
supplemented with 2.0 mg/l BAP to give the highest
shoot induction percentage (88.00%), there was maxi-
mum number of shoots (4.25) as well as the longest shoot
(4.85 cm). In contrast, Wan Nurul Hidayah et al. [24] re-
ported that the highest number of shoots per explant
(32.93 ± 3.93) was obtained from full strength MS media
supplemented with 0.25 mg/L BAP. Moreover, they stated
that the interaction between hormones and the strength of
media was also highly significant to the number of shoots
produced per explant.
3.2. Rooting of Shoots and Transplantation of
Plantlets
In order to obtain complete plantlets, in vitro shoot buds
were separated and transferred to the rooting mediums.
Half or full strength MS mediums fortified with different
concentrations of auxins (IBA and NAA) were used for
the rooting experiment. Table 3 shows the effect of dif-
ferent concentrations of IBA (0.5 and 1.0 mg/L) and
NAA (0.5 and 1.0 mg/L) on the root formation of vanilla
shoot buds. The shoot buds produced roots in both
Table 3. Effect of auxin on root f o r m ation from regenerat ed shoots of V. planifolia.
MS strength Treatment of media Rooted explants (%) Number of root per explants Length of roots (cm)
MS basal (MSO) 100 ± 5 2.8 ± 0.21 4.52 ± 0.21
MSO + charcoal 80 ± 7 2.7 ± 0.45 4.31 ± 0.23
0.5 IBA 95 ± 5 2.4 ± 0.34 3.14 ± 0.34
1.0 IBA 95 ± 8 1.8 ± 0.11 2.16 ± 0.09
0.5 NAA 75 ± 3 1.9 ± 0.34 2.16 ± 0.12
1.0NAA 80 ± 5 2.5 ± 0.32 2.45 ± 0.15
0.5 IBA + 0.5 NAA 85 ± 7 2.3 ± 0.15 2.34 ± 0.18
Full
strength
1.0 IBA + 1.0 NAA 85 ± 3 2.1 ± 0.39 2.56 ± 0.16
MS basal (MSO) 100 ± 8 2.9 ± 0.12 4.32 ± 0.11
MSO + charcoal 100 ± 5 2.8 ± 0.45 4.11 ± 0.17
0.5 IBA 95 ± 9 2.9 ± 0.09 2.81 ± 0.17
1.0 IBA 95 ± 4 2.5 ± 0.06 2.78 ± 0.12
0.5 NAA 85 ± 6 2.6 ± 0.17 2.89 ± 0.14
1.0 NAA 80 ± 8 2.1 ± 0.06 3.21 ± 0.13
0.5 IBA + 0.5 NAA 85 ± 3 2.2 ± 0.11 2.32 ± 0.15
Half
strength
1.0 IBA + 1.0 NAA 85 ± 9 2.3 ± 0.12 2.16 ± 0.09

A Simple and Efficient Protocol for the Mass Propagation of Vanilla planifolia 1691
strengths of MS mediums tested, ranging from 75% -
100%. Moreover, root induction was strongly stimulated
by a growth regulator-free MS medium (Figures 1(g)-(h).
Results showed that an auxin-free medium gave the high-
est percentage of rooting, number of roots and root length
in both strengths of MS mediums. Among the auxins
tested for root initiation of vanilla, IBA had the greatest
effect on rooting, with the high responses up to 95%, as
compared to NAA with only 80% - 85%. The results also
showed that the medium containing IBA did not produce
very different results, compared to NAA with regard to
the number and length of roots.
In the present study, a growth regulator-free MS me-
dium was found to be more effective in root induction.
This is in agreement with studies carried out on several
plant species, such as A. maximus [25], Phyllanthus spe-
cies [26], Astragalus adsurgens [27] and A. aquilonius
[28]. Abebe et al. [20] observed no difference between
growth regulator-free medium and medium supplemented
with different levels of NAA (0.5 - 1.5 mg/L) for rooting
and elongation. Furthermore, Soledad Diaz et al. [15]
suggested that NAA incorporated into the medium was
not necessary as they found a rooting rate of 90% was
attained when the plantlets were placed in a hormone-
free medium. These observations notwithstanding, it is
known that auxins, especially NAA, have an inducing ef-
fect on rooting under tissue culture conditions [29]. More-
over, according to Majumder et al. [30], the response of
shoots to rooting was very much dependent on the con-
centrations and combination of auxins used. They found
that the combination of 1.0 mg/l IBA + 0.5 mg/l IAA was
the best medium for rooting. The influence of IBA and
IAA for root proliferation has been reported in medicinal
plants [31,32].
The rooted plantlets were acclimatized successfully
when transferred to pots containing a top soil-compost
mixture (2:1). The survival rate was 90% after being kept
for a month in a glasshouse (Figure 1(i)).
4. Conclusion
A simple and efficient protocol for mass propagation of
vanilla was developed by testing various concentrations
of growth regulators and nutrition conditions. The proc-
ess was carried out on in vitro cultures without using
expensive materials such as coconut water. Half strength
MS mediums supplemented with 1 mg/L BAP proved
conclusively more satisfactory and were sufficient for
shoot propagation as well as differentiation at initial
stages. It was also concluded that, compared to shoot
apices, the stem nodal segments showed higher potential
to regenerate. Our investigation also revealed that no
auxin supplementation was necessary for rooting differ-
entiation (100% achieved) in the multiplication of regen-
erated shoots. Hence, it is a cost effective technique for
shoot proliferation of vanilla using nodal segments. This
protocol for rapid micro propagation can be utilized for
large-scale production of Vanilla planifolia.
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