Annals of Botany 87: 5359, 2001 doi:10.1006/anbo.2000.1299, available online at http://www.idealibrary.

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Micropropagation of Potato: Evaluation of Closed, Diusive 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
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. Growth was substantially enhanced and vitrication (stunting and epinasty of leaves and hooking of stem apices) was reduced by increasing the eciency of ventilation, the eects being greatest with forced ventilation. In the conventional diusive treatment (via a polypropylene membrane), leaf epinasty occurred but the leaves were not stunted unless ACC had been added. AgNO3 prevented vitrication 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 diusive AgNO3 treatment. Ethylene removal was clearly an important factor contributing to the better growth found with diusive 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 diusive ventilation. Callus developed on the stem bases in all sealed (airtight) and diusive 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, vitrication.

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 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 l 1 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 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 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. zobayed@biosci.hull.ac.uk

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 eects. To this end we explored the eects of improving the eciency of headspace ventilation and thereby of reducing the concentrations of ethylene. Explants were grown under three types of ventilation: the sealed condition, diusive 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 eciency 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

0305-7364/01/010053+07 $35.00/00

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Zobayed et al. Ventilation Aects Micropropagation of Potato

Types of ventilation To achieve the diusive ventilation, a disc of polypropylene membrane (thickness 25 mm; oxygen transmission rate, 51.8 10 2 m3 m 2 d 1 MPa 1: 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 modied form of a prototype described by Armstrong et al. (1997) and ts directly onto the culture vessel ( for details see Zobayed et al., 1999a). Briey, the mechanism which creates the pressurized ow depends upon the humidity-induced diusion of atmospheric gases (O2 , N2 and CO2) into the ventilator through an inow Nuclepore membrane ( pore diameters 0.030.05 mm). Diusion occurs under the inuence 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 humidication under the membrane and the resistance to back ow aorded by its micro-porous nature. A sterile stream of humidied air (5 cm3 min 1) passes into the culture vessel and comparatively free venting occurs through an outow membrane ( pore diameter 0.2 mm).

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 l 1 sucrose, 8.0 g l 1 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 m 2 s 1) 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 (6080 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 identied by a retention time of about 1.4 min. Nitrogen was used as the carrier gas at a rate of 60 cm3 min 1. The identication of the ethylene peak was separately conrmed 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 typetip 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 computed 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 l 1 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.

Eects 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 1 agar, 20 g l 1 sucrose, and no growth regulators, and grown with or without additives (AgNO3 , 3.0 mmol l 1 or ACC, 2.0 mmol l 1) 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) diusive ventilation, vessel capped by polypropylene membrane; (e) diusive ventilation AgNO3 ; ( f ) diusive ventilation ACC; ( g) forced-ventilation apparatus (5 cm3 min 1); (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 1) in sealed vessels. Growth was optimal at 3.0 mmol l 1 AgNO3 ; above this, there was some toxic eects 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 5065 %. PPF at shelf level was 100 mmol m 2 s 1. 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. Ventilation Aects Micropropagation of Potato

In vitro tuberization of potato aected by dierent 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 1), sucrose (80 g l 1) and agar (8.0 g l 1). The concentration of BAP and sucrose for optimal growth and tuberization under diusive ventilation (vessels capped with polypropylene membrane) was previously determined by experimenting with a range of BAP (0.02.0 mg l 1) and sucrose concentrations (40 g l 1, 80 g l 1 and 120 g l 1). 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 ventilation apparatus ( ow rate 5.0 cm3 min 1). Five replicates were prepared for each treatment. The cultures were kept at 258C under cool-white uorescent lamps (PPF 100 mmol m 2 s 1) and a 16 h photoperiod. Plantlets were harvested after 8 weeks; fresh mass and numbers of tubers were recorded.

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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 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 signicantly. 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 diusive counterparts (Fig. 1C) may be accounted for by ethyleneinduced swelling of the shoots. With diusive 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 eect noted in this experiment was the development of callus from the base of the stem in the sealed and diusive 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 associated with vitrication (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 humidied, 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 dierences 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 diusive treatments than in the forced ventilation treatments. Consequently, photosynthetic rates in the forced ventilation treatment would have been greater and the positive feedback eects 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 1 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 dierences 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. Diusive ventilation resulted in much lower ethylene accumulation and even with ACC addition the concentration did not exceed 0.4 ml l 1 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 eective 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 dierences 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 diusive and sealed ventilation they declined at varying rates from a little above atmospheric. The initial concentrations presumably reected some photosynthetic enhancement of oxygen within the headspace. Also, within the diusive and sealed treatments, and presumably due to their eects on the

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Zobayed et al. Ventilation Aects Micropropagation of Potato

33 30 27 24 21 18 15 12 9 6 3 0 A
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ax ay ay bx cx

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F I G . 1. The inuence 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 the mean s.e. of six replicates. Signicant dierences 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 signicance was determined by Student-Newman-Keuls test. Sealed, Vessels sealed with silicone rubber bungs (h); diusive, vessels capped with polypropylene discs (D); and forced ventilation rate 5.0 cm3 min 1 (E).

plantlets, AgNO3 or ACC additions can be seen to have inuenced 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 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, it seems likely that the gradual depression in the oxygen

Zobayed et al. Ventilation Aects Micropropagation of Potato

3.0 Callus volume (cm3 per plantlet) 2.5 2.0 1.5 1.0 0.5 0.0 ax 20
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F I G . 2. The inuence of dierent 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. Signicant dierences between ventilation treatments at P40.05 indicated by a, b and between respective controls, ACC and AgNO3 treatments by x, y, z. Statistical signicance was determined by StudentNewman-Keuls test. Sealed, Vessels sealed with silicone rubber bungs (h); diusive, vessels capped with polypropylene discs (D).

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F I G . 4. Eects of dierent 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 5065 %, temperature was 258C and the PPF was 100 mmol m 2 s 1. 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 min 1.

0.6

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concentrations in the sealed and diusive 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 aecting 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 cauliower 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 25 % 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 eects here and in the sealed (control), diusive ACC, and diusive (control) are probably

F I G . 3. Eects of dierent 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 2 s 1. 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 min 1.

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4.0 3.5 CO2 concentration (%) 3.0 2.5 2.0 1.5 1.0 0.5 0 A

Zobayed et al. Ventilation Aects Micropropagation of Potato

Sealed + ACC

In vitro tuberization of potato aected by dierent ventilation treatments The numbers and fresh mass of tubers were very low in the sealed condition compared with those produced with diusive or forced ventilation. Compared to diusive ventilation, forced ventilation did not signicantly increase the number of tubers but it did increase their fresh mass which was almost double that of tubers in the diusive 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 eect 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 specic attempt was made to nd out whether ethylene aected tuberization. However, since the numbers of tubers were similar with diusive and forced ventilation, it seems likely that the low concentrations of ethylene in vessels with diusive ventilation were insucient 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 eect 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 diusive 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. 68 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 Huee 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

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F I G . 5. Eects of dierent 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 5065 %, temperature was 258C and the PPF was 100 mmol m 2 s 1. 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 min 1. 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 diusive 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 1; 50.01 %) by day 21. CO2 concentrations were improved with diusive ventilation and after 21 d CO2 concentration was 200 ml l 1 (0.02 %) in the diusive AgNO3 treatment. In all the forced ventilation treatments, the CO2 concentrations remained above 300 ml l 1 (0.03 %) despite the greater CO2 demand associated with the increased productivity. Again, the results conrm the benets of forced ventilation.

Zobayed et al. Ventilation Aects 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
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Jackson MB, Abbott AJ, Belcher AR, Hall KC, Butler R, Cameron J. 1991. Ventilation in plant tissue culture and eects of poor aeration on ethylene and carbon dioxide accumulation, oxygen depletion and explant development. Annals of Botany 67: 229237. 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: 381386. 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: 480485. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15: 473479. Paque M, Boxus P. 1987. `Vitrication': review of literature. Acta Horticulturae 212: 155166. Ziv M. 1991. Vitrication: morphological and physiological disorders of in vitro plants. In: Debergh PC, Zimmerman RH, eds. Micropropagation. The Netherlands: Kluwer Academic Publishers, 4569. Zobayed SMA, Armstrong J, Armstrong W. 1999a. Evaluation of a closed system, diusive and humidity-induced convective throughow ventilation on the growth and physiology of cauliower in vitro. Plant Cell Tissue and Organ Culture 59: 113123. Zobayed SMA, Armstrong J, Armstrong W. 1999b. Cauliower shootculture eects of dierent types of ventilation on growth and physiology. Plant Science 141: 221231. 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: 587592.