ann bot 2001 zobayed 53 9

7/27/2019 Ann Bot 2001 Zobayed 53 9

http://slidepdf.com/reader/full/ann-bot-2001-zobayed-53-9 1/7

Micropropagation of Potato: Evaluation of Closed, Diusive and Forced Ventilation onGrowth and Tuberization

S. M. A. ZO BAYED *, J. AR MSTRON G and W. ARMST RON 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

Dierent 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 eects 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. Growthwas substantially enhanced and vitri®cation (stunting and epinasty of leaves and hooking of stem apices) was reducedby increasing the eciency of ventilation, the eects being greatest with forced ventilation. In the conventionaldiusive 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 theother 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 diusive AgNO3 treatment. Ethylene removal was clearly animportant factor contributing to the better growth found with diusive and especially with the forced ventilationtreatment; with the latter, ethylene concentrations in the culture vessels were virtually zero. In addition, enhancedCO2 supply probably contributed to the better performance under forced ventilation compared to diusiveventilation. Callus developed on the stem bases in all sealed (airtight) and diusive treatments except where AgNO3was used. No callus was observed in any treatment where forced ventilation was applied and in vitro tuberization(tuber size) was considerably improved by this treatment. # 2000 Annals of Botany Company

Key words: Callus, ethylene, potato, tuberization, vitri®cation.

INTRODUCTION

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 eective 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 lÀ1 or even less (Jackson et al ., 1987). Hussey and

Stacey (1981) reported that potato shoots become stoloni-

ferous 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 eects. 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 disease-

free potatoes (Levy et al ., 1993). Miniature tubers (micro-

tubers) formed on plantlets grown in vitro are useful

also because they are very convenient for the maintenance

and handling of disease-free material: microtubers areeasily stored, transferred and distributed (Akita andTakayama, 1994).

The purpose of this project was to improve cultureconditions of potato explants by means other than the useof ethylene absorbers, or antagonists, which can have toxiceects. To this end we explored the eects of improving theeciency of headspace ventilation and thereby of reducingthe concentrations of ethylene. Explants were grown underthree types of ventilation: the sealed condition, diusiveventilation (via a polypropylene membrane) and forcedventilation, each being applied with and without theethylene antagonist, AgNO3 , and the ethylene precursor

1-aminocyclopropane-1-carboxylic acid (ACC) in theculture medium.

The paper also describes ways of improving in vitrotuberization by means of increasing the eciency of headspace ventilation.

M A T 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 tap-water, cut into small pieces of approx. 15 mm3, eachbearing a sprout initial, and were placed in paper bags

inside an incubator at 218C to allow the rapid developmentof white etiolated sprouts which provided the source of the

Annals of Botany 87: 53±59, 2001doi:10.1006/anbo.2000.1299, available online at http://www.idealibrary.com on

0305-7364/01/010053+07 $35.00/00 # 2000 Annals of Botany Company

* For correspondence. Fax 44 (0) 1482 465458, e-mail [email protected]

7/27/2019 Ann Bot 2001 Zobayed 53 9


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

diusive ventilation (capped with polypropylene ®lms)

and containing Murashige and Skoog (MS) medium(Murashige and Skoog, 1962) and 20 g lÀ1 sucrose,

8.0 g lÀ1 agar and no growth regulator. The cultures were

kept in a growth room at 258C under cool-white ¯uorescent

lamps (photosynthetic photon ¯ux 100 mmol mÀ2 sÀ1)

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 oxygenconcentrations

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 Â 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 minÀ1. 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 eect 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 com-

puted from the new IRGA reading after taking due account

of the dilution eect on the sample. Before injection, the

analyser had been calibrated using a 350 ml lÀ1 CO2 supply,

and the subsequent injection samples were added after the

removal of 1.0 cm

3

of gas from the circuit and scavengingthe IRGA CO2 to zero.

Types of ventilation

To achieve the diusive ventilation, a disc of polypro-

pylene membrane (thickness 25 mm; oxygen transmissionrate, 51.8 Â 10À2 m3 mÀ2 dÀ1 MPaÀ1: 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 wassimple and non-mechanized; it is a more convenient andmuch modi®ed form of a prototype described byArmstrong et al . (1997) and ®ts directly onto the culture

vessel (for details see Zobayed et al ., 1999a). Brie¯y, themechanism which creates the pressurized ¯ow depends

upon the humidity-induced diusion of atmospheric gases(O2 , N2 and CO2) into the ventilator through an in¯owNuclepore membrane (pore diameters 0.03±0.05 mm).

Diusion occurs under the in¯uence of a concentrationgradient across this membrane which is induced and

maintained by the higher humidity under the membranerelative to the outside air. Pressurization occurs because

of the continued humidi®cation under the membrane andthe resistance to back ¯ow aorded by its micro-porousnature. A sterile stream of humidi®ed air (5 cm3 minÀ1)

passes into the culture vessel and comparatively free ventingoccurs through an out̄ ow membrane ( pore diameter 0.2 mm).

Eects of ventilation types and the ethylene inhibitor(AgNO3) and the ethylene precursor (ACC) on the growthof nodal stem cuttings

One explant (nodal segment with one unfolded leaf andwith 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 lÀ1 agar, 20 g lÀ1

sucrose, and no growth regulators, and grown with orwithout additives (AgNO3 , 3.0 mmol lÀ1 or ACC,

2.0 mmol lÀ1) to the medium under the following ventila-tion conditions: (a) sealed with silicone rubber bung; (b)sealed AgNO3 in the medium; (c) sealed ACC in the

medium; (d ) diusive ventilation, vessel capped by poly-propylene membrane; (e) diusive ventilation AgNO3 ;

( f ) diusive ventilation ACC; ( g) forced-ventilationapparatus (5 cm3 minÀ1); (h) forced ventilation

AgNO3 ; (i ) forced ventilation

ACC. There were sixreplicates per treatment. The choice of AgNO3 concentra-tion was made after testing a range of concentrations (0.6± 5.0 mmol lÀ1) in sealed vessels. Growth was optimal at

3.0 mmol lÀ1 AgNO3 ; above this, there was some toxiceects on growth.

Ethylene, CO2 and oxygen concentrations were measured

at intervals during the ®rst 21 d of the experiment. Plantletswere grown with continuous illumination in a growth room

where the air temperature was 258C and relative humidity50±65 %. PPF at shelf level was 100 mmol mÀ2 sÀ1.Plantlets were harvested on day 25; growth measurementsincluded leaf number, area and fresh mass, stem fresh mass

and length, root number and maximum length and volumeof callus.

54 Zobayed et al.Ð Ventilation Aects Micropropagation of Potato

7/27/2019 Ann Bot 2001 Zobayed 53 9


In vitro tuberization of potato aected by dierent types of ventilation

For tuberization, nodal segments were inoculated in60 cm3 culture vessels (one per vessel) on MS mediumsupplemented with 6-benzylaminopurine (BAP)(1.0 mg lÀ1), sucrose (80 g lÀ1) and agar (8.0 g lÀ1). The

concentration of BAP and sucrose for optimal growth andtuberization under diusive ventilation (vessels capped withpolypropylene membrane) was previously determined byexperimenting with a range of BAP (0.0±2.0 mg lÀ1) andsucrose concentrations (40 g lÀ1, 80 g lÀ1 and 120 g lÀ1).To examine the eects of ventilation types on tuberization,each vessel was ®tted with either: (a) a silicone rubber bung;(b) a polypropylene membrane; or (c) a forced ventilationapparatus ( ¯ow rate 5.0 cm3 minÀ1). Five replicates wereprepared for each treatment. The cultures were kept at258C under cool-white ¯uorescent lamps (PPF100 mmol mÀ2 sÀ1) and a 16 h photoperiod. Plantlets wereharvested 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

Eects of ventilation types and the ethylene inhibitor(AgNO3) and precursor (ACC) on growth and headspaceatmosphere

Growth. After 25 d of culture, the best growth in thetreatments without additives was observed in explantsgrown 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-foldincrease in leaf area, while leaf fresh mass increased six-foldand the length of the roots also increased signi®cantly.

When plantlets were grown in the tightly-sealed con-dition viz. sealed without additives and sealed ACC,shoots were swollen and the leaves small with a tendency tobe folded. Stem apices became hooked in shape, and rootswere stunted. Some shoots became brown at the tips. Theseresults are consistent with earlier observations of Jacksonet al . (1987) and Hussey and Stacey (1984). In contrast,

plantlets grown under forced ventilation had well-developedshoot and root systems, and morphologically the plantletsappeared normal with normal stem apices. The higher stem

fresh mass found in the sealed (control) and in thesealed ACC treatments compared with their diusivecounterparts (Fig. 1C) may be accounted for by ethylene-induced swelling of the shoots.

With diusive ventilation, and due to the addition of AgNO3 , increased leaf area, leaf fresh mass and stem freshmass were observed and the root length was approximately

doubled (Fig. 1) compared with that of the control. Amajor eect noted in this experiment was the developmentof callus from the base of the stem in the sealed anddiusive 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

callus development, its production is commonly associatedwith 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 largerculture vessels. However, the forced ventilation systemdescribed here would enable the use of smaller vessels.

A further possible advantage of this type of forcedventilation is that the aerating gases are humidi®ed, andthis should help to reduce loss of water vapour from bothplantlets and medium.

It is likely that with longer-term growth under micro-propagation the dierences found in this experiment wouldbecome even more accentuated: for example, it is probablethat CO2 concentration would have been nearer to thecompensation point in the diusive treatments than in theforced ventilation treatments. Consequently, photo-synthetic rates in the forced ventilation treatment wouldhave been greater and the positive feedback eects of thismight well be cumulative beyond the 25 d growth period

adopted here. These ®ndings are in close agreement withthe ®ndings of Zobayed et al . (2000) where forcedventilation was found to improve the growth of Eucalyptusplantlets.

Headspace atmosphere

Ethylene. In sealed vessels, the addition of ACC to themedium resulted in high concentrations of ethylene: afteronly 12 d 1.45 ml lÀ1 had accumulated and this was2.3-times that of the sealed control (Fig. 3). Subsequentlythe ethylene concentrations in the ACC treatment declined

slightly, while in the sealed controls they continued to rise sothat by day 21 the dierences between the two were muchless than previously. In the sealed AgNO3 treatment theethylene concentrations were higher than those of the sealedcontrols; AgNO3 does not inhibit ethylene production and itis presumed that the higher ethylene concentration in thistreatment was a function of the larger plantlets or a lack of feed-back inhibition on biosynthesis. Diusive ventilationresulted in much lower ethylene accumulation and even withACC addition the concentration did not exceed 0.4 ml lÀ1

even at day 21. Nevertheless, it is clear that the poly-propylene membranes helped to reduce ethylene accumula-tion markedly. On the other hand, forced ventilation was

even more eective at minimizing ethylene accumulationand the gas was virtually undetectable even in the ACCtreatments.

Oxygen. In terms of the temporal patterns in headspaceoxygen regime, the results in Fig. 4 reveal very distinctdierences between forced ventilation and the other twoventilating treatments. Thus, with each of the forcedventilation treatments, concentrations remained constantand close to atmospheric for the whole period, whereas withdiusive and sealed ventilation they declined at varying ratesfrom a little above atmospheric. The initial concentrationspresumably re¯ected some photosynthetic enhancement of

oxygen within the headspace. Also, within the diusive andsealed treatments, and presumably due to their eects on the

Zobayed et al.Ð Ventilation Aects Micropropagation of Potato 55

7/27/2019 Ann Bot 2001 Zobayed 53 9


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 experi-

ment: 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 werevery much higher than those of the sealed ACC or sealed

(control) treatments and only a little lower than treatments

having forced ventilation.

The diusely ventilated treatments showed a similar

pattern to their sealed counterparts but the dierences 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, itseems likely that the gradual depression in the oxygen

Control ACC AgNO3Control ACC AgNO3

Control ACC AgNO3 Control ACC AgNO3

Control ACC AgNO3Control ACC AgNO3

0

20

60

40

80

120

100

21

18

15

12

9

6

3

0

50

45

40

35

30

25

20

15

10

5

0

50

40

30

20

10

0

6

5

4

3

2

1

0

33

30

27

24

21

18

15

12

9

6

3

0

L e a f F

M ( m

g )

L e a f a r e a ( c m 2 )

S t e m

h e i g h t ( m

m )

I n c r e a s e d s t e m F M ( m

g )

R o o t n u m b e r p e r p l a n

t l e t

T o t a l r o o t l e n g t h ( m

m )

E F

DC

A B

ax

bx

cx

ax

by

cx

ay

bz

cxcx

bz

az

cx

by

ay

cx

bx

ax

cx

bx

ax

by

ay

ay

cx

bx

ax

ax

bx

cxax

bx

cx

ay

by

cx

ax

bx

cx

ay

bycx

az

az

bx

ax

bx

cx

ay ay

bx

az

bz

cx

F IG . 1. The in¯uence of dierent 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 themean s.e. of six replicates. Signi®cant dierences between ventilation treatments at P4 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 sealedwith silicone rubber bungs (h); diusive, vessels capped with polypropylene discs (D); and forced ventilation rate 5.0 cm3 minÀ1 (E).


7/27/2019 Ann Bot 2001 Zobayed 53 9


concentrations in the sealed and diusive treatments lacking

AgNO3 were due to (a) increased respiratory demandsassociated with the production of varying quantities of non-

photosynthetic callus, and the development of the root

systems, and possibly (b) some degree of senescence aectingthe photosynthetic tissues.

These results are consistent with the ®ndings of some

other authors. In tightly-sealed vessels containing Ficusplantlets, oxygen concentrations of approx. 10 % were

observed (Jackson et al ., 1991). In sealed cauli¯owershoot-culture, Zobayed et al . (1999b), reported an oxygen

concentration of approx. 7.1 % whereas with forced ventila-tion 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 con-

centration in the sealed ACC treatment had reached0.9 %; by day 21 the concentration was nearly 4 %

(Fig. 5A). The eects here and in the sealed (control),diusive ACC, and diusive (control) are probably

Control ACC AgNO3

C a l l u s v o l u m e ( c m 3 p

e r p l a n t l e t )

3.0

2.5

2.0

1.5

1.0

0.5

0.0

ax

bx

ay

by

az az

F I G . 2. The in¯uence of dierent methods of capping of culture vesselson 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 dierences between ventilation treatments at P40.05 indi-

cated by a, b and between respective controls, ACC and AgNO3 treat-ments by x, y, z. Statistical signi®cance was determined by Student-Newman-Keuls test. Sealed, Vessels sealed with silicone rubber bungs

(h); diusive, vessels capped with polypropylene discs (D).

Days

2520151050

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

E t h y l e n e c o n c e n t r a t i o n ( µ l l 1 )

Diffusive+ACC

Diffusive+AgNO3

Diffusive control

Forced vent

Sealed control

Sealed+AgNO3

Sealed+ACC

F I G . 3. Eects of dierent types of ventilation and ACC (2.0 mM) andAgNO3 (3.0 mM) on ethylene concentrations in the headspace of a60 cm3 culture vessel containing in vitro-grown potato plantlets. Theambient relative humidity was 50-65%, temperature was 258C and thePPF was 100 mmol mÀ2 sÀ1. Each symbol represents the mean+ s.e.of ®ve replicates. Sealed, Vessels sealed with silicone rubber bungs;

diusive, vessels capped with polypropylene discs; and forcedventilation rate 5.0 cm3 minÀ1.

Days

252015105010

12

14

16

18

20

22

O x y g e n c o n c e n t r a t i o n ( % )

Sealed+ACC

Sealed control

Diffusive+ACC

Diffusive control

Forced vent

SealedAgNO3

Diffusive+AgNO3

F I G . 4. Eects of dierent types of ventilation and ACC (2.0 mM) andAgNO3 (3.0 mM) on oxygen concentrations in the headspace of a60 cm3 culture vessel containing in vitro-grown potato plantlets. Theambient relative humidity was 50±65 %, temperature was 258C and the

PPF was 100 mmol mÀ2

sÀ1

. Each symbol represents the mean + s.e.of ®ve replicates. Sealed, Vessels sealed with silicone rubber bungs;diusive, vessels capped with polypropylene discs; and forced

ventilation rate 5.0 cm3 minÀ1.


7/27/2019 Ann Bot 2001 Zobayed 53 9


attributable to the respiratory activity of the callus whichdeveloped only in these treatments. Thus, the balance

between photosynthesis and respiration was moved infavour of respiratory CO2 output.

In the other treatments ( forced control, forced

AgNO3 , diusive AgNO3 and sealed AgNO3

treatments) callus did not form and CO2 concentrationsremained relatively constant or declined with time (Fig. 5B)with the decline being greatest where ventilation waspoorest. Thus, in the sealed AgNO3 treatment, CO2

concentrations were at or close to the compensation point(45 ml lÀ1; 50.01%) by day 21. CO2 concentrations wereimproved with diusive ventilation and after 21 d CO2

concentration was 200 ml lÀ1 (0.02%) in the diusiveAgNO3 treatment. In all the forced ventilation treatments,

the CO2 concentrations remained above 300 ml lÀ1 (0.03%)despite the greater CO2 demand associated with the

increased productivity. Again, the results con®rm thebene®ts of forced ventilation.

In vitro tuberization of potato aected by dierentventilation treatments

The numbers and fresh mass of tubers were very low inthe sealed condition compared with those produced withdiusive or forced ventilation. Compared to diusiveventilation, forced ventilation did not signi®cantly increase

the number of tubers but it did increase their fresh masswhich was almost double that of tubers in the diusive

treatment. However, the shoots became swollen in placesafter 4 weeks of culture in forced ventilation. In contrast,very little tuberization occurred in the sealed condition.

Jackson et al . (1987) found no eect of ethylene on theinduction of tuberization. In contrast, Hussey and Stacey(1984) reported that the addition of potassium permang-anate to the culture vessel to absorb ethylene markedlyincreased tuberization in potato. They also reported thatthe presence of ethylene tended to make the shoots becomestoloniferous (Hussey and Stacey, 1981). Mingo-Castel et al .(1976) reported that ethylene inhibits tuberization. More-

over, they showed that increased CO2 concentrationpromotes tuberization. In the present investigation nospeci®c attempt was made to ®nd out whether ethyleneaected tuberization. However, since the numbers of tuberswere similar with diusive and forced ventilation, it seemslikely that the low concentrations of ethylene in vessels withdiusive ventilation were insucient to cause inhibition.The poor tuber initiation in sealed vessels might have beendirect, i.e. due to ethylene inhibition of tuber formation, orindirect i.e. growth inhibition of the plantlets may havedelayed their attainment of tuber-producing physiologicalage.

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 vesselheadspace and/or the lack of ethylene in the culture vesselheadspace. Since the results have shown a positive eect of CO2 enrichment on shoot growth over and above that of ethylene removal, it seems very likely that the greater yieldof tubers receiving forced rather than diusive ventilationcould have been largely due to the greater photosynthateproduction of the larger plantlets.

Finally, it should also be noted that in potato, short daysand low temperatures generally favour tuberization. Inthese experiments a 16 h photoperiod was provided andthe temperature was 258C. It is anticipated that tuberiza-

tion might be further improved by providing a shorterphotoperiod (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 aforced ventilation system.

ACKNOW LEDGEM ENTS

We are very grateful to Mrs Margaret Huee for technicalhelp and advice and especially with the work involving the

GLC. We are also grateful to Mr Mike Bailey forfabricating the ventilating systems, Dr M. B. Jackson of

A

B

2520151050

Time (d)

0

0.05

0.1

0.15

0.2

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

C O 2 c o n c e n t r a t i o n ( % )

C O 2 c o n c e n t r a t i o n ( % )

Sealed+ACC

Sealed control

Diffusive+ACCDiffusive control

Sealed+ACC

Sealed controlDiffusive+ACC

Diffusive control

Sealed+AgNO3

Forced controlForced+AgNO3

Diffusive+AgNO3

Forced+ACC

Forced vent

F IG . 5. Eects of dierent types of ventilation and ACC (2.0 mM) and

AgNO3 (3.0 m

M

) on CO2 concentrations in the headspace of a 60 cm

3

culture vessel containing in vitro-grown potato plantlets. The ambientrelative humidity was 50±65 %, temperature was 258C and the PPF was100 mmol mÀ2 sÀ1. Each symbol represents the mean+ s.e. of ®vereplicates. Sealed, Vessels sealed with silicone rubber bungs; diusive,vessels capped with polypropylene discs; and forced ventilationrate 5.0 cm3 minÀ1. A, CO2 concentration on a scale of zero to

4 %. B, CO2 concentration on scale of zero to 0.2 %.


7/27/2019 Ann Bot 2001 Zobayed 53 9


Long Ashton Research Station for advice on ethylenemeasurement 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 eects on culturegrowth. Physiologia Plantarum 78: 526±531.

Akita M, Takayama S. 1994. Stimulation of potato (Solanumtuberosum L.) tuberization by semicontinuous liquid mediasurface 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 ventilationsystem bene®ts Annona squamosa L. explants and coconut calloid.Annals of Botany 79: 31±40.

Armstrong W. 1994. Polarographic oxygen electrodes and their use inplant aeration studies. Proceedings of the Royal Society of Edinburgh 102B: 511±527.

Hussey G, Stacey NJ. 1981. In vitro propagation of potato (Solanumtuberosum L.). Annals of Botany 48: 787±796.

Hussey G, Stacey NJ. 1984. Factors aecting 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 RegulatorGroup, 57±71.

Jackson MB, Abbott AJ, Belcher AR, Hall KC, Butler R, Cameron J.1991. Ventilation in plant tissue culture and eects of pooraeration on ethylene and carbon dioxide accumulation, oxygendepletion and explant development. Annals of Botany 67:229±237.

Levy D, Seabrook JEA, Coleman S. 1993. Enhancement of tuberizationof axillary shoot buds of potato (Solanum tuberosum L.) cultivarscultured in vitro. Journal of Experimental Botany 44: 381±386.

Mingo-Castel AM, Smith OF, Kumamoto J. 1976. Studies on thecarbon dioxide promotion and ethylene inhibition of tuberizationin potato explants cultured in vitro. Plant Physiology 57: 480±485.

Murashige T, Skoog F. 1962. A revised medium for rapid growth andbioassays with tobacco tissue culture. Physiologia Plantarum 15:473±479.

Paque M, Boxus P. 1987. `Vitri®cation': review of literature. ActaHorticulturae 212: 155±166.

Ziv M. 1991. Vitri®cation: morphological and physiological disordersof in vitro plants. In: Debergh PC, Zimmerman RH, eds. Micro-

propagation. The Netherlands: Kluwer Academic Publishers,45±69.

Zobayed SMA, Armstrong J, Armstrong W. 1999a. Evaluation of aclosed system, diusive and humidity-induced convective through-¯ow ventilation on the growth and physiology of cauli¯ower invitro. Plant Cell Tissue and Organ Culture 59: 113±123.

Zobayed SMA, Armstrong J, Armstrong W. 1999b. Cauli¯ower shoot-culture eects of dierent types of ventilation on growth andphysiology. Plant Science 141: 221±231.

Zobayed SMA, Afreen F, Kubota C, Kozai T. 2000. Mass propagationof Eucalyptus camaldulensis in a scaled-up vessel under in vitrophotoautotrophic condition. Annals of Botany 85: 587±592.


ann bot 2001 zobayed 53 9

Documents