Annals of Botany 87: 53±59, 2001
doi:10.1006/anbo.2000.1299, available online at http://www.idealibrary.com on
Micropropagation of Potato: Evaluation of Closed, Di€usive and Forced Ventilation on
Growth and Tuberization
S . M . A . Z O B AY E D * , J . A R M S T RO N G and W. A R M S T RO N G
Department of Biological Sciences, University of Hull, Hull, HU6 7RX, UK
Received: 5 June 2000 Returned for revision: 8 August 2000 Accepted: 15 September 2000 Published electronically: 16 November 2000
Di€erent types of ventilation of the culture vessel headspace, each with and without the ethylene inhibitor AgNO3
(3.0 mM) or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (2.0 mM) in the culture medium,
were investigated in terms of their e€ects on the growth of potato cuttings (Solanum tuberosum L. `cara').
Concentrations of CO2 , O2 and ethylene in the culture vessel headspaces were monitored during the study. Growth
was substantially enhanced and vitri®cation (stunting and epinasty of leaves and hooking of stem apices) was reduced
by increasing the eciency of ventilation, the e€ects being greatest with forced ventilation. In the conventional
di€usive treatment (via a polypropylene membrane), leaf epinasty occurred but the leaves were not stunted unless
ACC had been added. AgNO3 prevented vitri®cation in the latter case and reduced it in the sealed treatment. On the
other hand, with all forced ventilation treatments, even with the addition of ACC, the plantlets grew well and some of
the growth parameters exceeded those in the di€usive ‡ AgNO3 treatment. Ethylene removal was clearly an
important factor contributing to the better growth found with di€usive and especially with the forced ventilation
treatment; with the latter, ethylene concentrations in the culture vessels were virtually zero. In addition, enhanced
CO2 supply probably contributed to the better performance under forced ventilation compared to di€usive
ventilation. Callus developed on the stem bases in all sealed (airtight) and di€usive treatments except where AgNO3
was used. No callus was observed in any treatment where forced ventilation was applied and in vitro tuberization
# 2000 Annals of Botany Company
(tuber size) was considerably improved by this treatment.
Key words: Callus, ethylene, potato, tuberization, vitri®cation.
I N T RO D U C T I O N
In vitro propagation of potato by the serial culture of
axillary shoots on separated nodes has been reported by a
number of researchers, and is now becoming established as
an e€ective means of rapidly multiplying new or existing
cultivars in disease-free conditions (Hussey and Stacey,
1984). However, a major drawback to the procedure is that
the potato plant is highly sensitive to ethylene, and ethylene
accumulation in vitro strongly inhibits the growth and
development of shoots. It is known that growth of potato
plantlets can be distorted by concentrations of ethylene of
0.1 ml l ÿ1 or even less (Jackson et al., 1987). Hussey and
Stacey (1981) reported that potato shoots become stoloniferous in tightly-closed culture vessels and leaves are small.
Jackson et al. (1991) found that shoot height of Solanum
tuberosum was 64 % of that of the control after 14 d of
culture in tightly-sealed vessels. They also concluded that
accumulated ethylene is responsible for these e€ects. To
remove ethylene from potato culture vessels, Jackson et al.
(1987) used mercuric perchlorate and thus increased shoot
height.
In recent years the in vitro tuberization phenomenon
has become important for the rapid propagation of diseasefree potatoes (Levy et al., 1993). Miniature tubers (microtubers) formed on plantlets grown in vitro are useful
* For correspondence. Fax ‡44 (0) 1482 465458, e-mail s.m.
[email protected]
0305-7364/01/010053+07 $35.00/00
also because they are very convenient for the maintenance
and handling of disease-free material: microtubers are
easily stored, transferred and distributed (Akita and
Takayama, 1994).
The purpose of this project was to improve culture
conditions of potato explants by means other than the use
of ethylene absorbers, or antagonists, which can have toxic
e€ects. To this end we explored the e€ects of improving the
eciency of headspace ventilation and thereby of reducing
the concentrations of ethylene. Explants were grown under
three types of ventilation: the sealed condition, di€usive
ventilation (via a polypropylene membrane) and forced
ventilation, each being applied with and without the
ethylene antagonist, AgNO3 , and the ethylene precursor
1-aminocyclopropane-1-carboxylic acid (ACC) in the
culture medium.
The paper also describes ways of improving in vitro
tuberization by means of increasing the eciency of
headspace ventilation.
M AT E R I A L S A N D M E T H O D S
Establishment of plantlets from tubers
Tubers of Solanum tuberosum L. `cara' were washed in tapwater, cut into small pieces of approx. 15 mm3, each
bearing a sprout initial, and were placed in paper bags
inside an incubator at 218C to allow the rapid development
of white etiolated sprouts which provided the source of the
# 2000 Annals of Botany Company
54
Zobayed et al.ÐVentilation A€ects Micropropagation of Potato
initial explants. These sprouts were sterilized with 10 % v/v
sodium hypochlorite solution and cut into 1.0 cm long
nodal sections each containing a single axillary bud. For
initial establishment and routine maintenance of cultures,
these sections were inoculated in a culture vessel with
di€usive ventilation (capped with polypropylene ®lms)
and containing Murashige and Skoog (MS) medium
(Murashige and Skoog, 1962) and 20 g l ÿ1 sucrose,
8.0 g l ÿ1 agar and no growth regulator. The cultures were
kept in a growth room at 258C under cool-white ¯uorescent
lamps ( photosynthetic photon ¯ux ˆ 100 mmol m ÿ2 s ÿ1)
on a 16 h photoperiod. Under these conditions a new shoot
developed from each node and at the four to ®ve node stage
these in turn were segmented into nodal sections to provide
the experimental explant material.
Measurement of ethylene, carbon dioxide and oxygen
concentrations
Ethylene concentration. For each experiment, ethylene
concentrations were determined by removing 500 ml samples
of gas from the culture vessels and analysing them using gas
chromatography (Pye Unicam). Poropack Q (60±80 mesh)
was used in a glass column (2500 mm 6.5 mm); the
temperatures of the column, injector and ¯ame ionization
detector were 100, 150 and 1508C, respectively. The
ethylene peaks were identi®ed by a retention time of
about 1.4 min. Nitrogen was used as the carrier gas at a rate
of 60 cm3 min ÿ1. The identi®cation of the ethylene peak
was separately con®rmed on other samples by repeating the
injection after exposing the vessel atmosphere to potassium
permanganate solution (0.1M), an ethylene absorber.
Oxygen concentration. Oxygen concentrations in the
culture vessels were measured at intervals by means of an
oxygen microelectrode (Clark typeÐtip diameter 10 mm:
Armstrong, 1994). Gas samples (1.0 cm3) from the culture
vessel headspace were removed using a hypodermic syringe
and injected into a small nitrogen-®lled chamber into which
the microelectrode protruded. Before injecting the sample,
1.0 cm3 of nitrogen was removed from the chamber.
Electrode calibration (electrolysis current vs. concentration)
was linear and the oxygen concentrations in the culture
vessels were obtained after taking due account of the
dilution e€ect on the sample.
CO2 concentration. The CO2 concentrations in the culture
vessels were obtained at intervals by injecting 1.0 cm3 gas
samples into a small chamber in a closed circuit system
(vol. 40 cm3), circulated through an infra-red gas analyser
(IRGA) (S. W. and W. S. Burrage, Hustingleigh, Ashford,
Kent, UK). Culture vessel CO2 concentrations were computed from the new IRGA reading after taking due account
of the dilution e€ect on the sample. Before injection, the
analyser had been calibrated using a 350 ml l ÿ1 CO2 supply,
and the subsequent injection samples were added after the
removal of 1.0 cm3 of gas from the circuit and scavenging
the IRGA CO2 to zero.
Types of ventilation
To achieve the di€usive ventilation, a disc of polypropylene membrane (thickness 25 mm; oxygen transmission
rate, 51.8 10ÿ2 m3 m ÿ2 d ÿ1 MPa ÿ1: Courtaulds Films,
Bridgwater, Somerset, UK) was secured over the mouth of
the tube by a rubber band.
The forced ventilation system employed in this study was
simple and non-mechanized; it is a more convenient and
much modi®ed form of a prototype described by
Armstrong et al. (1997) and ®ts directly onto the culture
vessel ( for details see Zobayed et al., 1999a). Brie¯y, the
mechanism which creates the pressurized ¯ow depends
upon the humidity-induced di€usion of atmospheric gases
(O2 , N2 and CO2) into the ventilator through an in¯ow
Nuclepore membrane ( pore diameters ˆ 0.03±0.05 mm).
Di€usion occurs under the in¯uence of a concentration
gradient across this membrane which is induced and
maintained by the higher humidity under the membrane
relative to the outside air. Pressurization occurs because
of the continued humidi®cation under the membrane and
the resistance to back ¯ow a€orded by its micro-porous
nature. A sterile stream of humidi®ed air (5 cm3 min ÿ1)
passes into the culture vessel and comparatively free venting
occurs through an out¯ow membrane ( pore diameter
ˆ 0.2 mm).
E€ects of ventilation types and the ethylene inhibitor
(AgNO3) and the ethylene precursor (ACC) on the growth
of nodal stem cuttings
One explant (nodal segment with one unfolded leaf and
with mean fresh mass of 40 mg) was transferred into each of
the glass culture vessels (volume 60 cm3) with MS medium
(10 cm3) supplemented with 8.0 g l ÿ1 agar, 20 g l ÿ1
sucrose, and no growth regulators, and grown with or
without additives (AgNO3 , 3.0 mmol l ÿ1 or ACC,
2.0 mmol l ÿ1) to the medium under the following ventilation conditions: (a) sealed with silicone rubber bung; (b)
sealed ‡ AgNO3 in the medium; (c) sealed ‡ ACC in the
medium; (d) di€usive ventilation, vessel capped by polypropylene membrane; (e) di€usive ventilation ‡ AgNO3 ;
( f) di€usive ventilation ‡ ACC; (g) forced-ventilation
apparatus (5 cm3 min ÿ1); (h) forced ventilation ‡
AgNO3 ; (i) forced ventilation ‡ ACC. There were six
replicates per treatment. The choice of AgNO3 concentration was made after testing a range of concentrations (0.6±
5.0 mmol l ÿ1) in sealed vessels. Growth was optimal at
3.0 mmol l ÿ1 AgNO3 ; above this, there was some toxic
e€ects on growth.
Ethylene, CO2 and oxygen concentrations were measured
at intervals during the ®rst 21 d of the experiment. Plantlets
were grown with continuous illumination in a growth room
where the air temperature was 258C and relative humidity
50±65 %. PPF at shelf level was 100 mmol m ÿ2 s ÿ1.
Plantlets were harvested on day 25; growth measurements
included leaf number, area and fresh mass, stem fresh mass
and length, root number and maximum length and volume
of callus.
Zobayed et al.ÐVentilation A€ects Micropropagation of Potato
In vitro tuberization of potato a€ected by di€erent types of
ventilation
For tuberization, nodal segments were inoculated in
60 cm3 culture vessels (one per vessel) on MS medium
supplemented
with
6-benzylaminopurine
(BAP)
(1.0 mg l ÿ1), sucrose (80 g l ÿ1) and agar (8.0 g l ÿ1). The
concentration of BAP and sucrose for optimal growth and
tuberization under di€usive ventilation (vessels capped with
polypropylene membrane) was previously determined by
experimenting with a range of BAP (0.0±2.0 mg l ÿ1) and
sucrose concentrations (40 g l ÿ1, 80 g l ÿ1 and 120 g l ÿ1).
To examine the e€ects of ventilation types on tuberization,
each vessel was ®tted with either: (a) a silicone rubber bung;
(b) a polypropylene membrane; or (c) a forced ventilation
apparatus ( ¯ow rate ˆ 5.0 cm3 min ÿ1). Five replicates were
prepared for each treatment. The cultures were kept at
258C under cool-white ¯uorescent lamps (PPF ˆ
100 mmol m ÿ2 s ÿ1) and a 16 h photoperiod. Plantlets were
harvested after 8 weeks; fresh mass and numbers of tubers
were recorded.
R E S U LT S A N D D I S C U S S I O N
E€ects of ventilation types and the ethylene inhibitor
(AgNO3) and precursor (ACC) on growth and headspace
atmosphere
Growth. After 25 d of culture, the best growth in the
treatments without additives was observed in explants
grown with forced ventilation (Fig. 1). The suppression of
ethylene activity by silver was also very evident: in the
sealed condition the addition of silver led to a ®ve-fold
increase in leaf area, while leaf fresh mass increased six-fold
and the length of the roots also increased signi®cantly.
When plantlets were grown in the tightly-sealed condition viz. sealed without additives and sealed ‡ ACC,
shoots were swollen and the leaves small with a tendency to
be folded. Stem apices became hooked in shape, and roots
were stunted. Some shoots became brown at the tips. These
results are consistent with earlier observations of Jackson
et al. (1987) and Hussey and Stacey (1984). In contrast,
plantlets grown under forced ventilation had well-developed
shoot and root systems, and morphologically the plantlets
appeared normal with normal stem apices. The higher stem
fresh mass found in the sealed (control) and in the
sealed ‡ ACC treatments compared with their di€usive
counterparts (Fig. 1C) may be accounted for by ethyleneinduced swelling of the shoots.
With di€usive ventilation, and due to the addition of
AgNO3 , increased leaf area, leaf fresh mass and stem fresh
mass were observed and the root length was approximately
doubled (Fig. 1) compared with that of the control. A
major e€ect noted in this experiment was the development
of callus from the base of the stem in the sealed and
di€usive treatments, with or without the addition of ACC;
however, ACC increased the quantity of callus produced
(Fig. 2). Silver ions prevented callus induction, as did forced
ventilation. It should be noted that, where (as in this case)
the culture medium has not been designed to stimulate
55
callus development, its production is commonly associated
with vitri®cation (Paque and Boxus, 1987; Ziv, 1991).
Jackson et al. (1991) acknowledged that the problem of
ethylene accumulation can be lessened by the use of larger
culture vessels. However, the forced ventilation system
described here would enable the use of smaller vessels.
A further possible advantage of this type of forced
ventilation is that the aerating gases are humidi®ed, and
this should help to reduce loss of water vapour from both
plantlets and medium.
It is likely that with longer-term growth under micropropagation the di€erences found in this experiment would
become even more accentuated: for example, it is probable
that CO2 concentration would have been nearer to the
compensation point in the di€usive treatments than in the
forced ventilation treatments. Consequently, photosynthetic rates in the forced ventilation treatment would
have been greater and the positive feedback e€ects of this
might well be cumulative beyond the 25 d growth period
adopted here. These ®ndings are in close agreement with
the ®ndings of Zobayed et al. (2000) where forced
ventilation was found to improve the growth of Eucalyptus
plantlets.
Headspace atmosphere
Ethylene. In sealed vessels, the addition of ACC to the
medium resulted in high concentrations of ethylene: after
only 12 d 1.45 ml l ÿ1 had accumulated and this was
2.3-times that of the sealed control (Fig. 3). Subsequently
the ethylene concentrations in the ACC treatment declined
slightly, while in the sealed controls they continued to rise so
that by day 21 the di€erences between the two were much
less than previously. In the sealed ‡ AgNO3 treatment the
ethylene concentrations were higher than those of the sealed
controls; AgNO3 does not inhibit ethylene production and it
is presumed that the higher ethylene concentration in this
treatment was a function of the larger plantlets or a lack of
feed-back inhibition on biosynthesis. Di€usive ventilation
resulted in much lower ethylene accumulation and even with
ACC addition the concentration did not exceed 0.4 ml l ÿ1
even at day 21. Nevertheless, it is clear that the polypropylene membranes helped to reduce ethylene accumulation markedly. On the other hand, forced ventilation was
even more e€ective at minimizing ethylene accumulation
and the gas was virtually undetectable even in the ACC
treatments.
Oxygen. In terms of the temporal patterns in headspace
oxygen regime, the results in Fig. 4 reveal very distinct
di€erences between forced ventilation and the other two
ventilating treatments. Thus, with each of the forced
ventilation treatments, concentrations remained constant
and close to atmospheric for the whole period, whereas with
di€usive and sealed ventilation they declined at varying rates
from a little above atmospheric. The initial concentrations
presumably re¯ected some photosynthetic enhancement of
oxygen within the headspace. Also, within the di€usive and
sealed treatments, and presumably due to their e€ects on the
Zobayed et al.ÐVentilation A€ects Micropropagation of Potato
50
45
40
6
cx
B
cx
Leaf area (cm2)
cx
cx
bz
bx
ay
by
Control
ACC
C
cx
ax
cx
ax
by
bx
ay
by
2
50
bz
az
bx
3
0
AgNO3
cx
bx
4
1
ax
ax
cx
cx
5
35
30
25
20
15
10
ay
ax
Control
D
ACC
AgNO3
cx
cx
by
40
bx
ay
bx
ay
30
ax
ax
20
10
5
0
21
Root number per plantlet
A
Stem height (mm)
33
30
27
24
21
18
15
12
9
6
3
0
Control
E
ACC
12
bx
ax
bx
cx
by
cx
az
az
9
6
3
0
Control
Control
ACC
AgNO3
F
ay
18
15
0
AgNO3
ACC
AgNO3
Total root length (mm)
Increased stem FM (mg)
Leaf FM (mg)
56
cx
120
bx
cx
100
bz
az
80
bx
60
40
20
0
ax
Control
ay ay
ACC
AgNO3
F I G . 1. The in¯uence of di€erent methods of capping of culture vessels on leaf fresh mass (A), leaf area (B), stem fresh mass (C), stem height (D),
root number (E) and root length (F) per plantlet of in vitro grown potato (Solanum tuberosum L.) after 25 d of culture. Each bar represents the
mean ‡ s.e. of six replicates. Signi®cant di€erences between ventilation treatments at P 4 0.05 indicated by a, b, c and between respective
controls, ACC and AgNO3 treatments by x, y, z. Statistical signi®cance was determined by Student-Newman-Keuls test. Sealed, Vessels sealed
with silicone rubber bungs (h); di€usive, vessels capped with polypropylene discs (D); and forced ventilation rate ˆ 5.0 cm3 min ÿ1 (E).
plantlets, AgNO3 or ACC additions can be seen to have
in¯uenced the rate of decline in the oxygen concentrations.
In the sealed (control) and sealed ‡ ACC treatments, the
oxygen concentrations fell substantially during the experiment: after 21 d of culture there was, respectively, only
14.8 % and 11.6 % oxygen in the headspaces compared to
approx. 20 % in the equivalent forced ventilation treatments
(Fig. 4). With AgNO3 in the culture medium the oxygen
concentrations in the headspaces of the sealed vessels were
very much higher than those of the sealed ‡ ACC or sealed
(control) treatments and only a little lower than treatments
having forced ventilation.
The di€usely ventilated treatments showed a similar
pattern to their sealed counterparts but the di€erences were
less. Thus, in the ACC treatment the oxygen concentration
had dropped to approx. 16 % over 21 d, while in the
control and AgNO3 treatments the values were approx.
17.5 % and 19.5 %, respectively.
In view of the growth parameters recorded in Fig. 1, it
seems likely that the gradual depression in the oxygen
Zobayed et al.ÐVentilation A€ects Micropropagation of Potato
22
ay
2.5
2.0
20
ax
Forced vent
Diffusive + AgNO3
1.5
1.0
Sealed–AgNO3
by
bx
0.5
0.0
az
Control
ACC
az
AgNO3
F I G . 2. The in¯uence of di€erent methods of capping of culture vessels
on callus volume of in vitro grown potato (Solanum tuberosum L.)
plantlets (25-d-old). Each bar represents the mean ‡ s.e. of 6 replicates.
Signi®cant di€erences between ventilation treatments at P40.05 indicated by a, b and between respective controls, ACC and AgNO3 treatments by x, y, z. Statistical signi®cance was determined by StudentNewman-Keuls test. Sealed, Vessels sealed with silicone rubber bungs
(h); di€usive, vessels capped with polypropylene discs (D).
Oxygen concentration (%)
Callus volume (cm3 per plantlet)
3.0
57
18
Diffusive control
16
Diffusive + ACC
Sealed control
14
12
Sealed + ACC
1.6
10
0
5
10
15
20
25
Days
1.4
Sealed+ ACC
Ethylene concentration (µl l–1)
1.2
Sealed+AgNO3
1.0
F I G . 4. E€ects of di€erent types of ventilation and ACC (2.0 mM) and
AgNO3 (3.0 mM) on oxygen concentrations in the headspace of a
60 cm3 culture vessel containing in vitro-grown potato plantlets. The
ambient relative humidity was 50±65 %, temperature was 258C and the
PPF was 100 mmol m ÿ2 s ÿ1. Each symbol represents the mean + s.e.
of ®ve replicates. Sealed, Vessels sealed with silicone rubber bungs;
di€usive, vessels capped with polypropylene discs; and forced
ventilation rate ˆ 5.0 cm3 min ÿ1.
Sealed control
0.8
0.6
0.4
Diffusive+ ACC
0.2
Diffusive+AgNO3
Diffusive control
0.0
Forced vent
0
5
10
15
20
25
Days
F I G . 3. E€ects of di€erent types of ventilation and ACC (2.0 mM) and
AgNO3 (3.0 mM) on ethylene concentrations in the headspace of a
60 cm3 culture vessel containing in vitro-grown potato plantlets. The
ambient relative humidity was 50-65 %, temperature was 258C and the
PPF was 100 mmol m ÿ2 s ÿ1. Each symbol represents the mean + s.e.
of ®ve replicates. Sealed, Vessels sealed with silicone rubber bungs;
di€usive, vessels capped with polypropylene discs; and forced
ventilation rate ˆ 5.0 cm3 min ÿ1.
concentrations in the sealed and di€usive treatments lacking
AgNO3 were due to (a) increased respiratory demands
associated with the production of varying quantities of nonphotosynthetic callus, and the development of the root
systems, and possibly (b) some degree of senescence a€ecting
the photosynthetic tissues.
These results are consistent with the ®ndings of some
other authors. In tightly-sealed vessels containing Ficus
plantlets, oxygen concentrations of approx. 10 % were
observed (Jackson et al., 1991). In sealed cauli¯ower
shoot-culture, Zobayed et al. (1999b), reported an oxygen
concentration of approx. 7.1 % whereas with forced ventilation it remained a little below atmospheric. Adkins et al.
(1990) found that in a sealed Petri dish containing rice callus
the oxygen concentration was 2±5 % after 24 d of culture.
Carbon dioxide. Changes in CO2 concentration were
barely noticeable until day 14 by which time the concentration in the sealed ‡ ACC treatment had reached
0.9 %; by day 21 the concentration was nearly 4 %
(Fig. 5A). The e€ects here and in the sealed (control),
di€usive ‡ ACC, and di€usive (control) are probably
58
Zobayed et al.ÐVentilation A€ects Micropropagation of Potato
4.0
A
CO2 concentration (%)
3.5
Sealed + ACC
3.0
2.5
2.0
1.5
Sealed control
1.0
0.5
Diffusive + ACC
Diffusive control
Forced vent
0
CO2 concentration (%)
0.2
B
Sealed control
Sealed+ACC
0.15
Diffusive+ACC
Diffusive control
0.1
0.05
Forced+ACC
Forced control
Forced + AgNO3
Diffusive + AgNO3
0
Sealed + AgNO3
0
5
10
15
Time (d)
20
25
F I G . 5. E€ects of di€erent types of ventilation and ACC (2.0 mM) and
AgNO3 (3.0 mM) on CO2 concentrations in the headspace of a 60 cm3
culture vessel containing in vitro-grown potato plantlets. The ambient
relative humidity was 50±65 %, temperature was 258C and the PPF was
100 mmol m ÿ2 s ÿ1. Each symbol represents the mean + s.e. of ®ve
replicates. Sealed, Vessels sealed with silicone rubber bungs; di€usive,
vessels capped with polypropylene discs; and forced ventilation
rate ˆ 5.0 cm3 minÿ1. A, CO2 concentration on a scale of zero to
4 %. B, CO2 concentration on scale of zero to 0.2 %.
attributable to the respiratory activity of the callus which
developed only in these treatments. Thus, the balance
between photosynthesis and respiration was moved in
favour of respiratory CO2 output.
In the other treatments ( forced control, forced ‡
di€usive ‡ AgNO3
and
sealed ‡ AgNO3
AgNO3 ,
treatments) callus did not form and CO2 concentrations
remained relatively constant or declined with time (Fig. 5B)
with the decline being greatest where ventilation was
poorest. Thus, in the sealed ‡ AgNO3 treatment, CO2
concentrations were at or close to the compensation point
(45 ml l ÿ1; 50.01 %) by day 21. CO2 concentrations were
improved with di€usive ventilation and after 21 d CO2
concentration was 200 ml l ÿ1 (0.02 %) in the di€usive ‡
AgNO3 treatment. In all the forced ventilation treatments,
the CO2 concentrations remained above 300 ml l ÿ1 (0.03 %)
despite the greater CO2 demand associated with the
increased productivity. Again, the results con®rm the
bene®ts of forced ventilation.
In vitro tuberization of potato a€ected by di€erent
ventilation treatments
The numbers and fresh mass of tubers were very low in
the sealed condition compared with those produced with
di€usive or forced ventilation. Compared to di€usive
ventilation, forced ventilation did not signi®cantly increase
the number of tubers but it did increase their fresh mass
which was almost double that of tubers in the di€usive
treatment. However, the shoots became swollen in places
after 4 weeks of culture in forced ventilation. In contrast,
very little tuberization occurred in the sealed condition.
Jackson et al. (1987) found no e€ect of ethylene on the
induction of tuberization. In contrast, Hussey and Stacey
(1984) reported that the addition of potassium permanganate to the culture vessel to absorb ethylene markedly
increased tuberization in potato. They also reported that
the presence of ethylene tended to make the shoots become
stoloniferous (Hussey and Stacey, 1981). Mingo-Castel et al.
(1976) reported that ethylene inhibits tuberization. Moreover, they showed that increased CO2 concentration
promotes tuberization. In the present investigation no
speci®c attempt was made to ®nd out whether ethylene
a€ected tuberization. However, since the numbers of tubers
were similar with di€usive and forced ventilation, it seems
likely that the low concentrations of ethylene in vessels with
di€usive ventilation were insucient to cause inhibition.
The poor tuber initiation in sealed vessels might have been
direct, i.e. due to ethylene inhibition of tuber formation, or
indirect i.e. growth inhibition of the plantlets may have
delayed their attainment of tuber-producing physiological
age.
The greater fresh mass of tubers grown under forced
ventilation may have been due to an increase of CO2
concentration during the photoperiod in the culture vessel
headspace and/or the lack of ethylene in the culture vessel
headspace. Since the results have shown a positive e€ect of
CO2 enrichment on shoot growth over and above that of
ethylene removal, it seems very likely that the greater yield
of tubers receiving forced rather than di€usive ventilation
could have been largely due to the greater photosynthate
production of the larger plantlets.
Finally, it should also be noted that in potato, short days
and low temperatures generally favour tuberization. In
these experiments a 16 h photoperiod was provided and
the temperature was 258C. It is anticipated that tuberization might be further improved by providing a shorter
photoperiod (e.g. 6±8 h rather than 16 h) and cooler
temperatures.
In conclusion, the growth, quality of plantlet and sizes of
microtubers produced during the micropropagation of
potato can be greatly enhanced simply by introducing a
forced ventilation system.
AC K N OW L E D G E M E N T S
We are very grateful to Mrs Margaret Hu€ee for technical
help and advice and especially with the work involving the
GLC. We are also grateful to Mr Mike Bailey for
fabricating the ventilating systems, Dr M. B. Jackson of
Zobayed et al.ÐVentilation A€ects Micropropagation of Potato
Long Ashton Research Station for advice on ethylene
measurement and Mr Victor Swetez of the University of
Hull Botanical Garden for supplying the potato tubers.
L I T E R AT U R E C I T E D
Adkins SW, Shiraishi T, McComb JA. 1990. Rice callus physiology:
identi®cation of volatile emissions and their e€ects on culture
growth. Physiologia Plantarum 78: 526±531.
Akita M, Takayama S. 1994. Stimulation of potato (Solanum
tuberosum L.) tuberization by semicontinuous liquid media
surface level control. Plant Cell Report 13: 184±187.
Armstrong J, Lemos EEP, Zobayed SMA, Justin SHFW, Armstrong W.
1997. A humidity-induced convective through-¯ow ventilation
system bene®ts Annona squamosa L. explants and coconut calloid.
Annals of Botany 79: 31±40.
Armstrong W. 1994. Polarographic oxygen electrodes and their use in
plant aeration studies. Proceedings of the Royal Society of
Edinburgh 102B: 511±527.
Hussey G, Stacey NJ. 1981. In vitro propagation of potato (Solanum
tuberosum L.). Annals of Botany 48: 787±796.
Hussey G, Stacey NJ. 1984. Factors a€ecting the formation of in vitro
tubers of potato (Solanum tuberosum L.). Annals of Botany 53:
565±578.
Jackson MB, Abbott AJ, Belcher AR, Hall KC. 1987. Gas exchange in
plant tissue cultures. In: Jackson MB, Mantell S, Blake J, eds.
Advances in the chemical manipulation of plant tissue cultures.
BPGRG Monograph 16. Bristol: British Plant Growth Regulator
Group, 57±71.
59
Jackson MB, Abbott AJ, Belcher AR, Hall KC, Butler R, Cameron J.
1991. Ventilation in plant tissue culture and e€ects of poor
aeration on ethylene and carbon dioxide accumulation, oxygen
depletion and explant development. Annals of Botany 67:
229±237.
Levy D, Seabrook JEA, Coleman S. 1993. Enhancement of tuberization
of axillary shoot buds of potato (Solanum tuberosum L.) cultivars
cultured in vitro. Journal of Experimental Botany 44: 381±386.
Mingo-Castel AM, Smith OF, Kumamoto J. 1976. Studies on the
carbon dioxide promotion and ethylene inhibition of tuberization
in potato explants cultured in vitro. Plant Physiology 57: 480±485.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiologia Plantarum 15:
473±479.
Paque M, Boxus P. 1987. `Vitri®cation': review of literature. Acta
Horticulturae 212: 155±166.
Ziv M. 1991. Vitri®cation: morphological and physiological disorders
of in vitro plants. In: Debergh PC, Zimmerman RH, eds. Micropropagation. The Netherlands: Kluwer Academic Publishers,
45±69.
Zobayed SMA, Armstrong J, Armstrong W. 1999a. Evaluation of a
closed system, di€usive and humidity-induced convective through¯ow ventilation on the growth and physiology of cauli¯ower in
vitro. Plant Cell Tissue and Organ Culture 59: 113±123.
Zobayed SMA, Armstrong J, Armstrong W. 1999b. Cauli¯ower shootculture e€ects of di€erent types of ventilation on growth and
physiology. Plant Science 141: 221±231.
Zobayed SMA, Afreen F, Kubota C, Kozai T. 2000. Mass propagation
of Eucalyptus camaldulensis in a scaled-up vessel under in vitro
photoautotrophic condition. Annals of Botany 85: 587±592.