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).

Shoot elongation in temporary immersion system: effect
of duration of shoot-formation stage and initial number of explants
For this experiment, a 10-l culture bioreactor was used. The shoot-
ing medium contained 1 mg/l PB (step 1). The medium container was
filled at a ratio of 200 ml per explant. Fifty explants were cultured
in each container. The medium was sterilised at 120°C, at a pressure
of 1 kg/cm2 for 45 min. Two strategies aimed at the reduction of the
shoot-formation period or a decrease in the number of initial ex-
plants. After shoot-multiplication periods of 4 or 7 weeks, the me-
dium was removed and changed with MS medium supplemented with
0.5 mg/l BA+1 mg/l GA3 (step 2). After 7 days, the medium was re-
placed again with an MS medium supplemented with 1 mg/l GA3
(step 3) to achieve shoot elongation. Plant height was determined
and grouped into classes (<4, 4–6, 6–8, 8–10 and >10 cm). Based on
previous results, plants longer than 4 cm were considered to be com-
petent for ex vitro rooting and acclimatisation.
Ex-vitro rooting and acclimatisation of the cultured plants
Elongated shoots, taken from the temporary immersion system, were
transplanted into a substrate consisting of compacted red ferralitic
soil mixed in a 1:1 ratio with sugarcane mill baggasse. The trays were
placed in a greenhouse under humidity tents to prevent desiccation
during the first 10 days. Survival was evaluated after 30 days.
Statistical analysis
Test of normality (Kolmogornov-Smirnov), ANOVA and the Dun-
can test were performed using the Complete Statistical System
(CSS/3A Copyright © Stat soft, INC, 1991). Analysis of correlation
and regression and calcula of standard error were also done. Three
culture vessels were used in each treatment. The averages of the three
replications are presented.
Results and discussion
An automated bioreactor functioning on the principle of
temporary immersion was designed for large-scale pine-
apple propagation. This micropropagation system enabled
a constant supply of nutrients and aeration to plants with-
out the use of sophisticated technology. In addition, the
culture system described in this paper was easier to set up
and use than more expensive liquid bioreactors (Fig. 1).
The temporary immersion culture combines the advan-
tages of solid and liquid medium. Solid cultures allow aer-
ation but do not provide full contact with nutrient media.
Liquid culture medium permits an efficient nutrient up-
take, but hyperhydricity is often present (Smith and
Spoomer 1995; Aitcken-Christie et al. 1995). However, hy-
perhydricity has been never reported in pineapple liquid
cultures and was not present in our automated microprop-
agation system.
To evaluate the effectiveness of temporary immersion,
we compared it with micropropagation using solid or liq-
uid culture (Fig. 2). Explants cultured under the temporary
immersion system had the highest multiplication rate of all
the systems tested. The multiplication rate increased by
300% and 400%, respectively, over that obtained from liq-
uid medium and conventional solid support systems. Fresh
weight was correlated to the increase in shoot prolifera-
tion. However, there were no differences in dry weight ac-
745
Fig. 2 Comparison of solid, liquid and temporary immersion on
pineapple multiplication rate, the fresh and dry weights. Each point
represents the mean ±SE (n =15). Each treatment consisted of three
culture vessels, each one with five proliferating shoots at the begin-
ning of the culture period. Fresh and dry weights are the average of
the mass per culture in each treatment
cumulation between the liquid and temporary immersion
culture. This apparent contradiction may be explained by
the reduced development of the leaves in the temporary
immersion culture. Shoot clusters produced during axil-
lary shoot bud proliferation in the bioreactor are nearly
spherical, with shoots produced all around a central region.
However, some shoots are not adequate for direct ex vitro
rooting and acclimatization because of their small size.
Such shoots need further elongation. Due to the high prop-
agation rate, this further elongation does not increase pro-
duction costs. The reason for the efficiency of the tempo-
rary immersion culture system is probably the ability of
the system to aerate plant tissue and provide contact
between entire explants and the liquid medium. These two
features are usually not combined in a classic liquid cul-
ture procedure (Alvard et al. 1993).
A higher multiplication rate was achieved when a ratio
of 200 ml medium per explant was used in the temporary
immersion system. Volumes higher than 200 ml medium
per explant decreased the multiplication rate and the fresh
and dry weights (Fig. 3).
The combination of temporary immersion systems with
paclobutrazol (1 mg/l) greatly increased the pineapple
multiplication rate (Table 1). A strong interaction was ob-
served between culture system and paclobutrazol concen-
tration. While growth retardants and inhibitors of gibbe-
rellin biosynthesis are used extensively in agriculture and
ornamental horticulture to control plant growth and struc-
ture, until recently they were used only to a limited extent
in micropropagation. The positive effect of paclobutrazol
on pineapple micropropagation has been described by Es-
calona et al. (1995) and confirmed here. Paclobutrazol con-
trolled shoot growth and induced axillary bud prolifera-
tion. In the temporary immersion system, the use of pac-
lobutrazol for pineapple micropropagation promoted the
formation of compact bud clusters with limited leaf devel-
opment, thereby avoiding unnecessary leaf growth during
the shoot-formation stage.
746

Figure 4 shows the dynamics of shoot formation and pH

values of culture medium with paclobutrazol in temporary
immersion. The highest multiplication rate was obtained
when explants formed shoots during the seventh week,
which was associated with the stabilisation of acid pH in
the culture medium. Culture for a longer period (8 weeks)
promoted shoot deformation. These results point out the
advantages of the temporary immersion system. In vitro
culture media are generally adjusted to a pH of 5.5–6.0
during preparation. This feature is partly determined by
the effect of extreme pH on the gelling agent. It is well es-
tablished that the pH changes both during media prepara-
tion, particularly autoclaving, and in the presence of plant
tissues. When plant material is added, there is a rapid
Fig. 3 Effect of volume of culture medium per explant on pineap- change until equilibrium is reached (Williams 1993, 1995).
ple multiplication rate, the fresh and dry weights. Each point repre- This equilibrium pH in pineapple shooting seems to ap-
sents the mean ± SE (n =15). Each treatment consisted of three cul- proach 3.5.
ture vessels, each one with five proliferating shoots at the beginning
of the culture period. Fresh and dry weights are the average of the In these results there are several considerations: higher
mass per culture in each treatment proliferation rates are associated with pH around the equi-
librium point, which might facilitate the availability of
some ions. One of the advantages of temporary immersion
culture on in vitro nutrition may be that temporary immer-
sion limits the movement of ions associated with pH
change out of the plants. There is an initial net decrease in
the mineral content of plants following the transfer to fresh
medium during each subculture in conventional micro-
propagation. This ion loss can be sustained for many days
if the pH imbalance is maintained (Williams 1993). Acid
pH in pineapple culture medium may be attributed to the
differential uptake or excretion of NO3– and NH4+ ions
and/or carbonic acid accumulation as a consequence of
crassulacean acid metabolism (Osmond 1978).
A 7-week interval resulted in the best multiplication
rate, but the plant container was not large enough to allow
shoot elongation. A large number of plants were etiolated.
The use of MS medium and MS medium with gibberrellic
Fig. 4 Effect of shooting duration on multiplication rate and medi-
acid (1 mg/l) was the first strategy used to elongate bud
um pH. The bars represent the mean of three replicated smaples clusters from the temporary immersion system. This ap-
(mean ± SE) proach showed the necessity of achieving differentiation
of bud clusters before gibberrellic acid acts on the shoots.
The combination of BA and GA 3 7 days before the use of
1 mg/l GA3 provided for the best shoot elongation and plant
uniformity. In this experiment, two strategies were fol-
Table 1 Effect of paclobutrazol and culture system methods on mul-
tiplication rate lowed: reduction of the shoot-formation period or a de-
crease in the initial number of explants (Table 2). The com-
Culture methods Paclobutrazol Multiplication bination of a 4-week shoot-formation period and 5.0 ex-
(CM) (PB) rate ± SE a plants per liter as initial inoculation achieved the highest
(mg · l–1)
number of plants ready for ex vitro rooting and acclimat-
Conventional micropropagation 0.0 8.8±0.86 isation.
0.5 9.0±0.46 Plant survival percentage is shown in Figure 5. Plants
1.0 11.1±1.63 smaller than 4 cm had the lowest survival levels. Plants
Temporary immersion system 0.0 22.2±4.66 with a height range between 4 and 6 cm appeared to re-
0.5 37.7±2.12
1.0 68.8±5.70 quire an additional ex vitro treatment prior to the normal
Significance acclimatisation period. However, plants larger than 6 cm
Interaction CM × PB – ** could be grown directly in greenhouse.
One key requirement to introduce a new propagation
** Significant at the 1% level, using Duncan’s Multiple Range Test
for comparisons among culture methods and paclobutrazol concen- method using tissue culture techniques is the evaluation of
tration the genetic variability of regenerated plants. The best stud-
a
Each value represents the means ± SE (n = 3) ied qualitative trait in pineapple is leaf-margin type (Leal
 747
Table 2 Effect of duration of shoot-formation stage and initial number of explants on plant elongation in a 10-l temporary immersion
system

Duration of Explant · l–1 Classes Total number Number of Plant Competent

shoot-formation (initial (cm) of plants ± SE a competent percentage plants
stage (weeks) inoculation) plants (% ±SE) a (%)

4 <4 1115.6±62.3 32.3 ±6.16

5.0 4–6 569.0±118.4 (1918) 17.5 ±2.25 (65.6)

6–8 567.0±26.4 18.8 ±4.03
8–10 444.6±103.6 14.8 ±2.75
>10 337.6±9.96 14.53±1.58

<4 482.0±43.8 24.1 ±3.26

2.5 4–6 511.0±70.4 (1483) 24.1 ±6.16 (76.4)

6–8 463.0±98.0 23.2 ±1.31
8–10 229.0±28.3 12.7 ±2.92
>10 280.0±97.2 16.14±6.46

7 <4 1377.0±74.3 57.1 ±2.87

5.0 4–6 464.0±43.2 (1035) 19.0 ±5.52 (42.9)

6–8 346.0±38.7 14.35±1.6
8–10 129.0±49.0 5.42±2.91
>10 96.0±3.51 4.2 ±1.51

<4 1050.0±9.56 42.7 ±1.58

2.5 4–6 554.6±85.8 (1632) 22.2 ±2.58 (63.10)

6–8 486.0±86.4 22.3 ±5.51
8–10 349.6±98.7 9.42±2.29
>10 243.3±18.5 9.18±1.91
a
The data represents the means±SE of three repetitions

pineapple propagation scheme initially established by

Fig. 5 Pineapple plant survival after a 30-day acclimatisation peri-
od. r2 = 0.99, y = –20.34 + 18.83, x – 0.72 x2
and Coppens d’Eeckenbrugge 1996). The evaluation of
the frequency of spiny leaves in vegetatively propagated
plants and plants regenerated from temporary immersion
has been encouraging. To date, a frequency of about 6% of
phenotypic variation, different to the “spiny tip” Cayene
type of leaf, has been found in both groups of plants. A
field test for clonal fidelity is in progress.
New methods for pineapple micropropagation have
been developed to reduce the cost for commercial applica-
tions (Firoozabady et al. 1997). Significant changes to the
these authors are reported in this paper. First, the use of
paclobutrazol to increase axillary multiplication rate dur-
ing the shooting stage in temporary immersion system re-
sults in enhanced multiplication. Second, shoot elongation
is carried out in the same container, using two stages; the
first to achieve bud-cluster differentiation and the second
one to develop shoots. Third, shoots were separated from
clusters and placed directly on substrate for ex vitro root-
ing and acclimatisation over a period of 4 months.
In our production scheme it is possible to obtain approx-
imately 120 shoots from eight crown buds in about 10
weeks. After a further 8 weeks, using the above prolifera-
tion and elongation method in the temporary immersion
bioreactor, we produced a total of 6000 competent shoots
ready for ex vitro rooting and acclimatisation. An addi-
tional 5000–6000 shoots were also obtained which require
an additional in vitro treatment prior to the normal accli-
matisation period. In total, we obtained a 100-fold increase
in the 4-month period following the initial culture of the
crown buds.
The production costs were estimated taking into con-
sideration the efficiency and duration of this process by
steps. The following areas are key targets for reducing cost:
(1) reduction in number of containers (this index has an
exponential increase during shooting stage); elimination
of shelves in culture room; reduction in material manipu-
748
lation; elimination of cutting and planting [this is the most
expensive part of the micropropagation process (Chu
1995)]; biological optimisation (the use of growth retar-
dant and new culture method improves multiplication rate);
elimination of in vitro rooting and reduction of contami-
nation levels. This system is presently being introduced for
the large-scale production of pineapple. Most of the obsta-
cles currently found in conventional micropropagation
have been overcome. This protocol reduces by 20% the
production cost per pineapple plant in comparison with the
conventional method.
Acknowledgements The authors wish to thank Dr. W. Parrot for
critical reading of the manuscript and to those students and techni-
cians who have contributed to the experimental work.
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