Mm

GW

a

ARR1A

KNTDREO

1

sTlaat2Tgcse(mew(t

DS

0d

Scientia Horticulturae 127 (2011) 542–547

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

icropropagation of dahlia in static liquid medium using slow-release tools ofedium ingredients

eert-Jan De Klerk ∗, Jolanda ter Bruggeageningen UR Plant Breeding, PO Box 16, 6700 AA Wageningen, The Netherlands

r t i c l e i n f o

rticle history:eceived 16 August 2010eceived in revised form0 November 2010ccepted 17 November 2010

a b s t r a c t

Growth of dahlia shoots in vitro was ca. 4 times faster in liquid medium than on solidified medium. Inliquid standard medium (3% sucrose, macroelements according to Driver–Kuniyuki Walnut medium,microelements according to Murashige–Skoog medium, 0.44 �M benzylaminopurine), the majormedium ingredients were consumed for 75–80% during the first 6 weeks. Addition of extra ingredientsincreased growth, demonstrating that the amount of ingredients added at the start of culture was subop-

eywords:utrientsissue cultureahliaefractometer

timal. When the extra ingredients were given at the start of the culture, concentrations became too highand therefore inhibitory. When the ingredients were added during the subculture cycle by means of smallaliquots of a concentrated solution or by means of slow-release tools, growth was strongly increased.Osmocote gave satisfactory results as a slow-release tool for inorganics. For organic ingredients (sucroseand benzylaminopurine), a novel slow-release tool was developed.

C-metersmocote

. Introduction

The main ingredients of plant tissue culture media includeucrose, inorganic nutrients, plant growth regulators and vitamins.heir consumption during a subculture cycle has been examined iniquid medium with cell suspensions and organ cultures (Schmitznd Lorz, 1990; Desamero et al., 1993; Holme, 1998; Morard etl., 1999; Lian et al., 2002), and on solid medium with organ cul-ures (Leifert et al., 1995; Ruzic et al., 2001; Ramage and Williams,003). In liquid medium many of the nutrients are being used up.hus, addition of extra nutrients is expected to bring about morerowth. Because an increase of the initial doses may result in con-entrations that are unfavorable for growth, addition during theubculture cycle is desirable. This can be done by adding extra nutri-nts in a small volume of liquid medium with a high concentrationMaene and Debergh, 1985; Aitken-Christie and Jones, 1987). This

ethod, though, is inconvenient in a commercial lab, among oth-

rs because of the risk of contamination. In the present researche use slow-release tools for inorganic (minerals) and organic

carbohydrates and hormones) medium ingredients. Because ofhe techniques involved in producing these tools, inorganics and

Abbreviations: MS, Murashige–Skoog; BAP, 6-benzylaminopurine; DKW,river–Kuniyuki Walnut; OSRTs, organic slow-release tools; LM, liquid medium;M, solidified medium.∗ Corresponding author. Tel.: +31 317480797; fax: +31 317483457.

E-mail address: geertjan.deklerk@wur.nl (G.-J. De Klerk).

304-4238/$ – see front matter © 2010 Published by Elsevier B.V.oi:10.1016/j.scienta.2010.11.015

© 2010 Published by Elsevier B.V.

organics cannot be combined in one single device in large-scale production (L. Wagenaar, Katwijk, The Netherlands, pers.comm.).

In horticulture, granules enabling slow release of inorganic fer-tilizers are being used widely to reduce leaching losses (Maynardand Lorenz, 1979; Sharma, 1979). These granules may be usablein tissue culture. For organics no slow-release tools are available.Slow release may be achieved chemically. Carbohydrates may beadded, e.g., as starch which can be slowly broken down into small,usable units. Similarly, plant growth regulators may be addedas compounds in which the active regulator is conjugated withanother compound (Tsatsakis et al., 1995). In both cases, releaseis achieved by enzymes but control is difficult for various reasons,among others instability of the enzymes and pH dependence oftheir action. About the latter it should be noted that pH changesdrastically during tissue culture and is uncontrolled (De Klerk etal., 2008). Alternatively, slow release may be achieved physicallyby encapsulation within membranes that are little permeable ordegrade slowly. Wybraniec et al. (2002) studied the release of auxinand paclobutrazol encapsulated in granules together with inorgan-ics. We used another slow-release tool, porous tablets containingsucrose that is released by diffusion. Benzylaminopurine can alsobe included in this slow-release tool. In our experiments, slow

release of vitamins was not considered and the plantlets receivedonly the initial dose of vitamins. In this paper, we report thatgrowth in tissue culture can be extended using the granule-typerelease for inorganic nutrients and porous tablets for slow release oforganics.

tia Horticulturae 127 (2011) 542–547 543

2

2

wT5imKSws

leE

2i

EWsSa

wMtceJ

Str

7

2

(S

bc1

2

ededdtsnw

Table 1Composition of the inorganics released from Osmocote.

Osmocote (mg/l) DKW (mg/l) DKW/Osm

P 128 49 0.4NO3

− 612 387 0.6NH4

+ 512 200 0.4S 102 309 3.0K 591 624 1.1Na 24 59 2.5Mg 23 58 2.5Ca 5.9 299 50.7Mn 0.7 9 13.0

liquid medium was ca. 4 times higher than on solid medium(Figs. 2 and 3A). In the liquid medium, inorganic compounds andcarbohydrates were largely exhausted after 6 weeks (Fig. 3B). Onsolid medium, growth slowed down after 6 weeks and stopped fully

Fig. 1. Validation of carbohydrate determinations by a refractometer. Dahlia shoots

G.-J. De Klerk, J. ter Brugge / Scien

. Materials and methods

.1. Plant material and culture conditions

Established cultures of dahlia (Dahlia hybrida) cultivar 611025ere a generous gift of SBW International BV, Roelofarendsveen,

he Netherlands. To maintain the stock, 5 explants were cultured in0 ml static liquid medium at 50 �E m−2 s−1 (16 h per day) and 20 ◦C

n plastic containers (Wavin) with a diameter of 9 cm. The standardedium contained DKW-macronutrients according to Driver and

uniyuki (1984), MS-micronutrients according to Murashige andkoog (1962), 3% sucrose, 0.44 �M BAP. Subculture was every 4eeks. The explants were either single nodes (a node with ca. 1 cm

tem and two leaves) or apical sections (ca. 1 cm) of shoots.For the experiments, five single nodes were cultured in 50 ml

iquid medium in a Wavin container. Per treatment, three contain-rs were used. So in total, per treatment 15 explants were studied.xtra medium components were added as indicated.

.2. Measurements of sucrose, total amount of inorganics,ndividual elements, radioactive BAP and dry weight

The concentration of the total of minerals was estimated with anC-meter (315i, WTW Wissenschaftlich-Technische Werkstätten,eilheim, Germany). Individual minerals were determined by the

ervice laboratory of the Chemical Biological Laboratory for WUR –oil Centre (Wageningen, The Netherlands) using SFA-CaCl2 (NH4

+

nd NO3−) and ICP-AES (the other minerals).

Sucrose, and its hydrolysis products glucose and fructoseere determined enzymatically using an enzyme kit (Boehringer,annheim, Germany; sucrose is hydrolyzed by invertase to fruc-

ose and glucose; these sugars are measured). The total amount ofarbohydrates was measured chemically with anthrone (De Bruynt al., 1968) and with a digital refractometer (PAL-1; Atago, Tokyo,apan).

6-Benzylamino[8-14C]purine (2.02 GBq mmol−1) was fromigma (St. Louis, USA). Aliquots (200 �l) of the medium wereaken at the indicated intervals and to determine the amount ofadioactivity, 4.5 ml Aqua Gold (Packard) was added.

For dry weight determination, the plant material was dried at0 ◦C for 3 days.

.3. Slow-release tools

Osmocote Pro 3-4M was a generous gift of Scott InternationalGeldermalsen, The Netherlands). The characteristics are shown inection 3.

The slow release castings for sucrose and BAP were developedy Kiwi Farm (Katwijk, The Netherlands). Each weighed 5 g andontained 60, 40 or 20% (w/w) sucrose and each 0, 0.17, 0.56 or.11 �mol BAP. The characteristics are shown in Section 3.

.4. Statistics

The values of DW, and numbers of nodes are means from 15xplants ± SE. When EC and brix values of culture media wereetermined, the values are means from three containers ± SE. Allxperiments were repeated at least twice. In the other chemicaleterminations (Table 1, Figs. 1 and 4), the values represent single

eterminations. In this case, the experiment was repeated at leasthree times. The error bars in the figures are SEs. When no bar ishown, SE is smaller than the symbol. Values having same letter doot differ significantly at 0.05 level. The significances of differencesere evaluated by a Student-t test.

Fe 0.7 5 7.2

Released inorganics from Osmocote granules (2.5 g) in 500 ml water during 8 weeks.For comparison, the composition of DKW at the same concentration is shown.

3. Results

3.1. Validation of analytical techniques for carbohydrates

For estimation of the total amounts of sucrose and inorganics,quick methods are available, namely, determinations with a refrac-tometer (brix-meter) and with an EC-meter, respectively. Bothmethods were validated. We compared measurements of sucroseby a refractometer with an enzymatic test and the anthrone deter-mination. We used medium with explants after increasing periodsof culture. A determination with the refractometer took only a fewseconds. The three determinations gave very similar results (Fig. 1).

An EC-meter measures the electrical conductivity of a solution.The measured value depends on the dissociation of salts in anionsand cations. At high concentration, the conductivity is thereforeless. At full concentration DKW, the value determined by the EC-meter was 10% less than expected on base of 1/2 concentrationDKW (data not shown).

3.2. Growth in liquid and solid media and depletion of themedium

The increase of DW during a 6-week subculture cycle in

were grown in liquid medium. After 0, 2, 3, 4 and 6 weeks samples were takenand carbohydrates were measured by a refractometer, by the anthrone determina-tion (total carbohydrate) and by an enzymatic determination (sucrose, fructose andglucose). The values obtained by the anthrone and enzymatic determinations wereplotted as a function of the reading obtained with the refractometer. Note that inthis experiment the initial sucrose concentration was 4 g 100 ml−1.

544 G.-J. De Klerk, J. ter Brugge / Scientia Ho

Fp(

ao(mIa

kmMnw

ow

Fnm

ig. 2. Growth of dahlia in liquid medium (left) or on solid medium (right). Thehoto was taken 6 weeks after the start of culture. Note top necrosis on solid mediumarrows).

fter 8 weeks (data not shown), even though after 6 weeks ca. 75%f inorganic nutrients and 90% of carbohydrates were still availableFig. 3B). Thus, in liquid medium growth likely ceased because of

edium depletion and on solid medium because of other reasons.n liquid medium sucrose was hydrolyzed to glucose and fructosend after 3 weeks no sucrose occurred (data not shown).

Fig. 4 shows depletion of the various inorganic ingredients. Theinetics of depletion was different for various elements. In liquidedium, NH4

+, NO3−, P and K were rapidly taken up, Ca slowly and

g and S intermediate. The uptake of the DKW elements that are+

ot shown in Fig. 4 was also intermediate. On solid medium, NH4

as also taken up most rapidly.When the double amount of ingredients was added at the start

f the subculture cycle, growth increased (Fig. 5A, +st) showing thathen the standard level was applied, the level of one or more essen-

ig. 3. Growth of dahlia (A) and consumption of carbohydrates, inorganics and BAP (B)umber of nodes per initial explant. Carbohydrates and inorganics were determined by aedium, 14C-BAP was added and determined in an aliquot of medium.

Fig. 4. Consumption of the various inorganics in l

rticulturae 127 (2011) 542–547

tial components was not optimal. When three times the amountwas added at the start of the subculture cycle, though, growth waslittle probably because the concentrations were too high (Fig. 5A,+2st). When an extra dose of the standard amount of ingredientswas added 2 weeks after the start of the subculture cycle, growthwas more than when the extra dose was added at the start (Fig. 5B,+st). When a double extra dose of ingredients was given after 2weeks, growth was not inhibited as when it was added at the start,but growth doubled (Fig. 5B, +2st). It should be noted that after 2weeks, 60% of the inorganics and 25% of the carbohydrates had beentaken up from the standard medium (Fig. 3B).

3.3. Addition of extra nutrients by means of slow-release

For inorganic nutrition in horticulture, controlled release toolssuch as Osmocote are used. In tissue culture of most crops, theformulation of Murashige and Skoog (1962) is used. At the sametime, MS is a very general formulation and replacement by anothergeneral formulation as the one in Osmocote will probably allowsufficient growth (George and De Klerk, 2008). The same appliesto the DKW formulation. As a matter of fact, growth in MS andDKW was not very different (data not shown). The type of Osmocotewas selected on the basis of the speed of release (“hi-start” whichmeans fast release from the start). We determined the amount ofthe various inorganics released during 8 weeks in water (Table 1).In comparison with DKW, the most notable difference was the lowlevel of Ca in Osmocote (more than 50 times less). Other differencesincluded a higher level of N and P in Osmocote. The NH4-NO3 ratiowas 0.8 in Osmocote and 0.5 in DKW. Fig. 5a shows the release from

Osmocote. Obviously, in Osmocote the period for complete releaseis too long (Fig. 6A) since release is only needed for a period of 2–3months.

Slow release of organic compounds is used for pharmaceuti-cals (Ganderton, 1987), but the period of release occurs within

in liquid or on solid medium. Growth was monitored as dry weight (DW) and asrefractometer and an EC-meter, respectively. For measuring BAP depletion in liquid

iquid medium (A) and on solid medium (B).

G.-J. De Klerk, J. ter Brugge / Scientia Horticulturae 127 (2011) 542–547 545

Fig. 5. The effect of addition of extra medium ingredients at the start of culture (A) or two weeks after the start of culture (B). The extra ingredients (1 time (st) or 2 times(2st) the standard concentration in the medium) were given by adding a small volume with 10 times concentrated medium.

Fig. 6. Release of inorganics from Osmocote (A) and sucrose from “organic slow release tools” (OSRTs) (B). From OSRTs, 3 versions were tested containing 20, 40 or 60%sucrose, respectively. Note that full release requires ca. 24 weeks for Osmocote and 10 weeks for the OSRTs.

Fig. 7. The effect of addition of extra nutrients. Extra inorganics were added as 1 g Osmocote and extra sucrose as a concentrated solution after 2 weeks (A). Extra nutrientswere added as Osmocote (inorganics) and as four 60%-OSRTs (“organic slow release tools” for sucrose) (B). When the OSTRs were added, the initial sucrose concentrationwas halved because of the massive release of sucrose from the OSRTs during the first week.

Fig. 8. The effect of addition of extra BAP on the number of nodes per initial explant after 6 weeks culture. (A) 2.67 �mol BAP was added after 2 weeks or 0.44 �mol everyweek, i.e. after 1, 2, 3, 4, and 5 weeks. (B) Extra BAP (0.67, 2.22 or 4.44 �mol) was added using four 60%-OSRTs containing BAP. When OSRTs were added, the initial sucroseconcentration was halved because of the massive release of sucrose from the OSRTs during the first week.

5 tia Ho

hwdifitcs

siOrTo

egn(ni

4

4

seimaoms(hoocanf2(abattdMoco9ii

owImnt

46 G.-J. De Klerk, J. ter Brugge / Scien

ours–days and in tissue culture, release during a period ofeeks–months is needed. Thus novel slow release tools had to beeveloped. We have used newly developed tablets (actually cast-

ngs) that released sugar by diffusion. Release was fast during therst period and slower after that (Fig. 6B). In the experiments theablets nevertheless turned out to be usable but the initial con-entration of sugar was halved to compensate the fast release ofucrose during the first week.

When Osmocote or extra sucrose was added separately totandard medium, no additional growth occurred, showing themportance of the ratio of inorganics and sucrose (Fig. 7A). Whensmocote was added together with extra sucrose or with slow

elease tablets for sucrose, growth increased ca. 3 times (Fig. 7B).his shows the effectiveness of both Osmocote and the newly devel-ped OSRTs for sucrose.

Addition of an extra dose of BAP during the subculture cycle,ither as one dose after 2 weeks or as 5 small doses every weekreatly increased propagation as shown by the increase of nodeumber (Fig. 8A) but had hardly any effect on dry weight increasenot shown). When BAP was added via the OSRTs for sucrose, theumber of nodes increased depending on the dose that had been

ncluded in the OSRTs (Fig. 8B).

. Discussion

.1. Growth in liquid and solid media

For survival in static liquid medium, the level of the mediumhould not be high. A large portion (1/4) of the explant shouldmerge from the medium, otherwise, the explants die by drown-ng (data not shown). Growth in static liquid medium was much

ore than growth on solid medium (Figs. 1A and 2). This was prob-bly caused by better translocation of ingredients to the regionsf utilization (growing tissues) in liquid medium than on solidedium. There are two reasons for this. First, shoots cultured on

olid medium take up medium ingredients through their cut endLeifert et al., 1995; Guan and De Klerk, 2000). In dahlia, the cut endas only a small surface so uptake will accordingly be little. A sec-nd reason is insufficient translocation in the shoot, which dependsn water flows in xylem and phloem (Taiz and Zeiger, 2002). Vas-ular flows have not been examined in tissue-cultured plants butre probably much reduced compared with plants growing underatural conditions for the following reasons. Because transpiration

rom the leaves is low, flow in the xylem is reduced (De Klerk,010). Moreover, the driving force in phloem transport, the sourcein particular leaves) that loads the phloem with sucrose (Taiznd Zeiger, 2002), does not function adequately in tissue cultureecause of a low level of photosynthesis. In addition, dahlia shootsre long and thin which is unfavorable for mass movement. Onhe other hand, in liquid medium translocation of ingredients fromhe medium to the regions of utilization is facilitated by the shortistance from the site of uptake (leaves) to the areas of growth.edium ingredients are taken up by leaves via stomata and aque-

us pores (Schönherr, 2006). In an experiment with apple shootsultured in liquid medium with 14C-IAA, Guan and De Klerk (2000)bserved that 5% of the label was taken up via the cut end and5% via the leaves. Similarly, in temporary immersion bioreactors

n which uptake also occurs via the leaves, growth is usually muchmproved (Etienne and Berthouly, 2002).

Poor translocation of nutrients within the shoots during culturen solid medium gets also obvious from top necrosis. This disorder

as observed on solid medium and not in liquid medium (Fig. 2).

n other crops, top necrosis is reversed by increasing Ca2+ in theedium (Busse et al., 2008; Bairu et al., 2009), indicating that at the

ormal concentration, the amount of Ca2+ that reaches the apex isoo low. Top necrosis can also be reversed by aeration. Under such

rticulturae 127 (2011) 542–547

condition, water flow in the xylem (which is driven by transpira-tion) is increased so that more Ca2+ is translocated (Bairu et al.,2009). Dahlia is cultured not with MS but with DKW which con-tains 3 times as much Ca2+. Nevertheless, too little Ca2+reaches theapex. In liquid medium, necrosis of the apex does not occur, likelybecause of uptake via the leaves after which translocation to theapex is easier. We have measured the levels of inorganics in thetissues and observed that the level of Ca2+ in shoots cultured inliquid medium was ca. twice the level of shoots cultured on solidmedium (data not shown). For other minerals, e.g., K, the level inliquid-medium cultured shoots is about 20% higher.

The much improved growth in liquid medium is profitable onlywhen there are no disadvantageous effects on performance afterplanting in soil. We examined this and observed similar growthrates of plantlets form solid and liquid media in the glasshouse (J.ter Brugge and G.J. De Klerk, unp. results).

4.2. Measurement of carbohydrates and inorganic nutrients

The results of carbohydrate determination with a brix metergave reliable data despite various expected pitfalls. A major draw-back may be that in tissue culture media, sucrose is hydrolyzed toglucose and fructose by invertases excreted by the tissue. In liq-uid medium with dahlia explants, after 3 weeks most sucrose hasbeen hydrolyzed (J. ter Brugge and G.J. De Klerk, unp. results). Arefractometer for sucrose-determinations underestimates the totalconcentration of carbohydrates by ca. 30% when all sucrose hasbeen hydrolyzed (data not shown). Nevertheless, the effect on theactual determinations was minor. A second drawback of the refrac-tometer determination is that inorganic nutrients also interfere:full DKW has a brix reading of 0.4% and full MS a reading of 0.3%.

An EC-meter measures the electrical conductivity of a solution.The measured value depends on the dissociation of salts in anionsand cations. At higher concentration, the conductivity is thereforeless. At full concentration MS, the value determined by the EC-meter is 10% less than expected on base of 1/2 concentration MS(data not shown). Over 99% of the conductivity is brought about bythe macroelements.

4.3. Depletion results in reduced growth in liquid medium

The main medium ingredients include sucrose, a number ofinorganic compounds and plant growth regulators. These ingre-dients are rapidly depleted in liquid medium, but at different ratesNH4

+ being used up most rapidly. The rate of diffusion of the variousingredients in the medium is similar since they have compara-ble diffusion coefficients, so the differences are related to differentuptake rates by the tissue, a process that occurs at the interfaceof explants and medium. It is supposed that most ammonium-nitrogen is not taken up in the form of NH4

+, but that NH3 alsopermeates the plasma membrane after deprotonation, leaving H+

in the external solution (Bertl et al., 1984). Ca2+ uptake is slowest.This might be related to the difference between Ca2+ concentrationsin the cell wall and the apoplastic spaces which are usually 1000times higher than in the cytosol. So a high Ca2+ concentration ismaintained in the apoplast against a steep gradient that drives Ca2+

into the cell (Bush, 1995). Since there is no intermediate membrane,exchange between apoplast and medium is easy.

Extra ingredients given at the start of culture resulted in addi-tional growth (Fig. 5A). When too much was added (resultingin three times the standard concentration), the concentrations

became so high that growth was strongly reduced (Fig. 5A). Whenthe same amount was added not directly but after 2 weeks of cul-ture, growth was not inhibited and additional growth occurred(Fig. 5B). It should be noted that after 2 weeks ca. 60% of theinorganics and 25% of sucrose had been taken up so that after addi-

tia Ho

tr

4

gaiiTcrclct

rtkspiastg

cwFte

A

tfKs

R

A

B

Roubelakisangelakis, K.A., 1995. 1-Naphthylacetic acid slow-release polymeric

G.-J. De Klerk, J. ter Brugge / Scien

ion of extra medium at this time no adverse concentration waseached.

.4. Slow release tools

An increased amount of medium ingredients resulted in morerowth. Extra ingredients may be applied, among others, by addinghigh concentration at the start, but high concentrations are

nhibitory (Fig. 5A). Additional ingredients may also be added dur-ng the culture as a small aliquot of highly concentrated solution.his resulted in high growth, but is laborious and may introduceontaminations. We have therefore chosen for addition by slowelease. Osmocote could be used as inorganic fertilizer in tissueulture. Producing relatively small amounts of slow-release toolsike Osmocote is expensive, so Osmocote with Murashige–Skoogomposition is not feasible. The present formulations of Osmocote,hough, are satisfactory to obtain additional growth.

There are no commercial tools for slow release of organic mate-ial over a sufficiently long period. We have used tablets (castings)hat slowly released compounds by diffusion. Unfortunately, theinetics of release was not optimal for growing cultures. Releasehould be parallel to the growth curve, in the case of dahliaarallel to linear growth. As a matter of fact, the castings used

n our experiments showed fast release directly after the startnd after that slow release. We have also used granules withucrose from which the membranes degraded after 1–2 weeks, buthere were major technical problems in the production of theseranules.

We also used castings with increasing doses of BAP, but did notharacterize the release. Because of the low amounts of BAP, thisould require quantification using HPLC or 3H-/14C-labeled BAP.

ig. 8b, though, shows that adding BAP through the slow-releaseools used for sucrose strongly enhanced outgrowth of axillary budsvidenced by the increase of the number of nodes.

cknowledgements

We are indebted to Rebeca Palacios and Zacarias Torbado forheir excellent help in some experiments. The slow release toolsor organics were developed by Loek Wagenaar MSc (Kiwi Farm,atwijk, The Netherlands). We thank Loek Wagenaar for his enthu-iastic interest and support.

eferences

itken-Christie, J., Jones, C., 1987. Towards automation: Radiata pine shoot hedgesin vitro. Plant Cell Tissue Organ Cult. 8, 185–196.

airu, M.W., Jain, N., Stirk, W.A., Dolezal, K., Van Staden, J., 2009. Solving the problemof shoot-tip necrosis in Harpagophytum procumbens by changing the cytokinintypes, calcium and boron concentrations in the medium. S. Afr. J. Bot. 75,122–127.

rticulturae 127 (2011) 542–547 547

Bertl, A., Felle, H., Bentrup, F.-W., 1984. Amine transport in Riccia fluitans: cytoplas-mic and vacuolar pH recorded by a pH-sensitive microelectrode. Plant Physiol.76, 75–78.

Bush, D.S., 1995. Calcium regulation in plant cells and its role in signaling. Annu.Rev. Plant Physiol. Plant Mol. Biol. 46, 95–122.

Busse, J.S., Ozgen, S., Palta, J.P., 2008. Influence of root zone calcium on subapicalnecrosis in potato shoot cultures: localization of injury at the tissue and cellularlevels. J. Am. Soc. Hortic. Sci. 133, 653–662.

De Bruyn, J.W., Van Keulen, H.A., Ferguson, J.H.A., 1968. Rapid method for the simul-taneous determination of glucose and fructose using anthrone reagent. J. Sci.Food Agric. 19, 597–601.

De Klerk, G.J., 2010. Why plants grow in tissue culture. Prophyta Annu. 2010, 42–44.De Klerk, G.J., Hanecakova, J., Jásik, J., 2008. Effect of medium-pH and MES on adven-

titious root formation from stem disks of apple. Plant Cell Tissue Organ Cult. 95,285–292.

Desamero, N.V., Adelberg, J.W., Hale, A., Young, R.E., Rhodes, B.B., 1993. Nutrientutilization in liquid/membrane system for watermelon micropropagation. PlantCell Tissue Organ Cult. 33, 265–271.

Driver, J.A., Kuniyuki, A.H., 1984. In vitro-propagation of paradox walnut rootstock.HortScience 19, 507–509.

Etienne, H., Berthouly, M., 2002. Temporary immersion systems in plant microprop-agation. Plant Cell Tissue Organ Cult. 69, 215–231.

Ganderton, D., 1987. Current innovations in drug delivery. Drug Des. Deliv. 2, 1–7.George, E.F., De Klerk, G.J., 2008. The components of plant tissue culture media I:

macro- and micro-nutrients. In: Plant Propagation by Tissue Culture. Volume 1:The Background, third ed. Springer, Heidelberg, pp. 65–113.

Guan, H., De Klerk, G.J., 2000. Stem segments of apple microcuttings take up auxinpredominantly via the cut surface and not via the epidermal surface. Sci. Hortic.86, 23–32.

Holme, I.B., 1998. Growth characteristics and nutrient depletion of Miscant-hus × ogiformis Honda ‘Giganteus’ suspension cultures. Plant Cell Tissue OrganCult. 53, 143–151.

Leifert, C., Murphy, K.P., Lumsden, P.J., 1995. Mineral and carbohydrate nutrition ofplant cell and tissue culture. Crit. Rev. Plant Sci. 14, 83–109.

Lian, M.L., Chakrabarty, D., Paek, K.Y., 2002. Growth and uptake of sucrose and min-eral ions by bulblets of Lilium Oriental Hybrid ‘Casablanca’ during bioreactorculture. J. Hortic. Sci. Biotechnol. 77, 253–257.

Maene, L., Debergh, P., 1985. Liquid medium additions to established tissue culturesto improve elongation and rooting in vivo. Plant Cell Tissue Organ Cult. 5, 23–33.

Maynard, D.N., Lorenz, O.A., 1979. Controlled release fertilizers for horticulturalcrops. Hortic. Rev. 1, 70–140.

Morard, P., Fulcheri, C., Henry, M., 1999. Mineral nutrition of Gypsophila in vitro rootculture. J. Plant Nutr. 22, 717–730.

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assayswith tobacco tissue cultures. Physiol. Plant. 15, 473–497.

Ramage, C.M., Williams, R.R., 2003. Mineral uptake in tobacco leaf discs during differ-ent developmental stages of shoot organogenesis. Plant Cell Rep. 21, 1047–1053.

Ruzic, D., Saric, M., Cerovic, R., Culafic, L., 2001. Changes in macroelement contentof the media and in sweet cherry Inmil GM 9 shoots during in vitro culture. J.Hortic. Sci. Biotechnol. 76, 295–299.

Schmitz, U., Lorz, H., 1990. Nutrient-uptake in suspension-cultures of Gramineae. 2.Suspension-cultures of rice (Oryza sativa L.). Plant Sci. 66, 95–111.

Schönherr, J., 2006. Characterization of aqueous pores in plant cuticles and perme-ation of ionic solutes. J. Exp. Bot. 57, 2471–2491.

Sharma, G.C., 1979. Controlled-release fertilizers and horticultural applications. Sci.Hortic. 11, 107–129.

Taiz, L., Zeiger, E., 2002. Plant Physiology, third ed. Sinauer Associates, Sunderland.Tsatsakis, A.M., Paritsis, K.N., Shtilman, M.I., Shashkova, I.B., Alegakis, A.K.,

formulations – auxin type effect in tobacco leaf segments is affected by structureand hydrolysis. Plant Growth Regul. 17, 167–175.

Wybraniec, S., Schwartz, L., Wiesman, Z., Markus, A., Wolf, D., 2002. Release char-acteristics of encapsulated formulations incorporating plant growth factors. J.Environ. Sci. Health B 37, 235–245.