Influence of explant, temperature and different culture vessels on in vitroculture for germplasm maintenance of four mint accessions

Md. Tariqul Islam1,*, D. Philibert Dembele2 & E.R. Joachim Keller31Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute (BARI), Joydebpur,Gazipur-1701, Bangladesh; 2Institut d’ Economie Rurale, URG, BP 30, Bamako, Republic of Mali; 3Instituteof Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany(*requests for offprints; E-mail: tariqulislam_1999@yahoo.com)

Received 3 March 2003; accepted in revised form 15 September 2004

Key words: culture vessel, explant, medium depletion, Mentha, micropropagation, temperature variation

Abstract

Two experiments were conducted with four accessions of mint (Mentha spp.) on MS medium for theirin vitro performance. In the first experiment apical and nodal explants were cultured at both 20 �C and25 �C. Data was recorded at second and at fifth week. Both apical and nodal explants of mint showedbetter leaf production at 25 �C than 20 �C. Nodal explants of mint cultured at 25 �C in both cultivationperiods exhibited the highest number of leaves. In the second experiment apical explants were cultured infour different culture vessels viz., industrial glass jar (IG), magenta vessel (MV), Erlenmeyer flask (EF) andculture tube with 1(CT1) and 2(CT2) explants at 25 �C for 6 weeks. The highest weight loss from the media,evapo-transpiration and fresh weight gain were recorded in IG and next in MV. The lowest weight lossfrom the media and fresh weight gain both were found in CT2. However the lowest evapo-transpiration wasnoted in EF. The highest numbers of leaves were recorded from MV. Without explants, depletion ofmedium and increase of headspace were both higher in IG than in the other vessels. Overall, Magenta vesselGA 7 showed the best in vitro performance.

Abbreviations: CT – culture tubes; EF – Erlenmeyer flasks; IG – industrial glass jars; MV – Magentavessels; MS – Murashige and Skoog

Introduction

The application and economic importance of mints(Mentha spp.) are increasing due to the productionof mint oil as raw material for pharmaceutical andcosmetic uses as well as for flavouring food, bev-erages and tobacco. In vitro culture is usuallyperformed in closed vessels. Plants grow hetero-trophically on an agar-based medium containing acarbon source. Amongst other factors, cultureduration and different types of explant can influ-ence growth and development of a particular spe-cies. Many authors work on mint tissue culture(Bhaumik and Datta, 1989), multiplication(Ruseva, 1999), in vitro conservation (Islam et al.,

2003) and cryopreservation (Towill, 1990). Nor-mally, apical and nodal explants of mint are usedfor micropropagation, but their in vitro perfor-mances are scanty. With this view in mind, it isnecessary to identify the suitable explant type andoptimal temperature for micropropagation ofmint. The covering of the culture vessels protectsthe culture medium from microbial infections andprevents excessive evaporation of water from theculture medium. The type of vessel closure affectsthe gaseous composition (Lentini et al., 1988) aswell as the light environment and hence growth oftissues in culture such as shoot elongation, prolif-eration, and fresh weight increase as well as pos-sibly hyperhydric degradation processes (Mackay

Plant Cell, Tissue and Organ Culture (2005) 81:123–130 � Springer 2005

and Kitto, 1988; Mcclelland and Smith, 1990).Growth and development are affected by the gasexchange in plant tissue culture (Jackson, 2003).Lai et al. (1998) reported increased growth as aresult of improved ventilation of cultures in pa-paya while Murphy et al. (1998) and Jackson et al.(1994) reported no increase in growth of Delphin-inum and Solanum tuberosum, respectively. Venti-lation also improved acclimatization of plants afterremoval from in vitro conditions (Gribaudo et al.,2001a,b). Loose closures were reported to be betterthan tight ones for reducing hyperhydricity inGypsophila paniculata (Dillen and Buysens, 1989)and for promoting the growth of strawberryplantlets (Kozai and Sekimoto, 1988). Informationon the gaseous compounds accumulated in in vitrocultures focuses on ethylene, which compoundplays important regulatory roles in growth anddevelopment in vitro (Biddington, 1992). Differentculture vessels such as industrial glass jars (IG),Magenta vessels (MV), Erlenmeyer flasks (EF) andculture tubes (CT) are used for in vitro culture ofmint. A number of about 200 indigenous andexotic mint accessions are maintained at theInstitute of Plant Genetics and Crop Plant Re-search (IPK), Germany. Among them, fouraccessions are used as standards for long termexperiments. This is a long term study on slowgrowth culture (in vitro storage) of four mintaccessions. In the first attempt the accessions weresuccessfully in vitro conserved at +2 �C for6 months, and the plantlets were successfullyestablished (survival >90%) ex vitro (Islam et al.,2003). Now, in this study an attempt is made tofind out the influence of explant types and tem-perature levels on micropropagation and the typeof culture vessel suitable for in vitro culture forgermplasm maintenance of four mint accessions.

Materials and methods

Plant material and culture media

The work was conducted at the Institute of PlantGenetics and Crop Plant Research (IPK), Gater-sleben, Germany in 1999. Two experiments werecarried out with four accessions of mint, viz., theaccessions MEN 204 (Mentha · villosa Huds.),MEN 148 (Mentha · villosa Huds.) and MEN 186(Mentha · piperita L.), and MEN 166 (Mentha ·

piperita L.) which were diploid, tetraploid andoctoploid, respectively. MEN 148, MEN 166,MEN 186 and MEN 204 were obtained from Iraq,Germany, Cuba andCanada, respectively.MS salts(Murashige and Skoog, 1962) were supplementedwith 0.5 mg l)1 nicotinic acid, 0.5 mg l)1 pyridox-ine-HCl, 0.1 mg l)1 thiamine-HCI, 100 mg l)1

myo-inositol, 2 mg l)1 glycine and 3% sucrose. Noplant growth regulators (PGR) were used. Themedium pHwas adjusted to 6.8 with NaOH or HCland then autoclaved at 121 �C for 25 min.

Experiment I

Culture conditions

Apical and nodal explants, having one node andone leaf pair were collected from glasshouse grownplants. The explants were surface-sterilised with70% ethanol, sodium hypochlorite and Tween 20for 10 min. Apical and nodal explants of about10 mm in length were excised aseptically andinoculated into 10 · 6 cm MV GA-7 containing80 ml of medium, five explants in each vessel. Theexplants were cultured at 20 �C in a growth roomfor 1 week and then at 10 �C for 3 weeks, bothunder fluorescent light of 35–55 lM m)2 s)1 for16 h daily. Finally, the explants were cultured at+2 �C growth room for 6 months (under fluores-cent light of 35–55 lM m)2 s)1 for 16 h daily) aspreviously described by Islam et al. (2003). Apicaland nodal explants, having one node and one leafpair, were collected from in vitro ready stockplantlets. Before collecting the explants, the donorcultures were maintained at 20 �C for about1 week. Apical and nodal explants of about 10 mmin length were excised aseptically and inoculatedinto MV containing 80 ml of MS medium provid-ing five explants in each vessel. Fifty apical and fiftynodal explants were cultured both at 20 �C and25 �C growth rooms for 5 weeks under fluorescentlight of 35–55 lM m)2 s)1 for 16 h daily.

Data collection and statistical analysis

Numbers of leaves (leaf size at least 2 mm inlength) were counted after 2 weeks. After 5 weeks,numbers of leaves and root development (few andprofuse) were recorded. The experiment was laidout in a Completely Randomized Design and the

124

treatment means were separated using LSD(Gomez and Gomez, 1984).

Experiment II

Culture vessels and closure

The internal volumes of four types of culturevessels, viz., IG, MV, EF and culture tubes (CT1and CT2, with one or two explants, respectively)were 330, 380, 163 and 53 ml, respectively. In IG, athin ring of cotton felt was attached with the clo-sure. The closure was attached on the mouth of theglass jar by a clip so that ventilation occurredeasily through the felt ring. In MV, a ventilationsystem is provided by the manufacturer in theclosure. Aluminum foil was used as closure in bothEF and CT. This aluminum foil was attached byhand pressure.

Culture conditions

Each culture vessel of IG, MV, EF, CT1 and CT2contained 50, 50, 30, 10 and 10 ml MS media,respectively. Apical explants of about 10 mm longwith one node and one leaf pair, were excised fromin vitro plantlets of Experiment I and inoculatedinto four different culture vessels containing MSmedia without hormones. Number of apicalexplants per vessel were five, five, three, one andtwo in IG, MV, EF, CTI and CT2, respectively,resulting in a total of 30, 30, 30, 18 and 36 explants.The explants were cultured at 25 �C for 6 weeksunder fluorescent light of 35–55 lM m)2 s)1 for16 h daily. First, empty culture vessels wereweighed, then re-weighed with media and thenmedia and explants to determine the initial explantand medium fresh weights. After 6 weeks culture,the vessels were again weighed first with and thenwithout plantlets to allow estimation of the finalfresh weight of the plant tissue, medium weight andhence water losses from the medium. The freshweight gain was measured by subtracting the initialexplant weight from the final fresh weight. Weightloss due to evapo-transpiration was calculated asthe differences between total loss and fresh weightgain.

For measuring medium depletion and increaseof headspace over the media, 50, 50, 30 and 10 mlMS media (without explant) were poured into six

IG,MV, EF and CT, respectively, as control. Usingthree control vessels from each group, the initialheadspace over the media was determined by usingwater.With the remaining three vessels, the amountof fresh media weight was measured by subtractingthe empty vessel from the vessel with media. Thenthe vessels were kept at 25 �C in the same growthroom. After 6 weeks, the culture vessels were againweighed first and then the final headspace wasmeasured by water. Mean medium depletion andincrease of headspace were measured.

Data collection and statistical analysis

Number of leaves, weight of fresh biomass andremaining MS media (g) in the vessels wererecorded.

Results

Experiment I

Numbers of leaves developed from apical andnodal explants at 20 �C and 25 �C after 2 and5 weeks of culture in MS are shown in Table 1.Nodal explants produced the highest number ofleaves at 25 �C. On average, both explant typesproduced 14% more leaves at 25 �C than at 20 �Cafter 2 and 11% more after 5 weeks. Apical ex-plants of MEN 166 exhibited the maximumnumber of leaves at both temperature levels.However, for nodal explants the maximum num-ber of leaves was obtained from MEN 148. After5 weeks, however, MEN 186 produced the highestnumber of leaves (Table 1). After 5 weeks, bothexplant types produced more roots at 25 �C thanat 20 �C irrespective of accessions (data notshown).

Experiment II

Plants developed from MEN 186 after 6 weeksculture of apical explants are shown in Figure 1.On average, IG lost more weight (55, 76, 55 and77%) than MV, EF, CT1 and CT2, respectively,irrespective of accessions (Table 2). However,across the culture vessels, MEN 204 lost moreweight (11, 2 and 14%) than MEN 148, MEN 166and MEN 186, respectively. The lowest weightreduction was found in CT2 with all the accessions

125

Table 1. Numbers of leaves developed from apical and nodal explants at 20 �C and 25 �C after 2 and 5 weeks culture in MS

Accession Two weeks Five weeks

Apical Nodal Mean Apical Nodal Mean

20 �C 25 �C 20 �C 25 �C 20 �C 25 �C 20 �C 25 �C

MEN 148 8.72b 10.04c 10.80a 12.72a 10.57 14.96c 17.68b 18.92b 21.08b 18.16

MEN 166 9.68a 11.96a 9.20b 11.40b 10.56 13.08d 16.08b 15.52c 19.44b 16.03

MEN 186 9.12ab 10.80b 10.48a 12.64a 10.76 21.56a 22.00a 21.18a 24.08a 22.21

MEN 204 8.80b 9.44d 9.28b 9.48c 9.25 15.20b 17.12b 16.92c 16.84c 16.52

Mean 9.08 10.56 9.94 11.56 16.20 18.22 18.14 20.31

LSD (5%) 0.81 0.56 0.62 1.06 1.72 2.18 1.62 1.95

aIn a column any two means having a common letter are not significantly different at 5% level.bMean of five replications and mean of ten observations represent one replication.

Figure 1. Plantlets of mint MEN 186 developed from apical explants onMSmedium after six weeks culturing in: (a) Industrial glass jar,(b) Magenta vessel GA-7, (c) Erlenmeyer flask, (d) Culture tubes with one explant per test tube and (e) with two explants per test tube.

126

(Table 2). In all cases, weight loss of the mediacorresponded with weight gain of the plantlets. Onaverage, the explants cultured in IG gained moreweight (10, 33, 42 and 58%) than those in MV, EF,CT1 and CT2, respectively, irrespective of acces-sions. On average, the maximum (0.97 g) and theminimum (0.56 g) fresh weight gain were foundwith MEN 148 and MEN 186, respectively, acrossthe culture vessels. The highest fresh weight gainwas observed in all the vessels with MEN 148. Thelowest one in all the vessels with MEN 186, exceptwith CT1. The highest evapo-transpiration wasfound in IG with all accessions. On average, in IGmore weight loss occurred (66, 87, 59 and 82%)than in MV, EF, CT1 and CT2, respectively,irrespective of accessions. Except MEN 148, thelowest evapo-transpiration was observed in EFwith all accessions. On average, MEN 166 lostmore weight (33, 14 and 6%) than MEN 148,MEN 186 and MEN 204, respectively (Table 2).

After 6 weeks, vessels without plants of IGlost significantly more weight from the media (53,58 and 63%) than MV, EF and CT, respectively(Table 3). Furthermore, IG and EF had a

significantly higher increase in headspace than theremaining two vessels. Plants developed fromapical explants after 6 weeks (Figure 1) had, onaverage, a greater increase in leave numbers,which was 6, 2, 5 and 23% more than those withIG, EF, CT1 and CT2, respectively, irrespectiveof accessions (Table 4). However across the ves-sels, MEN 148 produced more leaves (69, 12 and22%) than MEN 166, MEN 186 and MEN 204,respectively. The highest number of leaves wasobtained from all the vessels with MEN 148 ex-cept IG with MEN 186. However the lowest leafnumbers were found in CT2 with all accessions.

Discussion

Experiment I

Both explants types grew slower at lower temper-ature. In terms of leaf numbers the growthresponse of nodal explants was better than that ofapical ones. This may also be genotype dependent.We used 10 mm long apical and nodal explants

Table 2. Weight loss from the culture media, fresh biomass gain of plantlets and calculated loss of water by evapo-transpiration after6 weeks culture in different vesselsa

MEN 148 MEN 166 MEN 186 MEN 204 Mean

Weight loss (g)

IG 4.46 ± 0.14 5.24 ± 0.21 4.84 ± 0.16 4.73 ± 0.21 4.82

MV 1.61 ± 0.06 2.56 ± 0.06 1.73 ± 0.08 2.87 ± 0.09 2.19

EF 1.29 ± 0.20 1.11 ± 0.25 1.02 ± 0.21 1.27 ± 0.25 1.17

CT1 2.37 ± 0.01 2.10 ± 0.02 2.00 ± 0.02 2.17 ± 0.01 2.16

CT2 1.16 ± 0.02 1.06 ± 0.02 0.94 ± 0.03 1.25 ± 0.02 1.10

Mean 2.18 2.41 2.11 2.46

Fresh weight gain (g)

IG 1.33 ± 0.06 0.79 ± 0.08 0.77 ± 0.03 1.14 ± 0.22 1.01

MV 1.29 ± 0.06 0.77 ± 0.08 0.55 ± 0.02 1.01 ± 0.11 0.91

EF 0.90 ± 0.05 0.63 ± 0.05 0.50 ± 0.02 0.67 ± 0.11 0.68

CT1 0.81 ± 0.04 0.40 ± 0.02 0.63 ± 0.01 0.51 ± 0.04 0.59

CT2 0.50 ± 0.03 0.43 ± 0.06 0.35 ± 0.01 0.42 ± 0.03 0.43

Mean 0.97 0.60 0.56 0.75

Evapo-transpiration (g)

IG 3.13 4.45 4.07 3.59 3.81

MV 0.32 1.79 1.18 1.86 1.29

EF 0.39 0.48 0.52 0.60 0.50

CT1 1.56 1.70 1.37 1.66 1.57

CT2 0.66 0.63 0.59 0.83 0.68

Mean 1.21 1.81 1.55 1.71

aEach value represents a mean ± standard error.

127

but their initial weights were not the same. Nodalexplants had more weight than apical ones. Nodalexplants initiated normally a pair of shoots firstand then further leaves, whereas in apical explantsthe tip simply continued growing. Therefore, nodalexplants produced more leaves than apical ones.Pierik (1998a) reported that larger explantssometimes regenerate easier than smaller ones andthat larger explants produce more shoots in vitro.Islam et al. (2003) reported that the nodal explantsdevelop more leaves and branches than apical onesconserved in vitro.

Experiment II

The four mint accessions responded differently todifferent culture vessels. The highest fresh weightgain and increase in leaf number were always ob-tained with MEN 148. The in vitro growth mightbe related to many factors like culture vessel types,headspace, ventilation through the closure and thenumber of explants per vessel. So, the maximumfresh weight occurred in IG where the ventilationwas best and the second highest headspace incomparison with the other vessel types. The max-imum number of leaves occurred in MV where themaximum headspace and the ventilation werecomparatively better than EF and CT. With or

without explants, the highest weight loss of mediawas found in IG and next in MV after 6 weeks.Correspondingly, maximum ventilation occurredin IG followed by MV and EF or CT. The resultsindicated that the environment of the culture ves-sel’s headspace has a strong effect on growth anddevelopment of mint culture. On the other hand,after 6 weeks culture in IG, we observed moredegradation like older leaves dropping down andmedium cracking. The leaves were looking morehealthy in MV then in IG. This may be due tohigher water loss in IG. Therefore, MV were betterthan IG for culturing mint. In carnation (Majadaet al., 1997) and rose (Ghashghaie et al., 1992),ventilation induced resistance to water loss. Jack-son et al. (1991) did not find any differences infresh weight due to ventilation and they reportedno differences in the growth of either Gerberajamesonii or Ficus lyrata growing in vessels with awide range of gas exchange rates. Reduction inshoot length, number of leaves, and also leaf areawas reported for potato plantlets grown underreduced relative humidity (Kozai et al., 1993). Thetype of closure of the vessel has been shown toaffect the internal gas composition (De Proft et al.,1985; Lee and Shuler, 1991). In general, loosefitting closures were better than tighter ones forimproving growth and morphogenesis and for

Table 3. Medium depletion and increase of headspace of different culture vessels without explant after 6 weeks

Type of vessel Depletion of

50 ml vessel medium

after six weeks (g)

Percent of medium

depletion after

six weeks

Increase of head space

after six weeks (ml)

Percent of head space

increase after six weeks

IG 9.41 ± 1.08 20.01 28.00 ± 0.88 10.49

MV 4.44 ± 0.05 9.44 17.33 ± 0.67 5.42

EF 2.27 ± 0.39 8.36 13.00 ± 0.33 11.11

CT 0.68 ± 1.41 7.37 1.67 ± 0.33 3.79

Table 4. Number of leaves developed after 6 weeks culture on different culture vessels of four mints accessions

Type of vessel MEN 148 MEN 166 MEN 186 MEN 204 Mean

IG 31.00 ± 1.00 10.40 ± 0.72 37.47 ± 1.56 30.40 ± 2.76 27.32

MV 41.60 ± 1.62 11.50 ± 0.72 31.20 ± 1.08 31.93 ± 1.74 29.06

EF 43.33 ± 1.70 13.20 ± 1.01 30.17 ± 1.23 27.73 ± 2.36 28.61

CT1 36.89 ± 1.41 11.06 ± 0.47 34.94 ± 0.90 27.83 ± 1.17 27.68

CT2 29.56 ± 1.36 9.65 ± 0.51 26.39 ± 0.90 23.94 ± 0.92 22.39

Mean 36.48 11.16 32.03 28.37

128

overcoming hyperhydricity. Beneficial effects oflarger air volumes or larger vessels (Pevalek-Koz-lina, 1990; McClelland and Smith, 1990) wereinterpreted as causing a better composition of thegaseous components as carbon dioxide, oxygen,ethylene, etc. mainly by retarding accumulation ofunfavourable gases. Depletion of culture mediamay depend on the closure type, which affects invitro growth (Pierik 1998b). Furthermore, thenumber of explants in vitro was an importantfactor. CT1 and CT2 are the same type of vessel,CT1 containing one, the other variant two ex-plants. Woltering (1986) cultured Gerbera inpolypropylene units, closely sealed systems withhigh concentration of both ethylene and CO2,which caused conditions detrimental to growth.

Conclusion

This analysis was performed in order to find themost suitable conditions for the maintenance ofmint germplasm in the warm phase of medium-term culture cycles without forcing the multipli-cation rate by hormone application. The resultsindicate that an increase of the cultivation tem-perature from 20 �C to 25 �C can stimulate the invitro growth of mints and that nodal explants weremore productive than apical ones. Growth anddevelopment of mint were mainly depending onthe vessel closure, ventilation and the headspaceper culture unit. High evaporation of mediacaused adverse effect on growth of in vitro plants.MV GA-7 was better than the other vessels for invitro culture of mint germplasm.

Acknowledgements

We would like to thank the German Foundationfor International Development (DSE) and theInstitute of Plant Genetics and Crop Plant Re-search (IPK, Gatersleben, Germany) for providingfinancial and technical supports to carry out theresearch work.

References

Bhaumik C & Datta PC (1989) Development of Japanese mint

tissue culture method. Indian-Perfumer 33: 165–168

Biddington NL (1992) The influence of ethylene in plant tissue

culture. Plant Growth Regul. 11: 173–187

De Proft MP, Meane LJ & Debergh PC (1985) Carbon dioxide

and ethylene evolution in the culture atmosphere ofMagnolia

cultured in vitro. Physio. Plant. 65: 375–379

Dillen W & Buysens S (1989) A simple technique to overcome

vitrification in Gypsophila paniculata L. Plant Cell Tiss. Org.

Cult. 19: 181–188

Ghashghaie J, Brenchkmann F & Saugier B (1992) Water

relations and growth of rose plants cultured in vitro under

various relative humidities. Plant Cell Tiss. Org. Cult. 30: 51–57

Gomez KA & Gomez AA (1984) Statistical Procedures for

Agricultural Research, 2nd ed. John Wiley and Sons, (pp.

187–215), Singapore

Gribaudo I, Novello V & Restagno M (2001a) Improved

control of water loss from micropropagated grapevines (Vitis

vinifera cv. Nebbiolo). Vitis 40: 137–140

Gribaudo I, Restagno M & Novello V (2001b) Vented vessels

affect growth rate ofVitis vinifera cv. Nebbiolo. 1st Int. Symp.

on ‘‘Acclimatization and Establishment of Micropropagated

Plants’’ Sani-Halkidiki, Greece. 19–22 Sept. 2001

Islam MT, Leunufna S, Dembele DP & Keller ERJ (2003) In

vitro conservation of four mint (Mentha spp.) accesssions.

Plant Tissue Cult. 31: 37–46

Jackson MB (2003) Aeration stress in plant tissue cultures.

Bulg. J. Plant Physiol., Special Issue 2003: 96–109

Jackson MB, Abbott AJ, Belcher AR, Hall KC, Butler R &

Cameron J (1991) Ventilation in plant tissue cultures and

effects of poor aeration on ethylene and carbon dioxide

accumulation, oxygen depletion and explant development.

Ann. Bot. 67: 229–237

Jackson MB, Belcher AR & Brain P (1994) Measuring the

shortcomings in tissue culture aeration and their conse-

quences for explant development. In: Lumsden PJ, Nicholas

JR & Davies WJ (eds) Physiology, Growth and Development

of Plants in Culture (pp. 191–203). Kluwer Academic

Publishers, Dordrecht, The Netherlands

Kozai T & Sekimoto K (1988) Effect of the number of air

exchange per hour of the closed vessel and the photosynthetic

photon flux on the carbon dioxide concentration inside the

vessel and the growth of strawberry plantlets in vitro.

Environ. Control Biol. 26: 21–29

Kozai T, Tanaka M, Jeong BR & Fujiwara K (1993) Effect of

relative humidity in the culture vessel on the growth and

shoot elongation of potato (Solanum tuberosum L.) plantlets

in vitro. J. Jap. Soc. Hort Sci. 62: 413–417

Lai CC, Yu TA, Yeh SD & Yang JS (1998) Enhancement of in

vitro growth of papaya multishoots by aeration. Plant Cell

Tiss. Org. Cult. 53: 221–225

Lee CWT & Shuler M (1991) Different shake flask closures alter

gas phase composition and ajmalicine production in Cath-

arathus roseus cell suspensions. Biotechnol. Techn. 5: 173–178

Lentini Z, Mussell H, Mutschler MA & Earle ED (1988)

Ethylene generation and reversal of ethylene effects during

development in vitro of rapid cycling Brassica campestris L.

Plant Sci. 54: 75–81

Mackay W A & Kitto SL (1988) Factors affecting in vitro shoot

proliferation of French tarragon. J. Am. Soc. Hort. Sci. 113:

282–287

Majada JP, Fal MA & Sanchez-Tames R (1997) The effect

of ventilation rate on proliferation and hyperhydricity

of Dianthus caryophyllus L. In vitro Cell. Dev. Biol. 33:

62–69

129

McClelland MT & Smith MAL (1990) Vessel type, closure, and

explant orientation influence in vitro performance of five

woody species. HortScience 25: 797–800

Murashige T & Skoog F (1962) A revised medium for rapid

growth and bioassays with tobacoo tissue cultures. Physiol.

Plant. 15: 473–497

Murphy KP, Santamaria JM, Davies WJ & Lumsden PJ (1998)

Ventilation of culture vessels I. Increased growth in vitro and

survival ex vitro of Delphinium. J. Hort. Sci. Biotechnol. 73:

725–729

Pevalek-Kozlina B (1990) Influence of the container size on the

rate of Prunus avium shoot multiplication. Acta Bot. Croat.

49: 49–52

Pierik RLM (1998a) In vitro culture of Higher Plants as a tool in

the propagation of horticultural crops.ActaHortic. 226: 25–40

Pierik RLM (1998b) Closure of test tubes and flasks. In: In vitro

culture of Higher Plants. (pp. 83–85). Kluwer Academic

publishers, The Netherlands

Ruseva R (1999) In vitro cultivation of mint (M. piperita)

with high multiplication rate. Plant Science (Bulgaria) 36:

201–203

Towill LE (1990) Cryopreservation of isolated mint shoot tips

by vitrification. Plant Cell Rep. 9: 178–180

Woltering EJ (1986) Ethylene and carbon dioxide accumulation

within various tissue culture systems. Acta Bot. Neerl. 35: 50

(abstract)

130