Plant Cell Reports (1999) 18: 743 – 748
M. Escalona · J. C. Lorenzo · B. González
M. Daquinta · J. L. González · Y. Desjardins
C. G. Borroto
Pineapple (Ananas comosus L. Merr) micropropagation
in temporary immersion systems
Received: 4 February 1998 / Revision received: 22 June 1998 / Accepted: 14 August 1998
Abstract A procedure for the mass propagation of pine-
apple plants (Ananas comosus L. Merr) using a temporary
immersion technique is described. This procedure involved
three distinct phases in the automated temporary immer-
sion system: shooting, bud differentiation and elongation.
To establish this protocol, we used in vitro shoots obtained
from established liquid culture as starting materials. Three
culture methods (solid, liquid and temporary immersion)
were compared. Temporary immersion increased the mul-
tiplication rate and fresh and dry weight after 42 days.
Conventional micropropagation (liquid medium) and tem-
porary immersion were compared in combination with
paclobutrazol. Paclobutrazol promoted the formation of
compact bud clusters with limited leaf development. The
highest multiplication rate (106) was found when ex-
plants were cultured in shooting medium (MS+2.1 mg/l
BA+0.3 mg/l NAA) supplemented with 1 mg/l PB for
7 weeks. A 10-l temporary immersion bioreactor was used
to test two approaches during elongation stage: reduction
of the shoot-formation period or decrease of the initial
number of explants. The highest number of competent and
uniform plants (191.8 plant/l) was achieved when shoots
were cultured for 4 weeks in shooting medium supple-
mented with PB.
Key words Pineapple · Temporary immersion ·
Automation · Micropropagation · Bioreactor
Communicated by W. Parrott
M. Escalona (½) · J. C. Lorenzo · B. González · M. Daquinta
J. González · C. G. Borroto
Centro de Bioplantas,
Universidad de Ciego de Avila Carretera a Morón,
Km 9 Ciego de Avila, CP 69450, Cuba
Fax: 53-33266340
e-mail: celulas@bioca.edu.cu
Y. Desjardins
Centre de Recherche en Horticulture,
Faculté des Sciences de l’Agriculture et de l’Alimentation,
Université Laval, Quebéc, Canada, G1K 7P4
© Springer-Verlag 1999
Abbreviations BA 6-Benzylaminopurine · NAA naphtha-
leneacetic acid · PB paclobutrazol · [(2RS,3RS)-1-4-chlo-
rophenyl 4,4-dimethyl-2-(1H-1,2,4-triazol-1-yL)pentan-
3-ol] · GA3 gibberellic acid · MS Murashige and Skoog
1962
Introduction
Micropropagation of pineapple plants has many advan-
tages over conventional methods of vegetative propaga-
tion. For example, this technique could allow for an effi-
cient and rapid increase of selected varieties. Commercial
pineapple micropropagation involves sequential culturing
in liquid medium (conventional micropropagation) for
meristem and axillary shoot-bud multiplication (Firooza-
bady et al. 1997; Daquinta and Benega 1997). Using this
approach, annual pineapple production is limited as a re-
sult of the number of pineapple plants needed annually to
start up new plantations. In general, the commercial use of
micropropagation is currently reduced because of high pro-
duction costs resulting primarily from high labour costs, a
low multiplication rate and poor survival rates during ac-
climatisation. Extensive expansion of pineapple micro-
propagation will not occur without new technology to pro-
vide automation and improved protocols for acclimatisa-
tion (Kitto 1997).
Much research has been conducted recently on automa-
tion in micropropagation. It has included the automation
of liquid medium preparation and feeding, plant image rec-
ognition and processing, microcutting and transplanting
(Aitken Christie et al. 1995; Kozai and Smith 1995; Smith
1995). Several liquid media immersion systems for micro-
propagation have been developed. Tisserat and Vander-
cook (1985) designed a system consisting of a large ele-
vated culture chamber that was periodically drained and
then refilled with fresh medium. Also, Aitken-Christie and
Davies (1988) developed a semi-automated system in
which plants were cultured on agar medium in a large con-
tainer, with the automatic addition and removal of liquid
744

medium on a periodic basis. Simonton et al. (1991) devel-

oped a programmable micropropagation apparatus using
cycled liquid medium which intermittently applied culture
medium to the plants according to a selected schedule.
Most recently, a temporary immersion system has been de-
scribed by Teisson and Alvard (1995) for plant propaga-
tion. This system, designated with the name RITA in com-
mercial settings, has been successfully used with several
plant species. Using the system of temporary immersion,
we report here an automated system for large-scale pine-
apple propagation.

Materials and methods

Plant material

Pineapple plants (Ananas comosus L. cv ‘Smooth Cayenne’) were

obtained from established liquid cultures grown on a shooting me-
dium, which consisted of MS salts supplemented with 2.1 mg/l BA
and 0.3 mg/l NAA, as recommended by Daquinta and Benega (1997).
The cultures were grown under cool-white fluorescent lamps provid-
ing 80 µmol photons · m–2 · s–1, with a 16-h photoperiod at 25°C.

Description of the automated temporary immersion system

The bioreactor system consisted of two containers; one for growing

plants and a reservoir for liquid medium. The two containers were
connected by silicone and glass tubes. In each case, the airflow was
sterilised by passage through 0.2-µm hydrophobic filters (Fig. 1).
Air pressure from an air compressor pushed the medium from one
container to the other to immerse the plants completely. The airflow
was reversed to withdraw the medium from the culture container. Fig. 1A–C Description of automated temporary immersion sys-
Electronic timers controlled the frequency and length of the immer- tems. A A solenoid valve is opened, and compressed air forces me-
sion period. Three-way solenoid valves provided on/off operation. dium into the plant container, immersing the plants B. C After a fixed
period of time a second solenoid valve is opened, and air pressure
forces medium back into the original container (A). 1 Air compres-
Comparison of micropropagation methods sor, 2 solenoid valve, 3 hydrophobic filter (0.2 µm)

Three micropropagation methods were compared: solid, liquid and

temporary immersion. The plant vessels used for the experiment had
a diameter of 8.5 cm, a height of 15 cm and a volume of 300 ml. The
amount of medium added to the culture vessel was 250 ml. In order
to avoid the complete submersion of explants in the liquid culture
medium, filter paper was used as supports to hold the plant materi-
al. Five explants (in vitro-cultured plants with two small shoots) of
approximately 2–3 cm in length were cultured in each container. For
the temporary immersion system, plantlets were immersed for 2 min
every 3 h. The shooting medium was as described before. Solid me-
dium contained 2.5 g/l Gelrite. The pH was adjusted to 5.8 before
autoclaving at 121°C and 1 kg/cm2 for 20 min. Cultures were incu-
bated at 25°C under cool white fluorescent tubes (80 µmol·m–2·s–1)
with a 16-h photoperiod. The multiplication rate and the fresh and
dry weights were determined after 42 days of culture. Multiplication
rates were determined by taking of the number of shoots at the end
divided by the initial number of explants. Dry weight was measured
after drying the plantlets for 72 h at 70°C.
Effect of paclobutrazol on shoot multiplication
Two culture systems were compared in combination with paclobu-
trazol at 0.0, 0.5 or 1 mg/l added to the shooting medium; conven-
tional micropropagation method (liquid culture) and the temporary
immersion method. The dimensions of the culture vessel for conven-
tional micropropagation were the same as those described above. The
volume of medium added to the culture vessel was 25 ml. Five ex-
plants were cultured in each container. Two 1000-ml vessels consti-
tuted the temporary immersion system. The culture medium reser-
voir included 1000 ml of medium. Five explants were also cultured
in each vessel. For the temporary immersion system, shoots were im-
mersed for 2 min every 3 h. Cultures were incubated at 25°C under
cool-white fluorescent tube (80 µmol · m–2 · s–1). The multiplication
rate was evaluated after 42 days of culture.

Effect of duration of shoot formation period in the temporary

Volume of medium needed for the temporary immersion system immersion system on multiplication rate and culture medium pH

The medium and experimental conditions were the same as described
above. The volume of culture medium in the reservoir for liquid me-
dium in the temporary immersion system was 50, 100, 150, 200 and
250 ml per explant. Five explants per temporary immersion system
were cultured. The multiplication rate and fresh and dry weights were
also determined after 42 days of culture.
Experimental conditions using the temporary immersion system de-
scribed above were used to carry out this experiment. Shooting me-
dium with 1 mg/l PB was used. The number of shoots was recorded
weekly between 4 and 8 weeks. Measurements of pH in the culture
medium were made at the same time using a potentiometer (ORION
American™ pH/ISE).