Acclimatization of micropropagated Silvan blackberry.pdf

8/22/2019 Acclimatization of micropropagated Silvan blackberry.pdf

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tACCLIMATIZATION OF MICROPROPAGATED

SILV AN BLACKBERRY

Laurence Tisdall

A thesis submitted to the Faculty of GraduateStudies and Research n partial fulfllmentof the requirements for the degree ofMaster of Science

Depanment of Plant ScienceMacdonald College ofMcGill UniversityMontreal Canada@ Nov 1989


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Suggcsted shan title:

ACCLIMATIZATION OF MICROPROPAGATEDSILV AN BLACKBERRY


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M.Sc.BSTR CT

Laurence TisdallAcclimatization of micropropagated

'Silvan' blackberry.

Plant Science

Tissue-cultured ;hoots and plantlets usually have leaves with non-functional, openstomata and little epicuticular and cuticular wax, resulting in excess evapotranspirationafter transplantation. Various strategies were evaluated to decrease ex vitro acclimatization difficulties for 'Silvan' blackberry, including transplanting unrooted shoots,increasing the medium agar concentration from 6 to 9 or 12 g/I and diluting the basalmedium. Increased medium agar concentrations and medium dilution did not improveaurvival or growth. Stomatal function resumed sooner in new leaves of plantlets thanshoots. High relative humidity (> 95 ) and low light :iltensty (90 pmol S 1 m Z)negatively affectrd stomatal closure bath on acclimatizing transplants and greenhousegrown plants. Guard cells developed on leaves in vitro were physiologically activebut had apparent anatomical abnOlmalities that inhibited closure. A rapid clearing andstaining method was developed for examinatioll o follar morphology using intact invitro blackberry (Rubus sp. 'Silvan') and strawberry f r a ~ a r i a x ananassa Duch. 'Totem')plantlets and sections of greenhouse-grown 'Silvan' and 'Totem' leaves. This methodinvolved tbree steps: 1) removing the chlorophyll by autoclaving in 80 ethanol; 2)dissolution of the protoplasm using 5 NaOH at 80 oC; 3) post-alkali treatment with75 bleach (4.5 NaCIO) at room temperature for tissue-cultured plantlets and atS oC for greenhouse-grown leaves. Aqueous safranin (10 mg/l) was used for staining.

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( R SUMM.Sc. Laurence TisdaIl Phytotechnie

Acclimatation de la mOre 'Silvan' sI . micropropagk

Les pousses et les plantules provenant de la culture de tissu ont gnralementdes feuilles sur lesquelles les stomates ne tonctionnent pas et sont ouvertes ainsi queune fonnation i n c o m p l ~ t e ou anormale des cires pidenniques. Ceci s aboutit unmanque de conttle sur l'vapotranspiration p ~ s leur transplantation. Des s t r ~ g i e svaries ont ~ values afin de r6duire les difficults d acclimatation de la mre Silvanex vitro, incluant la transplantation de pousses non-enracines. en augmentant laconcentration d agar dans le bouillon de culture de 6 9 ou 12 g/l et en diluant lebouillon de culture basale. Les concentrations d'agar augmententcs et la dilution dubouillon n ont amlior ni la survie ni la croissance. Le fonctionnement du stomatea repris plus tt pour les nouvelles feuilles des plantules que celles des pousses. Uneatmosphtt presque sature (> 95 ) et une intensit de lumire basse (90 )lmolS-I m-2) ont affect ngativement la fermeture des stomates sur le plantes provenantde la culture de ti lSU et les plantes venant de la serre. Les cellules de garde dveloppesin vitro ont fonctionn physiologiquement mais avec des anomalies anatomiques videntesqui ont rendu impossible la fermeture complte des stomates. Afin d examiner lamorphologie des feuilles. une mthode rapide d'clairciment et de coloration a tdveloppe en utilisant des plantules de mre


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cknowledlements

1 wish to acknowledge my sincere appreciation to my supervisor Dr. DanielleJ Donnelly for her guidance support and invaluable help throughout the course othis r e s e r ~ h and during the preparation of this thesis. The author would also liketo thank Professors J Peterson S Sparace and D Smith for advice given concemingexperimental procedus and Dr. D Buszard for editorial assistance

Thanles must also e extended to my fellow gaduate students: Caroline ConstabelJohanne Cousineau Susan Delafield Martine Korban Yvel Leclerc Boulo a andRichard Stahl for their ideas moral support and friendship. Helen Cohen Rimmeris acknowledged for her help conceming photographic work. Last but cenainly notleast thanles to my family for much support understanding and editing required tocomplett. this thesis.

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TABLE OF CONTENTSABSTRACf Il Il l l l Il l l iRESUME ................................................................................................ i iAKNOWLEOOEMENTS .......................................................................Il i i iLIST OF TABLESLIST OF FIGURES

................................................................................... vi V

ABBREVIA TIONS USED ............................................................................... viiiMANUSCRIYfS AND AUTIiORSHIP ............................................................. IXChapterl INrmoDucnoN .... ....................... .................................................... 12. LITERA TURE REVIEW .................. ............. ............ ........................... 4

2.1.2.2.2.3.2.4.

2 5

Inttoouction ............ 1 11111 111 1 1The culture induced environment 55Ex vitro transplantation .... ......................... ........................... 10Transplant phenotypes ex vitro ........... .................... .... ........... l2.4.1. The persistent leaves .................. ................... ...... ........ l2.4.2. The new leaves ......................................... .................. 13Acclimatization strategies for micropropagated plants 142.5.1. Acclimatization ex vitro ......................... ...................... 142.5.2. Hardening off in vitro ....................... ........................... 152.6. Conclusions and Prospects ......................................................... 182.7. Summary ................................................................................. 192.8. References cited .. ................ .............. ................... ......... .......... 22

3. A RAPID CLEARING AND STAINING METHOD FOR TISSUE-CULTURED PLANTLETS AND GREENHOUSE GROWN LEAVES

............................................................................................ 32

4. ACCLIMATIZATION OF MICROPROPAGATEDS ~ V A N BLACKBERRY ..................................................................... 364.1. Inttoouction ...............................................................................1 364.2. Materials and methoos4.2.1. Source Plants

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4.2.2.Transplan tation ........................................................................... 424.2.3. The effeet of medium agar concentration and in vitrorooting on Silvan following transplantation ...................... 424.2.4. Preliminary tests on stomatal funrtion .............................. 454.2.5. Evaluation of ex vitro stomatal function of Silvanplandets ......................................................................... ,.... 464.2.6. The effeets of high relative humidity and 10 N light in

tensity on ex vitro stomatal function of Silvan plandetsgrown on full and 1/4 strength l COting medium ................ 47

4.2.7. The effccts of high relative humidity and low lightintensity on stomatal function of greenhouse-grownSilvan plants ............................................ ............ ...... ..... 48

4.3. Results and Discussion .. .............................. ................ ....................... 494.3.1. be cffeet of medium agar concentration and in vitrorooting on Silvan following transplantation .................... 494.3.2. The effeet of r ~ h weight at transplantation (initial) and

persistent leaf number on final fresh weight 3 weeksafter transplantation ......................................................... S

4.3.3. The effeet of medium agar concentration and in vitrorooting on stomatal index .................................................. S94.3.4. Evaluation of ex vitro stomatal function of Silvanplandets ............................................................................... 614.3.5. Evaluation of stomatal c10sure of the fU St new leaves ofSilvan plandets three weeks following transplantation ..... 624.3.6. The effeets of high relative humidity and low lightintensity on ex vitro stomataJ function of Silvan plandets

grown on full or 114 su ength rooting medium .................. S4.3.7. The effeets of high relative humidity and low light

intensity on stomatal function of greenhouse-grownSilvan plants ...................................................................... 74

4.4. Conclusion ...... ............... ..... ................... ....... .............. ........ ............... 775. CONCLUSION ............................................................................................. 806. SUGGESTIONS FOR FURTIIER RESEARCH ..... ............ ................... ....... 837. REFERENCES CITED . ................................... .......... ..... ....... ...................... 84APPENDIX 1 .................................................................................................... 94

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lLIS[ O TABLIS

Table1 Summary of clearing method for intact tissue-cultured plandetsand greenhouse-grown plant leaves of 'Sil van' blackberry and

Totem strawben Y. . ........................................................................... . 322. Summary of medium type (M=multiplication medium R=rootingmedium) and agar concentration in the six treatments. ................... 423. Mean initial (wcek 0) and final (week 3) fresh weights (g) of 'Silvan'transplants fmm medium agar concentrations of 6 9 and 12 g/l ...................... 514. Mean persistent lea number al transplantation and the increase in growth

(%) of Sil van blackberry transplants grown on medium agarconcentrationsof 6. 9 and 12 gII from transplantation to week 3 ..... .............. ....... ........ ........... 595. Mean initial stomatal aperture standard error) in buffer and the stomatalapenure after replacing the buffer with a 1 M NaCI solution on Ieafpeels ofpersistent and new leaves of 3 wcek old Silvan blackberry plandets. Newleaves developedduring the fust. second and third week after transplantation. ...... 646. Mean stomatal apenures (pm) of 'Silvan' blackbeny plantlet leafpeels in thebuffer (B), after a 1 MNaCI solution was drawn over the peel (S) and oncethe salt solution had becn replaced with the buffer (B2). Stomata wereconsidered closed if the aperture was 2 Jlm. ............ ........................... ......... 73

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LIST OF nGURMFigure1. Photomicroscopic view of an in vitro 'Silvan' blackberry leaflet,focussing down from abaxial to adaxial surface. (A) Abaxial leaf

surface. (B) Internai view of vascular tissue and palisade layer. CCAdaxial surface. 0) Glandular trichome (colleter). Micrometerbar =5 pm (A-C); 10 pm 0). . ........................................................... 332. Mean height of 'SilVcUl' blackberry shoots (S) and plantlets (P), grown onmedium containing 6, 9 and 12 g/l Dco-bacto agar, during the fust 3weeksafter transplantation. ............................ .................................... ....... 523. Mean leafnumber of 'Silvan' blackberry shoots (S) and plantlets (P), grownon media containing6, 9 alld 12 g/lDifco-bacto agar, during the (ifSt 3weeksafter transplantation. ... ................... ................... ........................................... 534. Mean longest lea length of 'Silvan' blackberry shoots (S) and plantlets (P),grown on media containing 6, 9 and 12 g/l Difco-bacto agar, during the mt3 , ,'eeks after transplantation. .. .......... ................ ............ ............................. 545. Mean stomatal index of leaves from 'Silvan' blackberry shoots (S) andplantlets (P) grown on media containing 6, 9 and 12 g/l Difco-bacto agar.These leaves had developed in culture (P) and during the mt (wk 1), second(wk 2) and third (wk 3) week after transplantation. .. ................................... 606. Comparison within agar and in vitro-rooting treatments of meanstomatal index of 'Silvan' blackberry shoots and plantlets frommedia containing 6, 9 and 12 g/l Difco-bacto agar. Samplingincluded leaves which had developed in culture (Persis) and during the flfst

(1 wk), second (2 wk) and third (3 wk) week after transplantation. ............. 607. Photomicrographs of stomata of a 'Silvan' blackberry leaf, from plantletsgrown on Full MS , 3 days after removal from the dew chamber to thegrowth chamber. a) while on buffer; b) after application of the lM NaCIsolution; c) after replacement of the NaCI solution with buffer. Arrowsindicate functional or partially functional stomata. Bar = 50 pm. .... ............ 76

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"

RRBEyl TIONS USED

AbbreviatiQo - Common Na Growtb RlulatorsARA - Abscisic acid

BAP - BenzylaminQPurineIBA - Indole-3-butanoic acid

FunlicidesDenlateR - Benomyl

TrubanR - EtbazoleEertilizers14:14:1420:20:2010:52:10PQuinl mixturePromixR

Cbemical i l i

[s- z,E)-[S- I-hydroxy 2,6.6-trimetbyl-4-oxo-2-cyclohexen-l-yl)-3-methyl-2,4-pent-adienoic cidN- phenylmethyl)-IH-purin-6-aminel-indole-3-butanoic acid

I-methyl[l [ butylamine)carboxyl-lH benzimidazol-2yl] carbamateE-S-ethoxy-3-(tricbloI'O ethyl)-1 ,2,4-thiadiazole

14 N:14 P20,:1420 N:20 P20,:2010 N:52 P20,:10

(1 peat: 1perUte: 1 vennicuUte)

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Manuscripts and Authorship

The candidate has the option, subject to the approval of the Department,of including as part of the thesis the text. or duplicated published text.of an original paper, or papers. in this case the thesis must still conformto all other requirements explained in Guidelines Conceming Thesis Preparation. Additional material procedural and design data as weil as descriptionof equipment) must be provided in sufficient detail (e.g. appendices) toallow clear and precise judgment to be made of the importance and originalityof the research reported. The thesis should be more than a Mere collectionof manu scripts published or to be published. It must include a generalabstracto a full introduction and literature review and a final overall conclusion.Connecting texts which provide logical bridges between different manuscriptsare usually desirable in the interests of cohesion.

Il is acceptable for theses to include as chapters authentic copies ofpapers already published, provided these are duplicated clearly on regulationthesis stationery and bound as an integral part of the thesis. Pholographsor other materials which do not duplicate must be included in their originalfonn. n such instances. connecting texts are mandatory and supplementaryexplanatory material is almost always necessary.

The inclusion of manuscripts co-authored by the candidate and othersis acceptable but the candidate is required to make an explicit statementon who contributed to such work and to what extent, and supervisorsmust attest to the accuracy of the claims. e g before the Oral Committeo.Since the task of the Examiners is made more difficult in these cases.it is in the car.didate s interest to make the responsibilities of authors

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perfectly clear. Candidates following this option must inform the Depan-ment before l submits the thesis for review.

Conceming the manuscript titled Acclimatization strategies for micropropagated plants for the volume Micropropagation of Woody Plants , theauthor's contribution consisted of gathering and summarizing ail pertinentinfonnation as weil as submitting a fust draft of the J'eview. Subsequentrewriting and editing was done by both authors.

Conceming the publication titled U rapid clearing and staining methodfor tissue-cultured plantlets and greenhouse-grown leaves , the author devisedand completed the experiment and wrote the manu script. Dr. Donnellyprovided laboratory space and equipment and edited the manuscript.

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1. INTRODUCTION

Red raspberry are found in temperate zones around the world (McPheeters et al., 1988).Wma species belong to the Rosaceae family. Red raspberry is the best known and

most cultivated of this lenus (Snir, 1988). Total world production of red raspberrytotals 277 131 mt of which Canada produces 5.4 %. The production of raspben'ieshas more than doubled over the last 20 years, from an average yield in 1961-1965of 107 006 mt (pAO, 1976; cited in Snir, 1988) to the present 27 .31 mt (FAO,1984). The increase in production is due mainly to enlarged plantation areas and improvedyields. Yield increases have resulted from using new cultivars, specifie virus testedstocks and new growing and harvesting methods (Snir, 1988).

Blackberry species are native in many parts of the world but little domesticationor commercial production have occurred with these except in Nonh America. Thisis most likely due to the readily avail able fruit in the wild and the thomy, unmanageablecharacter of wild blackberry plants (Moore, 1984). Until the 194O's, most blackberryacreage resulted from chance seedings from the wil Most of the virus and viruslike diseases which occur in cultivated and wild blackberry are latent and moderatelydepress plant vigor and yield. Some diseases can be more serlous, causing recognizablesymptoms and severely weakening the plants (Converse, 1984).

Although tissue-culturing blackberry is more expensive than conventional vegetativepropagation methods, problems with disease May be considered more important thancost considerations (Caldwell, 1984). Tissue culture may be the only practical methodof redisuibuting virus-free material quickly if strict certification programs for RlIhIaare put into effeet (Converse, 1981). The tissue culture of blackberry also allowsfor rapid dissemination of new cultivar releases and year-round productior of plantlets(Converse, 1984). Blackberry multiplies very rapidly in culture providing ample clonal


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plants (3-6 x every 3-4 weeks) (Kyte, 1987) for production and research purposes.'Silvan' (Rubus sp.) is an outstanding blackberry culuvar from Australia, with

high yield, goad fruit quality and increased tolerance to disease compared with othercultivars (McGregor and Kroon, 1984). Il is also tolerant to environmental stress suchas wind and drought.

Tissue cultured plants have phenotypic characteristics which refleet acclimatization to the unique environmental conditions found in vitro. These phenotypic characteristics include a reduced foliar epicuticular and cuticular wax layer when comparedwith control, greenhouse-grown plant leaves (Conner and Conner, 1984, Fabbri et al.,1986, Sutter and Langhans, 1982, ~ t t e r 1984, 1985), non-functional, open stomata(Brainerd and Fuchigami, 1981, Donnelly et al., 1986, Short et al., 1987, Wardle andShon, 1983), and a mixotrophic mode of nutrition that relies principally on sucroseas a carbon source (Conner and Thomas, 1982). Together, these characteristics resultin limited control over evapotranspiration rates (Brainerd and Fuchigami, 1981, Marinet al. 1988, Sutter, 1988) and initially low rates of photosynthesis in transplants fromculture (Grout, 1988). High monality and severe dehydration may occur after transplantation ex vitro i proper steps re not taken to slowly acclimatize the transplantsto the greenhouse or field environment. This generally consists o placing the transplantsunder conditions o high relative humidity (9.3-100 ) and relatively low light intensity(approximately 150 Jlmol m l S 2), not exceeding three times culture levels (Donnellyand Vidaver, 1984b). Subsequently, the relative humidity is lowered and the light intensityincreased over a period of approximately one month. The acclimatization period exvitro may be reduced when certain in vitro acclimatization strategies are used. Theseinclude increased agar concentrations in the culture medium (Ziv et al., 1983) and rootingin vitro (Murashige, 1974).


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The objectives of this research werc to:

1. Develop a rapid foUar clearing and staining method to enable examinationof the extemal morphology of tissue cultured plandets and grcenhouse-grownplant leaves.

2. Determine the effects of both increased agar concentrations 6, 9 and12 g/l) n the culture medium and n vitro rooting on ex vitro survival, growthand stomatal characteristics of micropropagated Sil van blackberry shoots.

3. Determine the effeet of high relative humidity and low light intensity onstomatal function of greenhouse-grown Silvan plants and ex vitro plandetsfrom full nd 1/4 strength modified MS (1962) basal medium.

It is hoped that these experiments w ll lead to better methods of ex vitro acclimatizationas well as an improved understanding of stomatal function in vitro and during the importantinitial weeks following ex vitro transplantation.

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2. Llterature Revie

ACCLIMATIZATION STRATEGIES FORMICROPROPAGATED PLANTS

Danielle J. Donnelly and Laurence Tisdall

Manuscript for the volume Micropropagation of Woody PlantsTo e published by K1uwer Academic Publishers.

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2.1. Introductionhe succcssful ex vitro acclimatization of micropropagatcd plants dctcr

mines the quality of the end product and, in commercial production, the economicviabillty of the entcrprise [6]. When shoots or plantlets are ttansplantcd fromculture to greenhouse conditions they may desiccate or wilt rapidly and can dieas a result of the change in environment, unless substantial precautions are takento accomooatc them. In commercial micropropagation this stcp is often thelimiting factor [53] and at besl, is challenging, labour intensive and costly [6,7, 10]. Methods which work for ex vitro establishment of one spccies arcnot necessarily satisfactory to ensore the survival of another [47].

he following discussion was not intended to be an exhaustive surveyof the now extensive literature pertaining to acclimatization of micropropagatedplants. The reader is directed to several excellent reviews [6. 7 10, 39, 75].We feel that overcoming ex vitro acclimatizajon problems is contingent on animprovoo general understanding of how the environment affects the anatomy andphysiology of all plants subjected to environmental change. It is necessary tobegin with a better understanding of the unique effects of the in vitro andthe ex vitro environments on plant phenotype. It is infonnation on this subjectthat is summarized herein. In this way we hope to provide sorne new insightsinto modem acclimatization strategies, applied both in vitro and ex vitro.

2.2. The Culture-Induced PhenotypeTissue cultured shoots and plantlets share certain characteristic features that

are inconsistent with development under greenhouse or field conditions. Theculture-induced phenotype CIP) [16] reflects epigenetic variation [18]; accli-matization t environmental conditions which exist within the closed culturecontainen. In vitro environments are characterized by: a saturatcd atmospherc;

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relatively low light intensity (photosynthetic photon flux), averaging 12-70 umolm2 Sl; rclatively high and constant tempcraturc (20-28 OC); low rates of gasexchange bctween the containers and the external atmosphere and high concen

t r t i o ~ j s of carbohydrate and exogenous growth regulators in the medium.Although wc arc aware of some aspects of the CIP it can hardly h dcscribcdas weil defined Our knowledge is mostly limited to tempcrate species andalmost exclusively to angiospcrms. While in some cases the envirormentaldeterminants arc known, a direct relationship between certain aspects of the CIPand sorne component(s) of the culture environment rcmain obscure.

In vitro shoots and plantlets are invariably diminutive; much smaller thantheir greenhouse-grown counterparts. Blackberry leaves (Rubus sp.) in culturewere only 1-2 the area of greenhouse-grown control plant leaves [16]. Inminiature red raspberry (Rubus idaeus L.) plantlets the proportion of foliar celland tissue widths to total leaf width were the same in culture as for the largegreenhouse-grown control plants [18]. Mature leaves of red raspberry plantletsalways had palisade:epidennal cell ratios of 1:1 or 2: l typical of very youngcontrol leaves prior to epidermal cell expansion and palisade cell division [21].Microcultured Asian white birch BelUla plat)l>hylla var szechuanica (Schneid)Rehd.) was also shown to be small, more from decreased cell division thanreduced cell size [58]. The relatively high cytokinin concentration, especiallyin Stage II [52] mOOia and the low water potential of most media tend toinhibit apical dominance and affect stature. Media that are more dilute orlacking in cytokinins (many Stage m media) promote the development of largerorgan sizc (Donnelly, pers. obs.). In vitro shoots and plandets have increasedpercentage water content and reduced dry ltter accumulation per unit arcacompared to greenhouse-growll plants [2, 19]. This is reflected in fragile organswith reduccd mechanical support tissue and thin cell walls. Rcduced mechanical

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support tissue formation occurred in ail organs o re raspberry [21]. Thiscould have been nutritionally based but could also have been influenced by thetranquil in vitro environment which inhibited cell wall dcposition and sclerenchyma and collenchyma formation [21]. n sorne species vascular connectionswere fewer, thinner and poorly s t r u t u r e ~ as in the petioles of Asian whitebirch [58], the stems o carnation lDianthus c8I) o.phyllus L.) [45] or the motshoot interface o adventitious cauliflower CBl assica oleraceae var botrytis)plantlets [30].

The relatively low light levels and saturated internai atmosphere promoteleaves in vitro that anatomically resemble both shade leaves [4, 44, S ] andhydrophytic plant leaves [31]. They often have reduced or absent epicuticularor cuticular wax, which can lack the characterislic crystalline structure, or differin chemical composition from that o control plants [4, S 23, 25 28, 30, 5759 60, 63, 64, 73]. n vitro leaves had a thinner or somewhat collapsedepidermal layer [4, 21, 73, 74] with a clearly defined [13, S8] or an absentor limited [4, 23, 30, 31, 73] palisade layer, sometimes with obconically-shapedpaIisade cells [13, 18] and a loosely organized spongy mesophyll with anincreased percentage air space [4, 13, 18, 23, 73]. Palisade development isrelated to light levels [22] and is reduced in vitro as the Iight levels arerelatively low [4]. Increasing the light intensity in sweetgum (LiQuidambarstyraciflua) cultures increased Ieaf thickness, promoted paIisade differentiation anddecreased the percentage air space in the mesophyll [44]. In red raspberry,eaves in culture were simple, rather than compound. This may have resulted

froin incubation at relatively high, constant temperatures (27 OC) [21]. TheyaIso had fewer trichomes and an aItered distribution o glandular and thick-walledunicellular hairs compared to greenhouse-grown plants. Increased light intensityin culture promoted filiform but not other trichome formation [18].

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Stomatal frequency and density was higher [44, 74] or lower [4] in viarodependinl on the species, and the stomatal index was not greatly affected [5]or was lower [13] comparcd to control plants. Stomata on leaves n culturewere more circular n shape [50 51] larger [S 50 51 74] and had larlersubstomatal cavities [21] than stomata on control leaves. Sizc o the substomatalcavities has been conelated to the amount o water stress; largest when therelative humidity is highest [48]. Stomalal aperture is usually larger n vitrothan on control leaves [2] with guard cells raised above the cpidennal layer[16, 44, 73, 74] but can diffcr n vitro depending on the stage o culture orwhere the leal is situate< on the shoot. Stomatal aperture gradually decreasedin chrysanthemum leaves towards the less mature leaves o the shoot apex [72].In vitro stomata have slow response times or impaired function [2, sa 63, 64,72]; they do not close in response ta stimuli such as darkness, abscisic acidapplication, solutions with high osmolarity mannitol or sucrase) or whenexposed ta high levels of carbon dioxide [3, 72, 74, 77]. In chrysanthemum,funher opening was possible n COz-free air and higher light intensity or throughcytokinin exposure and was followed by closure to their original aperture [72].Interestingly, guard cell protoplasm was seen to react appropriately when leavesfrom culture were placed in solutions of various osmolarities or containingabscisic acid. So, impaired stomatal function may result from mechanical ratherthan physiological causes; reduced or altered distribution o cellulose microfibrils in the guard cell walls affecting cell wall elasticity [72, 77]. Guard cellwalls o in vitro Prunus cerasus L. were thinner [50, S ] and lacked invaginationso the anticlinal epidennal cell walls next to the inter-guard ceU wall ends,present in acclimatized or greenhouse-grown plants [51].

The hydathodes o rosaceous species in vitro were simpler and had fewerwater pores with larger apertures and reduced epithem, the tissue that recovers

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solutes from the ttacheids, compared to greenhouse-grown plants. This may resultfrom the high relative humidity or the low water potential of the medium invitro compared to ~ n h o u s conditions [14, IS 17]

In vitro plants rely principally on sugar as a carbon source [ ] and COluptake capability is low [19, 20, 33, 34, 38]. Microcultured birch photosynthesized at one third of control plant levels [58] and red raspberry at aboutone quarter of control plant levels [19] at saturating light intensities. In vitroshoots and plantlets are mixotrophic in their mode of nutrition; they apparentIyalternate between carbohydrate use and COl fixation. Carbohydrate use isstimulated by the high concentration of sugar and the presence of growthregulators in the medium and the relatively low light intensity during incubation. Carbon dioxide fixation is stimulated for a shon time each day; theC 2 concentration in the containers is rapidly depleted within about two hoursof the start of the photoperiod to at or below the compensation point for therest of the day [26, 38]. Mixotrophy contributes to the recycling of cellularrespiration and photosynthetic products and affects photosynthetic carbon metabolism

Pigment synthesis and ribulosebisphosphate carboxylase RubPcase) activitymay be impaired in culture; photosynthetic pigment content was low-nonnalin cultured red raspberry [19] and cauliflower, which also had low RubPcaseactivity [33, 35]. Some in vitro shoots and plantIets had starch in theirchloroplasts [13, 51, 54, 72], white others had littIe or no starch, as in Leucaenaleucocephala Lam) De Wit. [13] and sweetgum [43, 73]. As sucrose concentration of the medium was augmented, starch concentration in the chloroplastsincreased [54]. Little starch was exponed from the chloroplasts during the darkperiod and t tended to accumulate. In vitro Ieaves exhibited flattened, disorganized chloroplasts, in sorne cases with swollen thylakoids [43, 54, 73].


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( Accumulation of starch and disorganization of thylakoid structure was atttibutedto the rclatively low light levels in culture [S ] or altercd light spectrum resultingfrom glass containen [43]. However, cauliflower cultures had control levels ofphotosynthetic elcctron transpon, indicating nonnal thylakoid strJCture and function[33].

Propagates arc apparently acclimatized to in vitro conditions as theirgrowth is extrcmely prolific. Furthermore, the in vitro environment affects awide range of species in a similar way, morphologically and physiologically(Donnelly, pers. obs.). However, a change in a single climatic parameter ormedium component may affect one or more of the CIP characteristics, whichin tum affects in vitro, and s u s ~ u e n t ex vitro performance.

2.3. Ex Vitro TransplantationDifficulties in successfully transplanting tissue cultured shoots and plantlets

to soil rc weil documented [6, 2S 66]. They appear t he a direct resultof the culture-induced phenotype which reflects adaptation t in vitro conditionsbut is inappropriate when shoots or plantlets rc transferred to the greenhouseor field where the relative humidity tends t he less than 100 , the ambientlight levels are much higher than in culture, there rc fluctuating temperatures,the substrate has a much higher water potential and it is necessary to convertrapidly from a mixotrophic t a fully autotrophic mode of nutrition to survive.

Ex vitro plantlets have extreme evapotranspiration rates and may guttatecopiously, demonstrating impaired ability ta regulatc water loss. Excessiveevapotranspiration is affected by reduced or nonexistant stomatal control [2, 3S SI 61, 6S 71, 72, 77], and large cuticular water los ses [2, SO] possiblydue to poor epicuticular and cuticular wax formation [61] or reduced trichomenumbers [18, 60]. The major mechanism of water loss may depend on the

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spccies in question, as some ~ p i e s have large quantities of epicuticular waxn vitro but still bave water regulation problems ex vitro [60, 61, 65). Nocorrelations have been established betwcen ex vitro survival and the physical ormorphologieal characteristics of foUI ' wax [60, 65). The ex vitro guttation ratemily he affccted by the large increase in substrate water potential at transplantation nd tends to increase under conditions that augment the transpiration rate[15]. Ex vitro root f nction is uncertain at the time of transplantation, especiallyn adventitious propagation systems, and rnay contribute to water deficit intransplants [1, 6, 30).

To promote ex vitro survival and physiological competence; especiallyto gu rd against water stress and encourage autotrophy, a transitional environment is usually supplied for an acclimatization interval, ranging in duration fromone to several weeks [3, 4, 6, 23, 34]. In this transitional environment therelative humidity is kept in the range o 70-100 % via tenting, misting orfogging and the light level should not be too much greater than it was inculture. Red raspberry survival was optimal when the light intensity did notinitially exceed a three-fold increase over that found in culture [21]. Growthwas not limited by light since CO2 uptake was not different in transplants grownat light intensities two- to three-fold higher than in culture [19]. Gradually,as the plantlets acclimatize, the relative humidity can be decreased and the lightlevels can he increased towards ambienL

2.4. Transplant Phenotypes Ex Vitro2.4.1. The Persistent Leaves

Lcaves that developed in culture were retained after transplantation for aweek to several months prior to senescing [18, 19, 21, 32, 34]. Persistencedepended on the plant species and the degree o environmental stress ex vitro.

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These penistent leaves increased in size slightly, mainly due to il elongation[23, 31), and accumulated dry matter under cenain conditions [19). In somecases wax was deposited on the leaf surface after transplantation [23, 30, 50,64, 71, 7 i). Stomatal function open-closure mechanism) was either improved[ 1) or was not established in persistent leaves [70]. In most cases stomataliunction has been equated with closure [2-4, 18, 50 51, 61, 65, 74). However,stomatal closure may only indicate the collapse of the gu rd cell membranesin response to exposure to low levels of relative humidity 5) and need notindicate the stomatal capacity to reopen.

The role of the persistent leaves remains a controversial and importantissue. Photosynthetic capacity appears to vary with plant species in culture andmay detennine the x vitro contribution of persistent leaves. Cultured plantsare divisible into photosynthetically non-competent and competent species 29).For example, in the non-competent species group, cultured cauliflower [32] andstrawberry [34) were net respirers both in vitro and after transplantation. Inthese species, leaves that developed in culture deteriorated rapid1y after transplantation. These leaves contributed only those nulrients which could he resorbedby the transplant. Such leaves have been referred to as storage organs orpseudo-cotyledonary tissues [34, 69, 71]. Non-competence in strawberry has beenattributed to irreversibly reduced levels of RubPcase activity in leaves developedin the presence of sucrose Strawberry plandets defoliated in the absence ofsucrose in the medium were competent [29, 3S] Dieffenbachia Dieffenbachia

~ 3 3 as well as potato Solanum tuberosum) and chrysanthemumChr.ysanthemum morifolium) [29] were photosynthetically competent in vitro.

They achieved a positive carbon balance in culture and continued to conuibutephotosynthetically after transplantation. Leaves of competent species did notdeteriorate rapid1y after transplantation [29, 33, 69). Persistent leaves of Asian


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white birch [58] and m raspberry [19] seem to fall into the competent group.Red raspberry plantlets photosynthesized at a low level after transplantation.However, persistent leaves shifted to become both net respiren and sinks forphotoassimilates formed in the new leaves by one month ex vitro [19]. Retentionlime of these persistent leaves ranged up to three months ex vitro [18].

2.4.2. The New LeavesThe phenotype of new leaves formed ex vitto varies with the species,

the culture and transplant environments and the age of the transplant. Newleaves of cauliflower a non-competent species) that formed the second weekafter transplantation apparently exhibited greenhouse control levels of CO2 uptake[30]. However, new leaves of red raspberry a competent species) were transitional n the sense that weekly flushes of new leaves became progressivelylarger eventually with control-type anatomy, functional stomata and improved CO2uptake capability [18-21]. Measured five weeks after transplantation leavesformed during the fust week had activity levels much higher than in culture.but resembled the cultured leaf phenotype white those fonned the fifth weekwere operating at about half the control CO2 uptake rates and anatomicallyresembled greenhouse-grown control plant leaves [20]. Transitional leaves havealso becn observed in other plant species, both competent and non-competent[13, 23, 50 73, 74].

The number of transitional leaves produced by a transplant may dependon the number of immature leaf buds formed in culture. The degree oftransition of these leaves and how closely they resemble those of control plantsis probably a reflection of the stage of development of leaf primordia whenthe plantlet was transferred from culture and the conflicting stresses imposed onleaf development by both the culture environment and the new ambient

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environrnent [18. 19 21] It is likely too. that the retention of any culturetype organs on the transplant influences the physiological status of the rest ofthe plant [21].

2 5 Ac:climatization Strategies for Micropropagated Plants2.5.1. Acclimatizatlon Ex Vitro

Traditionally the acclimatization environment ex vitro is adjusted toaccomodate ttansplants from culture; gradually weaning them towanis ambientrelative humidities and Iight levels. As previously mentioned, transplants mustundergo a period of acclimatization, more specifically. a period of transitionaldevelopment in which both anatomical characteristics and physiological performance escape the influence of the in vitro culture conditions [19 21]. WhiteStage III plantlets are generally easier to transfer to soil than Stage n shoots.whenever possible shoots are preferred due to economic considerations [6].

There re inherent limitations in the efficiency of conventional transplantation protocols. For non-competent species the transplanting risks are muchgreater than for competent species. However. even the competent species canbe slow to adjust to lower relative humidity and take time to become photosynthetically efficient

Much has becn written on the optimization of ex vitro transplant environments for tissue cultured shoots and plandets [6 7, 10 75]. Novel approachesto ex vitro acclimatization include Oz enrichment without [42] or with supplementary lighting [11]. These reduced the ex vitro acclimatization interval incontrolled humidity chambers or greenhouses but failed to eliminate the requirement for habituation to low humidity [11] Among the most sophisticated exvitro acclimatization procedures utilizes the acclimatization unit [36, 40], anapparatus which emerged from the engineering science of climate-controlled green-

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houses. The micro-computer conb Olled acclimatization unit can determine therelative humidity temperature, light intensity COz concentration ir flow rateand even the temperature of the nutrient solution and has the potential to controlalmost every other feature of the environment. Ali facets o the environmentcan h made to change by increments over time; ranging from simulated invitro conditions at transplantation to that o the greenhouse or the open fieldweeks later. In the beginning changes are made in small increments whichare later increased. Special emphasis is placed on minimizing water stress inthe early stages ex vitro. It is not surprising that in such a unit both transplantsurvival and growth rates are significantly increased. For now this enviableresearch tool is beyond the reach o most scientists.

Antitranspirants have not proven useful in promoting ex vitro survival orperformance; phytotoxicity and interference with photosynthesis were both citedas possible reasons [62]. Other leaf surface-covering agents such s glyceml.paraffm wax and grease promoted ex vitro survival of several herbaceous speciesbut have not been evaluated over the long term or examined on woody species[55].

2.5.2. Hardening-off In VitroMurashige [52] was fU St to promote hardening o plants during Stage III.

This facilitates but does not of course preclude acclimatization ex vitro.Murashige recommended the reduction of medium nutrients, the use of auxinsfor rooting and increased light levels. Three major strategies have emerged thatfocus on substantially changing the in vitro environment especially in the laterstages of micropropagation in order to modify the CIP towards improved storagecapability photosynthetic competence or water relations and thus facilitatetransplantation.

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( The rUSI strategy assumes that larger persistent leaves, packed with greateramounts of storage compounds, would contributc mo after transplantation.Increasing the concentration of sugar in the medium might maximizc the numentfunction of persistent leaves [lI 34]. To some extent this strategy has beendiscounted as apt to heighten evapotranspiration losses in transplants [11 34].However, it seems to hold promise for some plants [46 54].

The second strategy assumes that autotrophic cultures will have persistentleaves that live longer and would be more photosynthetically productive ex vitro[34]. The objective is to madify the CIP towards autotrophy in culture. Todo this, the oxygen concentration can e reduccd in the culture environment,which depresses the photorespiration rate [56]. Alternatively, the sugar is reducedor completely eliminated (rom the me.dium [33-35, 38] while the photosyntheticphoton flux [12, 38, 41] and the carbon dioxide concentration [12, 41] areincreased [8]. Increasing the light intensity alone cannot raise the netphotosynthetic rate for cultures at their COz compensation point. Such aphotoautotrophic tissue culture system (PTCS) has the added advantage that

microbial contamination is less of a problem when sugar is eliminated from themedium [27]. In this system a gas permeable, c1ear plastic film is uscd asa vessel closure [38]. This plastic film improves gas exchange to the cultures;COz enrichment or 02 reduction; increases the light penetration to the containercontents and decreases the relative humidity of the vessels. Strawberry shootsrooted in the PTCS unit had dry weights 1.7-fold greater and photosyntheticrates 4-fold higher than plantIets in the control, Stage u treatment [27]. Nospecial ex vitro care was required for sorne plants [36, 37] although water stresswas still a problem for others [27]. In adopting the PTCS, in vitro culturehas been exchanged for a hydroponic system. This has the advantage that contamination by bacteria and fungi are no longer a problem in vitro. extensive

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climate conb Ol, Jarger vessels and robotization are possible and the ex vitroacclimatization stage is less of a problem [39]. However, one must acccpt theconcUITCnt loss of many of the advantages of micropropagation, such as rowthrates and miniaturization [8] and assume the problems of hydroponic systems,such as algae control and the requirements for chronic nutrient solutionadjustments (27).

The third strategy assumes that plants developcd under lower relativehumidity will have fewer transpiration and translocation problems ex vitro andpersistent leaves that look more like control plant leaves. The objective is tamodify the CIP away from the characteristic hydrophytic-type anatomy andpromote epicuticular and cuticular wax development, stomatal function andpossibly overcome other deficiencies. Lowering the relative humidity in vitrohas been done experimentally with varying results using desiccant in or aroundthe culture container, by coating the medium with oily materials, or both [57,64, 76), by opening culture containers into low relative humidity atmospheres(2), adjusting culture closures to reduce the relative humidity [57], using specialclosures that facilitate water loss [24] or by cooling container bottoms [67, 68].Generally the relative humidity could not he lowered to less than 80-85without culture in ury [57, 76]. A relative humidity of 85 decreased themultiplication rate of carnation but increased the number of glaucus leaves, thepigment and protein content, decreased the percentage water content and improvedex vitro survival [76]. At 80 relative humidity, growth rates of cauliflowerand chrysanthemum were similar to those of controls grown under 100 relativehumidity but ex vitro transplantation was greatly facilitated by functional stomataand greater epicuticular wax deposition in plantlets rooted at the lower relativehumidity [57]. Increasing the sugar or agar concentrations or adding osmoticagents such as polyethylene glycol to the medium will also lower the relative

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( humidity and in some cases served the same purpose as desiccants [45, 49,57, 76]. Cooling container bottoms created a temperature gradient of aboutOC condensing watcr vapor on the surface of the medium and lowering therelative humidity at plant height inside the containers [67, 68].

By decreasing the relative humidity in culture containers, both ttanspira-tion and translocation systems re presumably improved in cultured plants [7,8] with associated improvement in minerai ion uptake through the transpirationstream and other benetits [9]. The obvious disadvantage of more extreme relativehumidity reduction was to decrease the multiplication rate [57, 75, 76], posingan obvious dilemma [76]. Ziv [75] recommended that relative humidity reductionshould be considered in vitro, even at the expense of reduced propagation rates.As the propagation rate o cauliflower and chrysanthemum was not apparentlycompromised at 80 , relative humidity reduction was followed by theelimination of sucrose which successfully promoted autotrophy in Stage mcultures of both these plants [57]. This resulted in comparable photosyntheticrates for plantlets rooted at 80 relative humidity in vitro and seedling plants,and underlined the viability of this approach.

2.6. Conclusions and ProspectsIt is premature ta advocate any one of the several, not mutually exclusive,

acclimatization strategies presented above. Conventional ex vitro acclimatizationworks for many but not all micropropagated plants. When such protocols arerefined, they succeed in shortening the establishment interval and promotingsurvival, but do not usually eliminate the necessity for low humidity habitu-ation. The costs o facilities dedicated to ex vitro acclimatization can also bea limiting factor [29]. In order to deemphasize ex vitro acc1imatization andreduce associated costs il may he necessary , ) precede this stage by in vitro

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treatments. In vitro hardening-off procedures may e appropriate for some plantspecies, but are apparently not advantageous for ail. Modification to the CIPto promote improved photosynthetic competence and water regulation in shootsor plantlets at the later stages of micropropagation is deceptively simple. Asmore documentation results from the implementation of these varied acclimatization strategies. alone or in combination, it will e possible to make choicesmore accurately. These would probably e made on a plant by plant basiswith attention to economic considerations.

Tissue culturists are n an ideal position to evaluate the phenotypicplasticity of plants and to sort out the environmental determinants of plantphenotype, since they have access to large numbers of genetically identical plantsgrown under climate controlled conditions. In this way wc can learn moreabout the adaptational responses of all plants to specific environmental cues andclimatic changes. and can also meet an urgent industry objective, to successfullyaccomodate or manipulate the culture-induced phenotype in arder ta promotesurvival and performance ex vitro.

2.7. SummaryEx vitro acclimatization of micropropagated plants can e difficult, costly

and may Iimit commercial micropropagation. Solving ex vitro acclimatizationproblems is contingent on an improved understanding of the unique effects ofthe n vitro and ex vitro environments on plant phenotype.

Micropropagated plants possess a unique cuIture-induced phenotype CIP),reflecting acclimatization to in vitro conditions of: saturated environment; lowlight intensity; high and constant temperature; low rate of gas exchangebetween the containers and the extemal atmosphere and high concentration ofcarbohydrates and growth regulators in the medium. Many species are affected

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in a similar way, bath morphologically and physiologically, in vitro PlantlelSare tiny with increased percentage water content and have fragile organs withreduced mechanical suppon tissue, thin cell walls and, in SOlDe species, fewervascular connections. Leaves in vitro resemble those of shade and hydrophyticplants; with reduced or altered. epicuticular and cuticular wu thinner epidermallayers and loosely organized mesophyll with increased percentage ir spacecompared to control l e a v e ~ Stomata of cultured plants may vary in density,shape, size and function compared to those of control plants. Impaired stomataIfunction of persistent leaves appears to result from mechanical and notphysiological causes. In vitro plants are mixotrophic; altemating betweencarbohydrate use and C02 flXation. Pigment synthe sis and ribulosebisphosphatecarboxylase activity may be affected in culture.

The CIP is inappropriate when plants are transferred from culture togreenhouse or field conditions. Ex vitro transplants have extreme evapotranspiration rates due to reduced stomataI control, large cuticular water loss and, insome spe.cies, reduced trichome numbers. Water regulatory problems may beaffected by ex vitro guttation and in cenain adventitious propagates impaired rootfunction. Sorne species are photosynthetically non-competent both in culture andafter transplantation white others are competent. In non-competent speciespersistent leaves deteriorate rapidly after transplantation, behaving like storageorgans, while in competent species they do noL New leaves of transplants N Ctransitional; varying phenotypically from culture- to control-type with successiveflushes of new leaves. The degree of transition is affected by the stage ofdevelopment of leaf primordia before transfer from culture and conflicting stresseson leaf development imposed by the culture and transplantation environments.

Conventional ex vitro acclimatization involves a graduaI weaning oftransplants from culture conditions towards ambient relative humidity and light

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levels. This is more difficult for unrooted shoots and non-competent speciesthan for plantlets or competent species. Modem approaches involve supplementa1 COz with or without supplemental lighting, and use of a completely climatecontrolled acclimatization unit . Hardening-off in vitro plants facilitates but doesnot preclude acclimatization ex vitro. Three major strategies for in vitrohardening have focused on changing the culture environment to modify the CIPtowards improved storage capability, photosynthetic competence or water relations.Increasing the sugar concentration of the medium to maximize the storagecapability of persistent leaves holds promise for sorne species. The promotionof autotrophy via COz enrichrnent or Oz reduction, reduced levels of sugar andincreased light intensity is promising for sorne plants, although water stressremains a problem t transplantation. Lowering the relative hurnidity to 80S via several methvds did little to compromise the multiplication rate for

certain species and facilitated transplantation. Elimination of sugar from themedium, in conjunction with reduced relative humidity, promoted autotrophy forboth a competent nd a non-competent species.

In vitro hardening-off procedures are relatively new and have not yet beenwidely evaluated. It is premature to advocate any one of the several, nolmUlually exclusive, acclirnatization strategies outlined above. Tissue eulturistshave aeeess to large nurnbers of clonaI plants grown under defined environrnentalconditions. They are n an ideal position to define the environrnenlaldeterminants of phllt phenotype and eharaeterize plant phenotypic plasticity.

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2.8. References Citecl1. Rirchem, R., H.E. Sommer, and C.L. Brown. 1981. Seanning electtonmicroscopy of shoot and l OOt development in swectgum eallus tissue. ForestSei. 27:206-212.2. Brainerd, K.E. and L.H. Fuchigami. 1981. Acclimatization of asepticallycultured apple plants to low relative humidity. J. Amer. :Soc. Hon. Sei. 106:515-518.3. Brainerd, K.E. and L.H. Fuchigami. 1982. Stomatal functioning of inilm and greenhouse apple leaves in darkness, mannitol, ABA and COz J.

Expt. Bot. 33:388-392.4. Brainerd, K.E., L.H. Fuchigami, S. Kwaitkowsld, and C.S. Clark. 1981.Leaf anatomy and water stress of aseptically cultured 'Pixy' plum grown underdifferent environments. HonScience 16: 173-175.S. Conner, L.N. and A.J. Conner. 1984. Comparative water loss romleaves of Solanum laciniatum plants cultured in vitro and in vivo. Plant ScienceLett. 36:241-246.6. Conner, A.J. and M.B. Thomas. 1982. Re-establishing plantlets romtissue culture: a review. Proc. Inter. Plant Prop. Soc. 31:342-357.7. Debergh, P.C. 1986. Micropropagation of herbaceous plants. In: Micropropagation in horticulture. Practice and commercial problems. Alderson, P.O.and W.M. Dullforce (eds.) p. 27-36.8. Debergh, P.C. 1988. Control of in vitro plant propagation. Internationalsymposium on plant biotechnology. Piracicaba, Brazil. Oct. 25-28, 1988.9. Debergh, P. 1988. Improving mass propagation of in vitro plantlets.In: Horticulture in high technology era. Symp. Tokyo, Japan. May 10-11 p.47-57.


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10. Debergh, P. 1988. Micropropagation of woody species - stare of thean on in vitto aspects. Acta Hon. 227:287-295.11. Desjardins, Y., A. Gosselin and S. Velle. 1987. Acclimatization of exvitto sttawberry plantlets in CO 2- enriched environments and supplementary lighting. J. Amer. Soc. Hon Sei. 112:846-851.12. Desjardins. Y F. Laforge, C. Lussier and A. Gosselin. 1988. Effectof CO2 enriehment and high photosynthetie photon flux on the development ofautotrophy and growth of tissue-eultured strawberry, raspberry and asparagusplants. Acta Hon. 230:45-53.13. Dhawan, V. and S.S. Bhojwani. 1987. Hardening in vitro and morpho-physiological changes in the leaves during aeclimatization of micropropagatedplants of l ueaena leueocphala (Lam.) De Wit. Plant Sei. 53:65 72.14. Donnelly, D.1. and F.E. Skelton. 1987. Hydathode structure of micro-propagated plantlets and greenhouse-grown 'Totem' strawberry plants. J. Amer.Soc. Hon. Sei. 112:755-759.15. Donnelly, D.J. and F.E. Skelton. 1989. Comparison of hydathode structurein micropropagated plantlets and greenhouse-grown 'Queen Elizabeth' rose plants.J. Amer. Soc. Hon. Sei. 114: (In Press).16. Donnelly, D.1., F.E. Skelton and H.A. Daubeny. 1986. Externat leaffeatures of tissue-cuItured 'Silvan' blackberry. HortScienee 21 :306-308.17. Donnelly, D.J., F.E. Skelton and J.E. Nelles. 1987. Hydathode anatomyand adaxial water loss in mieropropagated 'Silvan' blackberry. J. Amer. Soc.Hon. Sei. 112:566-569.18. Donnelly, D.1. and W.E. Vidaver. 1984. Leaf anatomy of red raspberrytransferred from eulture to soil. J. Amer. Soc. Hort. Sei. 109:172-176.

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19. DonneUy, D.J. and W.E. Vidaver. 1984.exchange of red raspbcrry in mm and B mm109: 177-181.

Pigment content and guJ. Amer. Soc. Hon. Sei.

20. Donnelly, D.1., W.E. Vidaver and K. Colbow. 1984. Fixation of 142in tissue cultured red raspberry prior to and after transfer to soil. Plant Cell

Tissue Organ Culture 3:313-317.21. DonneUy, D.J. W.E. Vidaver and K.Y. Lee. 1985. The anatomy oftissue cultured red raspberry prior to and after transfer to soil. Plant CeU TissueOrgan Culture 4:43-50.22. Esau, K. 1977. Anatomy of seed plants.23. Fabbri, A., E. Sutter and S.K. Dunston.

2nd cd. Wiley, New York.1986. Anatomical changes n

persistent leaves of tissue-eultured strawbcrry plants after removal from culture.Scientia Hon. 28:331-337.24. Fari, M., A. Andrasfalvy and J. Nemeth. 1987. Thin PVC foil coveringTPFC), an efficient method for culture and preacclimatization of in vitro plant

cultures. Acta Hon. 212:371-374.25. Fuchigami, L.H. T.Y. Cheng and A. Soeldner. 1981. Abaxial transpirationand water loss in aseptically cultured plum. J. Amer. Soc. Hon. Sei. 106:519-522.26. Fujiwara, K., T. Kozai and I Watanabe. 1987. Fundamental studieson environments in plant tissue culture vessels. 3) Measurements of carbondioxide gas concentration in stoppered vessels containing tissue cultured plantletsand estimates of net photosynthetie rates of the plantlets. J. Agr. Met. 43:21-30 [Japanese with English summary).27. Fujiwara, K., T. Kozai and J. Watanabe. 1988. Development of aphotoautotrophic tissue culture system for shoots and/or plantlets at rooting andacclimatization stages. Acta Hort. 230:153-166.

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28. Grout, B.W.W. 1975. Wax dcvelopment on lea surfaces of Brassicaoleraceae var. Cumwong ~ g e n e r a t e d from meristem c u l t u ~ Plant Sei. Lett.5:401 4OS.29. Grout, B.W.W. 1988. Photosynthesis of regenerated plantlets in mand the stresses of transplanting. Acta Hon. 230:129-135.30. Grout, B.W.W. and M.J. Aston. 1977. Transplanting of cauliflower plantsregenerated from meristem culture. 1 Water loss and water ttansfer relate< tochanges n leaf wax and to xylem regeneration. Hon. Res. 17:1-7.31. Grout, B.W.W. and M.J. Aston. 1978. Modified leaf anatomy ofcauliflower plantlets regenerated from meristem culture. Ann. Bot. 42:993-995.32. Grout, B.W.W. and M.J. Aston. 1978. Transplanting o cauliflower plantsregenerated from meristem culture. Il. Carbon dioxide fixation and the devel-opment of photosynthetic ability. Hon. Res. 17:65-71.33. Grout, B.W.W. and M.E. Donkin. 1987. Photosynthetic activity of cauliflower meristem cultures in iWl and at transplanting into soil. Acta Hon.212:323-327.34. Grout, B.W.W. and S. Millam. 1985. Photosynthetic development ofmicropropagated strawberry plantlets following transplanting. Ann. Bot. 5S: 129131.35. Grout, B.W.W. and F. Priee. 1987. The establishment of pholosynlheticindependence in strawberry cultures prior to transplanting. Proc. Symp. Plantmicropropagation in honicultural industries. Ducate, G., M. Jacobs and A.Simeon (eds). Arlon, Belgium. p. 55-60.36. Hayashi, M. and T. Kozai. 1987. Development of facility foraccelerating the acclimatization of tissue cultured plantlets and the performanceof test cultivations. Pree. Symp. Plant micropropagation in horticultural industries.Ducale, G., M. Jacobs and A. Simeon. (eds). Arlon, Belgium. p. 123-134.

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37. Hayasm M. M Nakayama and T. Kozai. 1988. An application othe acclirnatization unit for growth o carnation explants, and for rootin andacclimatization o the plantlets. Acta Hort. 230:189-194.38. Kozai T. 1988. Autottophic micropropagation for mass propagation oin-vitro plantlets. Plant biotechnology seminar. Jakarta. 12-13 Dec. 1988.39. Kozai T. 1988. HiSh technology in protected cutlivation - fromenvironment control engineerins point o view. In Horticulture in mghtechnology era. Special Lecture. Tokyo. p. 1-43.40. Kozai T., M. Hayashi, Y. Hirosawa, T. Kodama and J. Watanabe. 1987.Environmental control for acclimatization o in vitro cultured plantlets. 1)

e v e l o p m ~ n t o the acclimatization unit for accelerating the plandet growth andthe test cultivation. J. Agr. Met. 42:349-358. 1apanese with Engtish summary).41. Kozai T., H. Oki and K. F u j ~ w a r a 1987. Effeets o COz-enrichmentand sucrose concentration under high photosynthetic photon fluxes on growth orissue-cultured Cymbidium plantIets during the preparation stage. Proc. Symp.Plant micropropagation in horticultural industries. Ducate, G., M. Jacobs andA. Simeon. Arlon Belgium p. 135-141.42. Lakso A.N., B.I. Reisch, J. Mortensen and M.H. Roberts. 1986. Carbondioxide enrichment for stimulation o growth o in vitro-propagated grapevinesafter transfer from culture. J. Amer. Soc. Hort. Sei. 111:634-638.43. Lee N., H.Y. Wetzstein and H.E. Sommer. 1985. Effeets o quantumflux density on photosynthesis and chloroplast ultrastructure in tissue-culturedplantIets and seed1ings of Liquidambar styraciflua L. towards improved acc1ima-tizarion and field survival. Plant Physiol. 78:637-641.44. Lee N., H.Y. Wetzstein and H.E. Sommer. 1988. Quantum flux densityeffects on the anatomy and surface morphology o in vitro- and in vivo-developedsweetgum leaves. J. Amer. Soc. Hort. Sei. 113:167-171.

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45. Leshem, B. 1983. Growth of carnation meristems iD mm anatomicalstructure of abnonnal plantlets and the effeet of agar concentration in the mediumon their formation. Ann. Bot. 52:413-415.46. Maene, L. and P. Debergh. 1985. Liquid medium additions to establishedtissue cultures t improve elongation and moting in vito. Plant Ccll TissueOrgan Culture. 5:23-33.47. Macne, L.J. and P.C. Debergh, 1986. Optimization of plant micropropa-gation. Med. Fac. Landbouw. Gent 51:1479-1488.48. Manning, C.E., D.O. Miller and J.D. Teare. 1977. Effeet of moislurestress on leaf anatomy and water use efficiency of peas. J. Amer. Soc. Hon.Sei. 102:756-760.49. Marin J.A. and R. Oella. 1987. Acclimatization of the micropropagatedcherry rootstock Masto e Montanana (Prunus cerasus L.). Acta Hon. 212:603-609.50. Marin J.A. and R. Oella. 1988. Is desiccation the cause of the poorsurvivaI rate in the acclimatization of micropropagated Prunus cerasus L. ActaHon. 230:105-112.51. Marin, J.A., R. Gella and M. Herrero. 1988. Stomatal structure andfunctioning as a response to environmental changes in acclimatized micropropagatedPrunus cerasus L. Ann. Bot. 62:663-670.52. Murashige, T. 1974. Plant propagation through tissue culture. Ann. Rev.Plant Physiol. 25:135-166.53. Poole, R.T. and C.A. Conover. 1983. Establishment and growth of inYilm-cultured Dieffenbachia. HortScience 18: 185-187.54. QueraIt, M.C. 1989. HistologicaI and ecophysiologicaI study ofthe changes occurring during the acclimatization cf n vitro cultured roses. Ph.D.Thesis. State Univ., Gent, Belgium. 98pp.

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55. Selvapandiyan A. 1. Subramani P.N. Shan and A.R. Mehta. 1988. Asimple method for direct transplantation o cultured plants to the field. PlantSei. 56:81 83.56. Shimada N. F. Tanaka and T. Kozai. 1988.concentration on net photosynthesis of C3 plantIets in 187.

Effects of low 02Acta Hon. 230:171-

57. Shon K.C. 1. Warbunon and A.V. Robens. 1987. In liIm hardeningo cultured cauliflower and chrysanthemum plantlets to humidity. Acta Hon.212:329-334.58. Smith M.A.L. J.P. Palta and B.H. McCown. 1986. Comparative anatomyand physiology o microcultured seedling and greenhouse-grown Asian whitebirch. 1. Amer. Soc. Hon. Sei. 111:437-442.59 Sutter E. 1984. Chemical composition o epicutieular wax in cabbageplants grown in Yiml Cano 1. Bot. 62:74-77.60. Sutter E.G. 1985. Morphologiea]. physieal and chemical characteristieso epicuticular wax on omamental plants regenerated in rltm. Ann. Bot. 55:321-329.61. Sutter E. 1988. Stomatal and eutieular water 10ss from apple cherryand sweetgum plants aCter removal from in Yilm culture. 1. Amer. Soc. Hort.Sei. 113:234-23rs.62. Sutter E.G. and M. Hutzell. 1984. Use of humidity tents and antitranspirants in the acclimatization of tissue-cultured plants to the greenhouse.Scientia Hon. 23:303-312.63. Sutter E. and R.W. Langhans. 1979. Epicuticular wax formation oncarnation plantlets regenerated from shoot tip culture. 1 Amer. Soc. Hon Sei.104:493-496.

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64. Sutter, E. and R.W. Langhans. 1982. Formation of epicuticular waxand its effect on water loss in cabbagc plants regeneratcd from shoot-tip culture.Cano J. Bot. 60:2896-2902.65. Sutter E.G., V. Novella and K. Shackel. 1988. Physiological andanatomical aspects o water stress of cultured plants. Acta Hon. 230:113-119.66. Timmis R. and G.A. Richie. 1984. Progress in Douglas frr tissue culture.Int. Symp. Recent advances in forest biotech. Traverse City, MT 1984. p. 37-46.67. Vanderschaeghe, A.M. and P.C. Debergh. 1987. Technical aspects ofthe control of the relative humidity in tissue culture containers. Proc. Symp.Plant micropropagation in horticultural industries. ducate, G., M. Jacobs and A.Simeon. (eds.) Arlon, Belgium. p. 68-76.68. Vanderschaeghe, A.M. and P.C. Debergh. 1988. Automation of tissueculture manipulations in the final stages. Acta Hart. 227:399-401.69. Wan11e K. V. Dalsou, 1 Simpkins and K.C. Shan. 1983. Redistributiono rubidium in plants of Cho santhemum morifolium Ram cv. Snowdon derivedfrom tissue cultures and transferred ta soil. Ann. Bot. 51:261-264.70. Wanlle K. E.B. Dobbs and K.C. Shon. 1983. In.Y.ilm acclimatizationo aseptically cultured plantlets to humidity. J. Amer. Soc. Hart. Sei. 108:386-389.71. Wardle K. A. Quinlan and 1 Simpkins. 1979. Abseisic acid and theregulation of water loss in plantlets of Brassica oleraceae L. var. botJytisregenerated through apical meristem culture. Ann. Bot. 43:745-752.72. Wan11e K. and K.C. Short. 1983. Stomatal response of in vitro culturedplantlets. 1 Responses n epidennal strips of Chrysanthemum to environmentalfactors and growth regulators. Biochem. Physiol. Pflanzen. 178:619-624.

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1

73. Wetzstein H.Y. and H.E. Sommer. 1982. Leaf anatomy of tissue culturedliQuidambar styraciflua Hamamelidaceae) dwing acclimatization. Amer. 1. Bot.69: 579158674. Wetzstein H.Y. and H.E. Sommer. 1983. Scanning electton microscopyof in Yi 2 cultured LiQuidambar styraciflul plantIets during acclimatization. 1.Amer. Soc. Hon. Sei. 108:475-480.75. Ziv M. 1986. In Yi tm hardening and acclimadzation of tissue cultureplants ln: Withers L.A. and P.O. Alderson eds.) Plant Tissue Culture andils Agricultural Application. Butterworth London 1986 p. 187-196.76. Ziv M. G. Meir and A.H. Halevy. 1983. Factors influencing theproduction o hardened glaucous carnation plantIets in mm Plant CeU TissueOrgan Culture 2:55-65.77. Ziv M. A Schwartz and D. Fleminger. 1987. Malfunctioning stomatain vitteous leaves o carnation pianthus cruyophyllus) plants propagated in vitro;implications for hardening. Plant Sci.52: 127-134.

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Clearing and staining leaves is useful for observing anatomieal detailsincluding stomatal index (AmOlt, 1959, O Brien and McCully, 1981). Themethod s main advantage over other techniques is the short preparation time andthe facility with whieh large sections of a leaf can he observed at one time.A variety of Il ethods were recommended in the literature, many requiring severaldays to complete with the results heing, at times, inadequate (Rodin and Davis,1967, Shobe and Lersten, 1967). An attempt was made to develop a clearingand staining method rapid enough to enable large numbers o samples to hecleared and stained within 1 day. It was also hoped that the method developedcould be used for a range of different plant species with varying leafmorphologies as well as for fragile tissue-cultured material.

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(

Rcpnnled (rom HORTSCICNCE. Vol. 23(6), [kcembcr 1988A pubheallon of the Amerlcan SocIC )' for Horllcuhural SClencc:. Alcundria, Virainia

HORTSCIENCE 23(6):1059-1061. 1988.A Rapid Clearing and StainingMethod for Tissue cultured Plantletsand Greenhouse grown LeavesLaurence Tisdall and Danielle J. DonnellyDepanment of Plant Science, Macdonald College o McGill University,21111 La ..cs/wre Road. Ste AI/ne de Bellevue, Quebec H9X ICa, CanadaAddlIIonal Index words. leaf c1earlllg, mlcroscopy, plant anatomyAbstract. A simple, rapid clearing and staining method was developed using intact invitro blackbury (Rubw spp. 'Silvaa') and sfrawberry (Fragana Xananassa Duch.'Tolem') plantlets and sections 01 greenhousegrown 'Silvan' and 'Totem' leaves. Thedc:aring mc:thod involved Ihree steps: 1) autodaving in 80% ethanol to remove thechlorophyll, whicb fook 15 fo 20 min for p'ant/eu and 25 and 35 min lor greenhousegrown 'Silvan' and 'Totem', l'tspectivtly; 2) dissolution or the protoplasm gsing 5NaOU at 80C, which tOOk 20 min lor plantle15 and 3S ancl 40 min for greenhousegrown 'S van' and 'Totem' respective/y; J) post-alkali treatlIIent wilh 75% bleach(4.5% NaCIO). For lissue-cullured planllets this look S to 10 min at room temperature,but grtc:nhousegrown malerial required 35 10 40 min at 55. Aqueous safranin (10mg liter- I ) was used for slaining. This method gave consislently good resulls and required a maximum of l hr for completion.

mIcrotome curlings by glVmg a better ideaof spatial relallonshlps between leaf tissues(1). Desplle 115 sefulness, tissue clearing 15a lechmque I h ~ ~ a s been largely underemployed. Clearing alltl slaming methods havebeen galhered and suml"1anzed (4, 7). Many

clearing melhods are excesslvely tlmccosummg, requlring severa/ days (9, 10),h3ve many steps wuh several differensomeumes tOXIC (I.e., pyndine), chemlC(5), while rhe final results are InadequateOur intention WOlS to develop a rapld. SIple, and reliable c1eanng method (or IIssucultured planllels and greenhousegrowleaves of raspberry and slrawbeny. To thend soughl 10 improve the most comprhensive threc-stcp clearing processes. The, volve: 1) pretrealment, 2) dlSsolulionprolOplasm, and 3) p o s \ - a l ~ a h IreJrmenPretreatment Involves removal of chlorphyll, usually wnh 70% or 80% ethanoleavlnS a white, opaque leaf. The prolplasm S subsequently dlssolved, rendennthe Icaf transparenl. Protoplasm IS removeusmg a slrong alka/i such as NaOH, whlCreacts wlth phenohc compounds, tumlng thleaf a uOIfonn brown. ThIs brown colonnis removed by bleachm8 the leaf in sodIuhypochloflte (NaCIO) (7). In vitro planlleof 'Silvan' blackbeny and 'Tolem' strawbeny were representatlve of very young Icavor fragIle matenal smce plaRllet leaves haalmost no cutle/e, verv thin cell walls, anfew cell layers. G r e ~ n h o u s e . g r o w n planleaves have more cUtlcle, thlcker cell walland larger, thicker leaves than tissue-cultured plantlels (2, 3) Greenhouse-grow'Sllvan' leaves have les . cuticle than 'Totem' strawberry and are sllghtly rhinner.

When pIani matenal IS c1eared. there IS aselective dlssolvmg of certam cell components. based on thelr chemlcal and/or phys.Ical propeTlles, for Ihe purpose of observmgothers. Organelles and nonhgmfied tissuesare the fir'it to be removed and dense, resls,tant Iignafled clements usually perSlStthroughout the c1eanng process (7).Lea( clearing has been used for morpho.10gICal IdentificatIon and anatomlcal 0bservat Ions, as \0 the exammallon of surfacestructures (stomales and halrs) or xylem (l,7). Cleared preparatIons may supplement

Table 1 Summal)' of clearmg mClhod for mlaCI IIssuecultured planllcls and grecnhousegrown pianIcaves of 'Sllvan' blackb.rry Ind 'Tolem' slnwbcny.

RCCClvcd for pubhc3uon 21 Dcc. 1987. Partialfinanclil support (rom Agncullure Canada (gnnl87027) acknowlcdgcd. Wc Ihank John BalR (orhls hclp IR prc:panng IhlS manuscrtpt. The cost ofp u b h ~ h t n g IhlS paper wu defrayc 1 n pan by thepaymenl of page chargcs. L'nder postal r(gulalions, Ihls paper Ihcrcfore musl bc hcrcbv marKcdad\eT1l. tmem solcly 10 Indlcalc h l ~ faci.HORTSCIENCE, VOL. :3(61, DECE'l.fOER 1988

TrcalmcnlPwrc3lmenl' Alkall PoslalkaltCultivar (mIR) (min) (mm)

TlSsueculturtdSllvan 10-20 20 5-10 (RnTotem 10-20 20 5-10 (RnG r t ~ n h o u . s t : g r o w nSllvan 2S 35 S (55'C)TOlem 35 '*0 ID (55'C1

'PrCIrCalmCnr iRvolvcd aUloclavmg IR 80% tlhanol.Alkali lrealmenl IRvolved clfp05urc 10 5 NaOH al BO'C.Postalklh trealmenl IRvolvcd cxposurc 10 7S blcach al toom Icmpcralurc (RT) or at 55C.

3 1059


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A

-c

_....."

- -,,

. ..:

-

,.:

B

1... _ .............

--_

Fig. 1 Phalomlcroscaplc VICW of an ln Vitro 'SlIvan' blackberry Icaller, focusslng down from JbaxlJI O JdJXI. 1 .,url.lce 1 \1 .\/1JXIJI k.1f IlLlLl 1111Inlernal VICW of vascul. r rl'isue and pahsJdc 1.lyer (Cl Adaxlal surface. (Dl Glandular trichome (collerer) ~ l l c . r n m l l c r hM = -Il ILnl I A - Jill) ~ r n(0).In vitro plantlets were grown on Wlcks rnhqUld Murashige and Skoog medium (6) Theplantlets were grown undcr 38 f.Lmol s l'mcool-whlle fluorescent hghts wrth J 16-hrphotoperiod. In vrtro plantlets were c1cJredrntact. Greenhouse-grown plJnts were keptat amblent temperatures wuh a 16hr photoperiod. Leaves were eut rnto 0 . 4 4 c m ~ drsk'iusrng a paper punch or rnto 1- or ~ c m ' ~ e c Irons. Dlsks were taken Jt random Jnu lnsorne cases rncluded the mrdnb area. Dlsk.,were used solely for rmtldl pretreJtment experrments whcre precrse area was e ~ s e n t l a l .Square sectIOns were used rn subsequent pretreatment, protoplasm dlssolutror., and postalkali stage expenments srnce they provldeda larger area for treatment evaluJtlon. Thesquare sections were cut Just below the trp

1060

of J leaf Jnd always rncluded J mldnb ~ e c -tian.Specimens were nnsed three to frve tlmesrn dlstllled water between each of the threecleanng stages. A small strarner was used toprevent specimen loss from the boules. Safranrn was used ta slarn the speCimen, afterthe post-al kali stage Jnd water SOJk.Pretreatment stage To ueterrnme the mosteffectIve concentrJtlon of ethJnol for removmg chlorophyll, 10 blacI.berry leaf d l s k ~ fromgreenhouse-grown plants were placed mto 50-ml ~ p e c l m e n boules m 40 ml of SOl(;, 6OC c,70l {; 80C:C 90c.c or 1 0 0 ~ ethanol. The contarners were autocl.lVeu at 103.4 x 10' PJand 120C far 15 mm, removcd from theJutoclave, and the caps closed tlghtly to prevent evaporatlon of ethanol. ThIs expenment

33

W J ~ repe.lleu three tlnlC\ The CI11.IIlill-L1i1orophyll .,olutlon., \ \ Ln : tlltered Jllli IfllnJedlJtely Jn.llyzetl 'peLlrophoIOf11ClrlL.lilv .It Ihechlorophyll JIN'lh.lnce p C . I ~ nt h : ; ~ 11111When u\lng Ihc "'qu.lre 'CLtIOn., reqlliredfor IJter SICp , Ihe thoroughne ., Il LhltHll'phyll removJI W.I' vl,u.llly .I."e, cd hj 'relImen colm. Succe\., ul pretre.tlnlCnt re.,ultedln the k.lt .,eLllon IUPlIng or"lJtll Re\ldll.dgrecn Indlc.lled Ih.tt ,dl thl l hloroplivll 11 Il1yet tn be remllvcu \\IhereJ., b r ) \ ~ 11Ing mC.IT1lthJt JutoclJvlng W.I\ eXle\\IVe or .111 II1.,utflclent 10111.11 volume ot clhJllol \\"., I l . , ~ d .c a u ~ l n g clilier .1 phcnolrc rC.ILllon or hurnIng

DIBOIl tlO f o pmlOplal l l l'n:lrl.tlld Ie.t~ e c t J o n were u.,cd III CVJlllJle the mo.,t ef-fective NJOH conccntr.l lon, tcmpu.tlllre . nd

H ( ) R T S U : ~ C . [ , VOl 23(6), DI (1 \IBl I I()X/{


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exposure tlme for complete protoplasm removal. NaOH was used al concentrallons of1%,2%,5%, and 10% al 20, 55 , and 80Cfor peflods rangmg from 15 mm 10 1 hr, 105-mm Incremenls. The effecl of aUloclavlOg8) was lesled. The lime for complete protoplasm removal could only be determlOedby mlcroscoplC observatIOn after the phenohc compounds had been bleached 10 thepost-alkah stage. The p r o t o p l a ~ m dissolu lionslage was deemed complete when mlcroSCOplC observa lion showed the spongy mesophyll and the paltsade cells to be free oforganelles.Posta/ka s t a g ~ . The optImum pretreatment and protoplasmdlssolutlOn stage treatmenlS were employed usmg new specimens.The most effecllve concentrallon of household blcach (6% NaCIO) for removIOg thebrown phenoltc OXldates was determlOed.Concentraltons of 2.5%. 5 0 ~ 7SC 'c. ar.d 100%bleach 1.510, 3%, 4.5%, and 6< ~ a C I O )were employed at 20, 55, 60, b5, and75C unlll the sampi es were clear Effectlveness of the postalkah stage was basedon the tune needed ta bl:ach specimens frombrown ta transparent and upon the mec hanical strength of samples after treatment. Aspecimen was deemed clear once every spotof brown had been bleached.Cleared specImens were stamed maqueoussaframn (10 mgliter l) and mounted 10glycerol for seml-permanent mounts. Permanent mounts reqUired dehydratmg cleared.unstamcd matenal through a senes of elhanolconcentrallons, stalOmg 10 95% or 100%ethanol. rehydratlOg lOto xylene, and mounting 10 Permount.Pretreatment stage. Spectrophotometncanalysls of chlorophyll extracted from 'SIIvan' leaf dlsks showed that 80% ethanol,wllhm the 15-mm Jutoclavmg mterval, extracted the most chlorophyll. Ethanol concentr:lIIons below 80% removed lesschlorophyll durmg thls IOterval. Ethanol at50% to 70% eventually dlssolved ail of thechlorophyll, but requued longer autoclavelimes and a much larger volume of ethanolslOce evaporallon was greater over the extended autoclave mtervals. Ethanol concentratIOns above 80% removed chiorophylliesseffecllvely. Chlorophyll removal from IOtactplant lets grown 10 vitrO took 15 to 20 mlO 1080% ethanol 10 the autoclave. For grecnhouse-grown leaves, chlorophyll removaillmewas 25 mm for 'Sllvan' and 35 mm for 'To-

HORTSCIE"CE. VOL. ~ 3 ( 6 ) , DECE\tOER 1988

tem'. The lime differences for chlorophyllextracllon were probably due to dlfferentamounts of eplcullcular and cUllcular wax(7).DlSso ullon ofprotop/asm. Thc most rapldtreatment for dlssolvmg the protoplasm was5% Na OH at 800e for va flOUS penods oflime dependmg on the type of IIssue. Intactplant lets grown 10 vitrO nccded 20 mlO; leafsquares (Jf mature greenhousegrown 'SIIvan' and 'To cm' Icaves needed 35 mm and40 mm, respecllvely. NaOH at 10% causedIIssue dlsmtegrauon, as dld autoclavmg lnNaOH for most samples.Posta ka I s l a g ~ . Immersion m 75% bleachat room temperature for 5 ta 10 mm was thepreferred postalkall trcatment for ln vitrOplant lets. Greenhousegrown matenal ln 7Se-;bleach at .55C becamc transparent ln between 35 to 40 mm. Bleach conccntratlOnsbelow 75t;C took longer to achleve the sameresults, but 100% bleach or temperatures ator above 60 macerated the tissues. The mldnb and pellole. due to thclr thlckness, werethc last arcas from whlch phcnolic cornpounds were totally bleached.Il was essentlal to remove die bleach com

pletely by soakmg for a few minutes 10 disIIl1ed water before stalnlOg wllh safranlObecause of the destrucllve interactIOn of safranln and sodium molecules. In vttro plantlets statned adequately in only 10 to 30 secwhereas grecnhouse-grown specimens took1 to 2 mm If superficlal Issues, such as hamor guard cells, \\-ere of I n t e r : ~ l or 510 7 mmIf IOternalllssues, such as X lem, were to beexammed.Smce ln vllro plantlets were c1eared Intact,mlcroscoplc observatIOn of the external andinternai structures of the root, stem, petiole,and leaves was poSSIble. Ali the cell layersfroOl ad axial to abaxlal leaf surfaces couldbe mlcroscoplcally observed (Fig. 1 A, B,and Cl. When mounted abaxlal slde up, theadaxlal epidermal layer was not always qUlleas clear as the upper surface due to follarwldth, but It was usually not difficult ta dlscern cell patterns or stomata. Differences 10cell shapes and palterns between palisade andspongy mesophyll layers were apparent.ln the case of greenhouse-grown leaves,secllons mcluding the mldnb were more likelyto exceed 2 to 3 mm m wldth. Mountedadaxlal slde up, the lower epldermal layerwas usually out of focus due ta the thlckepldermal ce Ils, the many celllayers and the

dlfficulty ln keeplOg the coversllp adhethe specimen. For optimum examlnalboth leaf surfaces. specimens wereand mounted bath adaxlal and abaxlaup, examlned and subsequently turnedor the mldnb was removed.ln summary, the cleanng and stmethod outhned 10 Table l wlth mlnolallons, gave rapld (maximum tlme ofand excellent results for the plants examThis c1earmg procedure was parllcularlcessful wllh m VitrO plant lets for whlcgans and celllayers werc made c1early VDue to liS speed and reproduclblhty,procedure may prove to be a use fui latorv tool to factlltate stomatal countmeasurements. It should be especldlly as a method for showmg the threedIslonal relallonshlps of cell Idyers: vJ5tissues, crystal deposlls. hydathodeschomes (Fig ID), Jnd other fohJr stures. We have found the method usefobtatnmg a general Vlew of an area thatbe exammed later uSlOg microtome cuttLlteralurt Cited

1. Arnon, H J 19 '9 Leaf cleanng, TNews 37192-19. -2. Donnelly, DJ and W E Vldaver 1984analomy of red raspberry Iransfcrredcuhurc 10 5011 J. Amer. Soc. Hon_109'172-176_3 Fabbn, A , E. Suner. and S.K Dun1986. Analomlcal changes 10 p c : ~ l s l e n r ,of tl UC cul'urcd , l r J \ I , ~ c r r y plan Jf-movallrom culrure SClcn:IJ Horl ~ s :3374 Gardner, R D 1975. An OVCrvlCW of blcal cleanng lechnrque Siam Technol 5\05.5. tchen. C 1952. AmClhud for c1earrng 1UPh}1opalhology 42 3526 Murashlge, T. and F Skoog 1962 Avlscd medium for rapld gro\\


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Having developed a satisfactory clearing and staining method for observingtissue-cultured and greenhouse-grown plant material, acclimatization experimentswere initiated in order to 1) detennine the effects of both increased agarconcentrations 6, 9 and 12 g/l) in the culture medium and in vitro rootingon ex vitro survivaI, growth and stomatal index o micropropagated Silvanblackberry shoots and 2) detennine the effect o high relative humidity and lowlight intensity on the stomatal function o leaves from a) ex vitro plantlets grownon full and 1/4 strength modified MS (1962) basal medium and b) greenhousegrown Silvan plants.

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4. Acc:Jimatizaion 1 mJc:ropropalated 'Silvan' blacl.berry4.1. Introduction

Ex v tro acclimatization can be one of the mos difficult stages of themicropropagation process. This is due to the impaired ability of transplants tocontrol water Joss as a result of poorly fonned epicuticular and cuticular waxlayers (Conner and Conner, 1984, Fabbri et al., 1986, SUUer and Langhans, 1982,Sutter 1984, 1985), non-functional, open stomata (Brainerd and Fuchigami, 1981,DonneUy et aL, 1986, Short et al., 1987, Wardle and Short, 1983) and, in sornecases, poorly formed root systems (Grout and Aston, 1977).

Generally, plants grown under culture conditions have reduced foliarepicuticular and cuticular wax. In vitro-formed foliar wax may he different inarrangement and chemical composition compared with that of greenhouse-grownplants (Sutter, 1984, 1985). Reducing the relative humidity level in vitro from100 to 35 by llsing a variety of closures for the cuhure containersincreased epicuticular wax fonnation in cauliflower (Brassica oleracea var botyrisand chrysanthemum (Chrysanthemum morifolium Ram.) (Short et al., 1987).Increasing the agar concentration from the usual 6-8 g/l to 15 gI1 resulted indecreased relative humidity in the culture containers; from 98 to 89

. Subculture to medium with an agar concentration of S g/l 2 weeks priorto transplantation promoted glaucous leaf production in carnauon (Dianthuscaryophyllus) plantlets and improved transplant survival from l to 72(Ziv et al. 1983). Similarly, increasing the agar concentration in thefooting medium from 6 g/l to 14 g/I increased the ex vitro survival ofmicropropagated cherry (Prunus cerasus) rootstock from 13 to 45.3 (Marinand Gella, 1987).

Stomata fonned on most in vitro plant leaves did not close in responseto stimuli snch as darknes

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