South African Journal of Botany 72 (2006) 191 – 194

www.elsevier.com/locate/sajb

Potential low-cost micropropagation of pineapple (Ananas comosus)

L.V. Be a,*, P.C. Debergh b
a
University of Can Tho, College of Agronomy, Department of Plant Science, 3/2 street, Can Tho City, Viet Nam
b
Gent University, Faculty of Bio-engineering Sciences, Department of Plant Production, Laboratory for Horticulture, Coupure links,
653 - 9000 Gent, Belgium

Received 12 May 2005; accepted 4 July 2005

Abstract

We developed a low cost micropropagation procedure for pineapple (Ananas comosus (L.) Merr). A liquid MS medium, supplemented with
0.8 – 1.0 mg BA l 1 of medium yielded the best multiplication, with about 8 to 9 shoots during a culture interval of 8 weeks. Proliferation and
rooting stages in vitro can be carried out under the natural ambience of a nethouse instead of growth chamber conditions. No statistical
differences were observed between the two environmental conditions for propagation and rooting, and survival after 10 weeks of
acclimatization approximated 100% for the nethouse plants. Nethouse plants were assessed to be about 20% cheaper to produce than growth
chamber plants.
D 2006 SAAB. Published by Elsevier B.V.

Keywords: Low-cost micropropagation; Pineapple

1. Introduction proliferation (stage II) and rooting in vitro (stage III) under
the natural conditions of a nethouse.
Pineapple is a vegetatively propagated crop of major
importance in tropical countries. Propagation in vivo is easy,
but the multiplication rate is low, and ranges from about 11 to 2. Materials and methods
17 plants over a period of 5 months (Lieu et al., 2004).
Moreover, suckers produced by this method are not disease- 2.1. Proliferation stage (stage II)
free, and especially viruses are a problem. In contrast,
micropropagated shoots are relatively expensive. Cultures were initiated from sucker meristems on liquid MS
To reduce costs of micropropagation, scientists have tried to (Murashige and Skoog, 1962) medium supplemented with
develop other procedures, for instance: automated subculturing 0.4 mg BA l 1 (benzyladenine). Shoots, over 2.5 cm and with
(Hartney, 1986), use of natural daylight to promote photoau- 5– 6 leaves, were used for propagation. The explants, with their
totrophic growth (George, 1993), large-scale production using leaves shortened were subcultured in jars on the same MS
a temporary immersion system (Escalona et al., 1998), periodic medium with different hormone supplements: 0, 0.2, 0.4, 0.6,
immersion bioreactor (Firoozabady and Gutterson, 2003), an 0.8, or 1.0 mg BA l 1 or 1.0 mg BA l 1 plus 0.5 mg IBA l 1
alternative for artificial light (Kodym and Zapata-Arias, 1999, (indolebutyric acid). The experiment was designed as a
2001), tubular skylight (Kodym et al., 2001), and natural-light randomized complete block with 4 replicates, 5 jars per replicate,
conditions of a nethouse (Tan, 2004). 2 explants per jar. The jars were of glass [6 cm diameter, 12 cm
The aim of our experiments was to lower the price of high, closed with a plastic cap and wrapped in polyvinylchloride
micropropagated plantlets by carrying out the stages of film. Thirty ml of liquid MS medium were used per container. As
the explants were not submerged in this amount of liquid
medium, the jars were kept stationary. Two locations with
different environmental conditions were used to grow the
* Corresponding author. cultures: a growth chamber [24 T 1 -C under a 12-h photoperiod
E-mail address: Lvbe@ctu.edu.vn (L.V. Be). provided by fluorescent tubes with a photosynthetic active
0254-6299/$ - see front matter D 2006 SAAB. Published by Elsevier B.V.
doi:10.1016/j.sajb.2005.07.002
192 L.V. Be, P.C. Debergh / South African Journal of Botany 72 (2006) 191 – 194

radiation (PAR) of about 30 Amolm 2s 1]; and a nethouse Table 2

(30 T 1 -C at 11:00 and 31 T1 -C at 15:00, under 8 – 10 h daylight Effect of hormonal treatments (HT) and environmental conditions (EC) on
chlorophyll a, chlorophyll b, and carotenoid content of axillary shoots after
with a PAR varying between about 95 – 125 A mol m 2s 1). As 8 weeks in culture
a radiometer was unavailable for recording light measurements,
Categories Chlorophyll Chlorophyll Carotenoid
a lux meter (ANA-325, Tokyo Photo-Electric) was used and the a (Ag g 1)- b (Ag g 1) (Ag g 1)
data converted to A mol m 2s 1 according to George (1993). 1
Hormonal treatments (mg l )
Data were analyzed by a two-way analysis of variance, in 0 BA 436.7 207.6 ab-- 69.3
which shoot number, clump weight, chlorophyll a, chlorophyll 0.2 BA 441.1 218.4 b 67.3
b, and carotenoid content after 8 weeks in culture were the 0.4 BA 474.4 165.6 a 78.8
dependent variables. The independent variables were the 0.6 BA 450.2 190.1 ab 75.9
0.8 BA 431.7 219.1 b 67.9
physical environments for multiplication (growth chamber or
1.0 BA 453.3 339.6 c 67.2
nethouse), as well as the 7 hormonal combinations. The content 1.0 BA + 0.5 IBA 435.1 307.5 c 37.5
of pigments was analyzed according to Wellburn (1994).
Environmental conditions
2.2. Rooting in vitro (stage III) Growth chamber 443.4 170.3 110.9
Nethouse 449.9 280.9 32.1
F test HT ns * ns
Shoots longer than 2.5 cm, obtained from stage II, were F test EC ns * *
used for rooting. Rooting treatments were liquid MS medium F test HT vs. EC * * ns
without plant growth regulators, supplemented with 3% or 4% Coefficient of variance (%) 9 16 23
sucrose, 4 replicates, 5 plastic containers (12 cm diameter, 7 cm - Ag g 1 fresh weight of leaf; --Values followed by the same letter are not
height) per treatment, with 15 shoots each. Data were analyzed significantly different at P  0.05 of Duncan’s test. The significance of the
by a two-way analysis of variance, in which individual weight calculated F values is: ns, not significant; *, significant at P  0.05.
of rooted plantlets, root number, leaf number and height of
plantlets were the dependent variables; the independent 1.0 BA plus 0.5 IBA. Generally, as the BA supply in the medium
variables were the environmental conditions (see proliferation increased, more axillary shoots were produced per inoculum
stage) for rooting, and two concentrations of sucrose. after 8 weeks in culture; the correlation coefficient was
R 2 = 0.86 ( P  0.05). There were statistical differences
3. Results ( P  0.05) amongst the aforementioned groups; addition of
0.5 mg IBA l 1 did not yield more axillary shoots than the
3.1. Multiplication stage higher concentrations of BA alone, but did stimulate shoot-
cluster development significantly, with an average mass of
As shown in Table 1, regeneration of new shoots was 6.5 g (Table 1). The latter produced many small shoots
dependent on the hormonal combination in the culture medium. (average height 0.9 cm). Only when the BA-concentration
The results can be divided into 3 groups (hormonal concentra- exceeded 0.4 mg l 1, did the height of the new shoots reach the
tions in mgl 1): (1) 0 BA; (2) 0.2– 0.6 BA; (3) 0.8 – 1.0 BA, and desired size (> 3 cm), except for the combination of BA and IBA.
Similarly, the weight of the shoot clusters increased with the
Table 1 concentration of BA, but only the treatment with 1 mg BA l 1
Effect of hormonal treatments (HT) and environmental conditions (EC) during was significantly better. Shoot height was significantly larger for
in vitro culture on the development of axillary shoots after 8 weeks in culture
the BA concentrations of 0.8 to 1.0 mg l 1 (over 3 cm high).
Categories Shoot number / Clump Average shoot Shoots shorter than 2 cm after 8 weeks in culture were not
cluster mass (g) height (cm)
1
employed in the experiment because they are not suited for
Hormonal treatments (mg l ) rooting or subculture. The optimal size of a shoot required at
0 BA 1 a- 2.1 ab 2.0 b
0.2 BA 4 b 1.7 a 2.0 b
this stage is dependent on many factors: plant species, and
0.4 BA 5 b 2.2 ab 2.4 b manner production (in liquid or on solid medium) or rooting (in
0.6 BA 5 b 2.4 b 3.4 c vitro or ex vitro). There does not appear to be a standard size of
0.8 BA 8 c 3.1 c 3.7 c shoots in vitro, but from the above studies it was concluded
1.0 BA 9 c 3.8 d 3.6 c that the useful length of shoots should be over 2 cm for
1.0 BA + 0.5 IBA 8 c 6.5 f 0.9 a
acceptable rooting and weaning success. In our experiment, we
Environmental conditions only used shoots of more than 2.5 cm (called useful shoots),
Growth chamber 5 3.1 2.5 both for rooting in vitro and subculture.
Nethouse 6 3.1 2.6 As reflected by Table 1, we concluded that 0.8 and
F test HT * * * 1 mg BA l 1 are optimal for shoot multiplication of pineapple
F test EC ns ns ns
F test HT vs EC1 ns ns ns
as they yield a significantly high number of axillary shoots of a
Coefficient of variance (%) 36 18 26 sufficient length under both environmental conditions. There
-Values followed by the same letter are not significantly different at P  0.05 of were no significant interactions between the BA concentrations
Duncan’s test. The significance of the calculated F values is: ns, not significant; and the different environmental conditions for the parameters
*, significant P  0.05. observed during this stage.
 L.V. Be, P.C. Debergh / South African Journal of Botany 72 (2006) 191 – 194 193

The physical environment during in vitro culture did not

affect the observed parameters (shoot number, shoot-cluster
weight, and shoot height) during the proliferation stage.
Neither did an increase in hormonal concentrations nor
environmental conditions have a significant effect on the
chlorophyll a (chl a) content of the leaves. However, the two
factors, hormone concentration and culture environment,
affected chl b content. The higher BA concentrations resulted
in significantly increased chl b in the leaves: 170 Ag g 1 for
growth chamber and 281 Ag g 1 for nethouse conditions,
which was statistically different ( P  0.05). The carotenoid
content of the cultures under nethouse conditions was
significantly lower ( F test at P  0.05) than under growth
chamber conditions (Table 2).
In general, the results (Table 2) show that nethouse
Fig. 1. Typical clusters of pineapple proliferating on liquid MS medium
conditions did not have a negative effect on chl a, but were supplemented with 1 mg BA l 1 after 8 weeks in culture. (a) under nethouse
positive for chl b and carotenoid content. The shootlets grew and (b) under growth room conditions.
well after 8 weeks in culture (Fig. 1).

3.2. Rooting stage environmental conditions, shoot development was satisfactory;

no statistical differences were observed in either plant weight
All new shoots produced on culture media with (in mg l 1) or root number per plant. Similarly, sucrose content (3 or 4%)
0, 0.2 or 0.4 BA, and 1 BA + 0.5 IBA were almost all rejected, did not affect performance. There was no interaction between
because their lengths did not satisfy rooting requirements environmental conditions and sugar content in terms of shoot
(either too short or too long). We used only shoots approxi- weight (Table 3). Conditions of high radiation in the nethouse
mately 2.5 cm in height from treatments of 0.6 or 1 mg BA l 1. did not inhibit root formation (5 roots per shoot) compared to 4
The morphological parameters of rooted plantlets which roots for growth chamber conditions; this difference was not
reached 2.5 cm are presented in Table 3. Under both statistically significant by the F test ( P  0.05).

Table 3 4. Discussion
Effects of sucrose content and environmental conditions (EC) on rooting of
plantlets in vitro after 4 weeks in culture Genetic variation in pineapple micropropagation has been
Treatments Plantlet Root number/ Leaf number/ Plantlet frequently reported (e.g. Wakasa, 1979; Mathews and Rangan,
weight (g) plantlet plantlet height (cm) 1979). High multiplication rates in conjunction with high levels
Growth chamber, 1.8 4 10 9.1 of hormones in the propagation medium can result in genetic
sucrose 30 g l- 1 instability and the production of ‘‘off-type’’ plants (Sharrock,
Growth chamber, 2.0 4 9 7.9 1992). To avoid this we chose to look for a low BA concentration
sucrose 40 g l- 1
Nethouse, 1.8 5 10 8.0
that could still induce a significantly improved propagation ratio.
sucrose 30 g l- 1 Under the two environmental conditions we obtained about one
Nethouse, 2.1 5 8 7.7 axillary shoot on 0 mg l 1, and 9 on 1.0 mg l 1 of BA from one
sucrose 40 g l- 1 explant during a culture interval of 8 weeks. Explants cultured
on medium with 0.8 –1.0 mg BA l 1 yielded optimal size shoots,
Environmental conditions
Growth chamber 1.9 4 9 8.5
with a height of about 4 cm (Table 1), and small shoots (less than
Nethouse 1.9 5 9 7.9 2 cm) were almost absent. A low concentration of hormones is
preferable for pineapple, because it allows the production of
Sucrose content healthy plants of desirable size in the growth chamber as well as
30 g l- 1 1.8 4 10 8.6 under the natural environment of a nethouse. These pineapple
40 g l- 1 2.1 4 8 7.8
F test EC ns ns ns ns
plantlets are easily rooted in hormone-free medium. The average
F test sucrose ns ns * * survival of plantlets was over 95% under both environmental
content conditions.
F test EC vs sucrose ns ns ns ns Our results and calculations (data not presented in the text)
content show that the cost-price of micropropagated plants under
Coefficient of 10 23 9 6
variance (%)
nethouse conditions are about 20% lower compared to growth
room conditions. The lower cost was almost entirely due to the
The significance of the calculated F values is: ns, not significant; *, significant
at P  0.05.
reduced energy costs for light and air conditioning. Debergh and
These plantlets come from the treatment containing 0.6 – 1 mg BA l 1 during Read (1991) reported that lighting costs account for 65% of the
stage II. total electricity bill, and are one of the highest non-labor costs in
194 L.V. Be, P.C. Debergh / South African Journal of Botany 72 (2006) 191 – 194

a tissue culture laboratory (Dooley, 1991). Compared with other chamber conditions. The procedures we propose may reduce
techniques, for example, the periodic immersion bioreactor (10 costs by up to 20%.
L Nalgene vessels) for propagation in vitro, the cost price of
FCayenne_ pineapple propagules was decreased by 35% Acknowledgments
(Firoozabady and Gutterson, 2003). An earlier study showed
that daylight instead of artificial light for banana propagation We would like to express our gratitude to the Coordinators of
allowed savings on costs for electricity of US$6 m 2 week 1, as the Program for Institutional University Cooperation between
compared to a standard growth room (controlled light intensity the Flemish Inter-University Council and Can Tho University
and temperature regimes) (Kodym et al., 2001). project (VLIR-IUC CTU project) for financial support.
Micropropagated plants produced under outdoor conditions
are not only cheaper, but the process is also environmentally References
friendly. Despite being economical, Kyte (1987) did not
recommend use of natural energy because of its fluctuations Debergh, P.C., Read, P.E., 1991. Micropropagation. In: Debergh, P.C.,
and difficulties to manage. However, southern Viet Nam is Zimmerman, R.H. (Eds.), Micropropagation Technology and Application.
Kluwer Academic Pulishers, Dordrecht, pp. 1 – 13.
located in a tropical region, with full sunlight over the whole year Dooley, J.H., 1991. Influence of lighting spectra on plant tissue culture.
and no large variations in temperature, so that natural light Presented at an ASAE meeting, Chicago, Illinois. In: Kodym, A., Zapata-
conditions can be used for micropropagation at lower cost. A Arias, F.J. (Eds.), Natural Light as an Alternative Light Source for the In
major remaining problem of our present protocol is reduction of Vitro Culture of Bananas (Musa acuminata cv, ’Grande Naine), Plant Cell,
the contamination ratio (3% infected under growth chamber and Tissue and Organ Culture, vol. 55, pp. 141 – 145.
Escalona, M., Lorenzo, J.C., González, B., Daquinta, M., Fundora, Z.,
16% under outdoor conditions). If this problem can be solved the Borroto, C.G., Espinosa, P., Espinosa, D., Arias, E.M., Aspiolea, E., de
cost-price of micropropagated plantlets might be even lower. Bioplantas, C., 1998. New system for in vitro propagation of pineapple
A cost-price between 162 and 132 VND/shoot (1 US (Ananas comosus (L.) Merr). In: Bartholomew, D. (Ed.), Pineapple News.
dollar = 15,000 VND) is acceptable for farmers in the Mekong Issue No. 5, April, 1998. Newsletter of the Pineapple Working Group,
International Society for Horticultural Science. http://www.tpss.hawaii.
Delta, because it is comparable with the price of plants
edu/PineappleNews/News 5/pnews5.htm.
produced in traditional ways. The cost of a plantlet is Firoozabady, E., Gutterson, N., 2003. Cost-effective in vitro propagation
dependent on the cultivar and its size. Recently, in Viet Nam, methods for pineapple. Plant Cell Reports 21, 844 – 850.
many commercial companies have sold FCayenne_ shoots to George, E.F., 1993. Plant Propagation by Tissue Culture (Part 1, 2), 2nd edn.
farmers at 500 VND/plant. This is not cheap because the Exegetics Ltd., England, pp. 582 – 794.
propagules are imported from Thailand. The cost of shoots Hartney, V.J., 1986. Commercial aspects of micropropagating eucalyptus. In:
George, E.F. (Ed.), Plant Propagation by Tissue Culture (Part 2), 2nd edn.
produced by splitting stem at the Southern Fruit Research Exegetics Ltd., England, p. 795.
Institute of Viet Nam is cheaper (287 – 297 VND/plant), but the Kodym, A., Zapata-Arias, F.J., 1999. Natural light as an alternative light source
availability of shoots is often limited because of a low for the in vitro culture of bananas (Musa acuminata cv, ’Grande Naine).
multiplication ratio (average being 11 over a period of 6 10 Plant Cell, Tissue and Organ Culture 55, 141 – 145.
months) (Lieu et al., 2004). After harvesting, mother plants of Kodym, A., Zapata-Arias, F.J., 2001. Low-cost alternatives for the micro-
propagation of bananas. Plant Cell, Tissue and Organ Culture 66, 67 – 71.
the FQueen_ pineapple cultivar give many suckers which are Kodym, A., Hollenthoner, S., Zapata-Arias, F.J., 2001. Cost reduction in the
collected by farmers, thus forming the cheapest source of micropropagation of bananas by using tubular skylights as source for
propagules. This procedure cannot be applied to FCayenne_ natural lighting. In vitro Cell Devision Biol-Plant 37, 237 – 242.
because the latter cultivar gives only a few suckers per plant. Kyte, L., 1987. Plants from test tubes: an introduction to micropropagation. In:
Firoozabady, E., Gutterson, N. (Eds.), Cost-Effective In Vitro Propagation
Moreover, the comparison is not always reliable because of
Methods for Pineapple, Plant Cell, vol. 21, pp. 844 – 850.
inappropriate price calculations. The production of sufficient Lieu, P.N., Tinh, N.N., Vui, P.V., Khai, T.P., Teisson, C., 2004. Study
amounts of propagation material by suckers requires a large of multiplication rate of conventional propagation in vivo of Cayenne
land area. For the reasons mentioned, natural conditions for (A. comosus L.). Impact de 10 années de coopération française sur
micropropagation are promising to reduce costs, and open up l’amélioration des productions fruitières au Vietnam - SOFRI.
possibilities for application in Viet Nam. Conférence, Mai 2004, pp. 1 – 11.
Mathews, V.H., Rangan, T.S., 1979. Multiple plantlets in lateral bud and leaf
explants in vitro cultures of pineapple. Scientia Horticulturae 11, 319 – 328.
5. Conclusions Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio
assays with tobacco tissue cultures. Physiologia Plantarum 15, 473 – 497.
With a low concentration of BA (0.8 – 1 m l 1) in liquid MS Sharrock, S., 1992. In vitro propagation of pineapple (Ananas comosus) and
medium we can achieve a micropropagation ratio of 8 to 9 plantain (Musa spp.) technical and economic considerations. Proceedings of
the Tenth Annual Conference, Grand Barbados Beach Resort, Barbados
shoots per explant with a culture interval of 8 weeks. Shoots West Indies, Nov. 11 – 13, 1992. BSTA, pp. 61 – 69.
proliferating on these media have an approximate height of 3 Tan, V.T., 2004. In vitro propagation of vetiver grass (Vetiveria zizanioides L.).
cm. Lower or higher BA concentrations yielded shoots of MSc. thesis of College of Agronomy, Cantho Univ. Vietnam (Vietnamese
lesser quality. language), pp 56 – 78.
Proliferation and rooting stages in the micropropagation of Wakasa, K., 1979. Variation in the plants differentiated from the tissue culture
of pineapple. Japanese Journal of Breeding 29, 13 – 22.
pineapple can easily be carried out under the natural ambience Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b, as
of a nethouse. The growth of plantlets produced under these well as total carotenoid, using various solvent with spectrophotometers of
conditions have a similar quality to those raised under growth different resolution. Journal of Plant Physiology 144, 307 – 313.