Comparative Growth Analysis and Acclimatization of Tissue Culture Derived Cocoyam (Xanthosoma Sagittifolium L. Schott) Plantlets

8/9/2019 Comparative Growth Analysis and Acclimatization of Tissue Culture Derived Cocoyam (Xanthosoma Sagittifolium L.

1/15

_____________________________________________________________________________________________________

*Corresponding author: Email: [email protected];

American Journal of Experimental Agriculture5(2): 94-108, 2015, Article no.AJEA.2015.011

ISSN: 2231-0606

SCIENCEDOMAINinternationalwww.sciencedomain.org

Comparative Growth Analysis and Acclimatization ofTissue Culture Derived Cocoyam (Xanthosoma

sagittifolium L. Schott) Plantlets

Anne E. Sama1, Mohamed A. Shahba2*, Harrison G. Hughes1and Mohamed S. Abbas2

1Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins,

Colorado, 80523-1173, USA.2Department of Natural Resources, Institute of African Research and Studies, Cairo University, Giza,

12613, Egypt.

Authors contributions

This work was carried out in collaboration between all authors. Authors AES and HGH designed thestudy and collected the data. Authors MAS and MSA managed the statistical analysis of the data and

wrote the first draft. Author MSA managed the literature searches. Author MAS managed the finalreport writing while Author HGH managed the final editing. All authors read and approved the final

manuscript.

Article Information

DOI: 10.9734/AJEA/2015/10379Editor(s):

(1)Marco Aurelio Cristancho, National Center for Coffee Research, CENICAF, Colombia.Reviewers:(1)Anonymous, National Centre for Genetic Resources and Biotechnology, Nigeria.

(2)Anonymous, Jomo Kenyatta university, Kenya.Peer review History:http://www.sciencedomain.org/review-history.php?iid=665&id=2&aid=6075

Received 26th

March 2014Accepted 24

thApril 2014

Published 12th

September 2014

ABSTRACT

The current study was carried out to compare the external leaf structure of tissue culture-derivedand conventionally-propagated Cocoyam [Xanthosoma sagittifolium (L) Schott] plantlets and todevelop an efficient acclimatization protocol for these plantlets. Acclimatization studies were carriedout during winter and summer to ascertain seasonal influence relative to plant survival upon transferfrom in vitro to natural conditions. Results indicated that, cocoyam leaves have few stomates onboth abaxial and adaxial surfaces with fewer on the adaxial surface. High levels of epicuticular wax(EW) found in vitro may have contributed to reduced transpiration rates. The reduced amounts ofEW on acclimatized plants could be attributed to the rapid cell enlargement in expanding leaves,more rapid than the rate of wax formation. Acclimatization using humidity tent decreased leaf wilting

Original Research Article


2/15

Sama et al.; AJEA, 5(2): 94-108, 2015; Article no. AJEA.2015.011

95

and damage compared with the control treatment or with the mist treatment. Mist-acclimatizedplantlets produced about 50% fewer leaves than those acclimatized in a humidity tent. Similarresults were obtained during winter acclimatization with a lower rate of leaf formation compared tosummer acclimatization. A relatively high humidity (60-80%) for approximately two weeks reducedleaf injury from wilting and desiccation.

Keywords: Tissue culture; cocoyam; epidermal cells; epicuticular wax; stomatal frequency; stomatalindex; acclimatization.

ABBREVIATIONS

MS: Murashige and Skoog (1962) medium; TDZ: Thidiazuron; BAP: Benzylaminopurine; BM: basalmedium; NAA: 1- naphthaleneacetic acid; AS: Adenine sulphate; EC: Epidermal cells;EW: epicuticular wax; SF: stomatal frequency; SI: stomatal index.

1. INTRODUCTION

Cocoyam [Xanthosoma sagittifolium (L) Schott] is

a monocotyledonous crop that belongs to theAraceae family. The stem has a starch richunderground structure, the corm, from whichoffshoots called cormels develop. Flowering israre, but when it occurs, the inflorescenceconsists of a cylindrical spadix of flowersenclosed in a 12-15 cm spathe [1]. It is a staplefood in the tropics and subtropics and one of thesix most important root and tuber crops world-wide [2]. The corm, cormels, and leaves ofcocoyam are an important source ofcarbohydrates for human nutrition, animal feed[3-5] and of cash income for farmers [6]. Africaproduces about 75% of the world production

which is about 0.45 million tons [7]. Cocoyambreeding and production is labor intensive andrequires large amounts of water [8]. It is highlysusceptible to diseases such as cocoyam root rotdisease caused by Pythium myriotylum [9] andDasheen Mosaic virus found in the leaves, cormand cormels [10].

Micropropagation is an efficient method to masspropagate good quality materials that maysubstantially improve production. It involves theuse of defined growth media supplemented withappropriate growth regulators that enablemorphogenesis to occur from naturally growing

plant parts [11]. Previous studies have shownthat shoot multiplication, somatic embryogenesisand tuberization can be induced in shoot tips ofcocoyam cultured in vitro on Murashige andSkoog medium [12] supplemented with variouscombinations of indol butyric acid (IBA), 1-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D),Benzylaminopurine (BAP) and kinetin [13].

The biochemical aspects of induction of in vitroorganogenesis have been investigated in anumber of plants including carrot [14], pea [15],

summer squash [16,17], winter squash [18],soybean[19], taro [20], watermelon [21],groundnut [22], asparagus [23], black pepper[24],canola[25], cotton [26], date palm [27], lentil[28], common bean [29], sunflower [30], rice [31]and banana [32]. However, the benefit of anymicropropagation system can only be realized bythe successful transfer of plantlets from tissue-culture vessels to the field conditions [33]. Mostspecies grown in vitro require an acclimatizationprocess in order to ensure that a sufficientnumber of plants survive and grow vigorouslywhen transferred to soil.

In spite of its importance in many countries,cocoyam has received very little researchattention [34]. The yield potential of cocoyam isseldom realized, mainly because of a lack ofknowledge concerning diseases, propermanagement practices, and physiologicaldeterminants that may limit plant growth anddevelopment [35]. The objectives of thisinvestigation were to determine an effectiveacclimatization protocol for micropropagatedcocoyam plantlets through a comparison of theexternal leaf structure of tissue culture-derivedplantlets and conventionally-propagated plants interms of epidermal cells, stomatal frequency and

stomatal index, and to determine an effectiveacclimatization protocol for cocoyam plantlets.

2. MATERIALS AND METHODS

2.1 Source of Explants

Cocoyam South Dade white plants wereobtained from the Tropical Fruit Company,Homestead, Florida as sprouted corm sections.


3/15


96

Sections were potted in polyethylene pots (100cm2) in a mix of peat, perlite and vermiculite(1:1:0.5 by volume). These plants weremaintained in a greenhouse under naturalphotoperiod. Temperature was maintained at 232oC. Plants were watered as needed with tapwater and fertilized with liquid fertilizer containingN:P:K at 20:10:20 by weight twice a week. Eightweeks after planting, sprouts were collected,trimmed to about 5 cm and washed underrunning tap water for 30-60 minutes. Shoot-tipsof 3-5 mm were excised and the apical meristemwith 4-6 leaf primordia and approximately 0.5mm of corm tissue at the base were disinfectedin a laminar flow hood using 1% (v/v) sodiumhypochlorite solution for 10 minutes beforetransferred onto the culture medium.

2.2 Basal Medium (BM)

A modified Gamborgs B5 mineral salts [36]supplemented with 0.05 M 1-naphthaleneaceticacid (NAA) was used throughout the study. Themodified component of B5 micro-salts wasMnSO4.4H2O at 10 mg L

-1. Organics consisted ofmyo-inositol (100 mg L-1), thiamine HCl (10 mg L-1), nicotinic acid (1 mg L-1) and pyridoxine HCl(10 mg L-1). Sucrose was provided at 30 g L-1.Whenever a semi-solid medium was desirable,agar (Sigma agar, type A) was added at aconcentration of 0.4%. The pH of the mediumwas adjusted to 5.7 0.02. A thidiazuron (TDZ)solution containing 0.01% dimethyl sulfoxide(DMSO) was used. Erlenmeyer flasks (125 ml)and test tubes (25 x 150 mm) were used forgrowing cultures. Aliquots of 25 ml and 15 mlwere dispensed into the flasks and test tubes,respectively. Flasks were stoppered with non-absorbent cotton plugs, and then covered withaluminium foil. Test tubes were covered withpolypropylene closures, Kaput caps (BellcoGlass, Inc., N. J). The media-containing vesselswere then autoclaved for 18 minutes at 121oC.

2.3 Acclimatizationof Cocoyam Plantlets

Acclimatization studies were carried out duringwinter and summer. Plants used for adaptationwere previously proliferated in vitro in 2.0 MTDZ multiplication medium. Beforetransplantation, agar was gently washed off theroots with tap water. Plants were transplantedinto 10 cm plastic pots containing pre-moistenednon-sterile soilless substrate composed of peat,perlite and vermiculite at a ratio of 1:1:0.5 byvolume. During transplantation, the number of

leaves, roots and plant height were recorded foreach plant. Plant height was measured from thebasal plate to the lamina tip of the youngest fullyexpanded leaf. The transplants were thensubjected to four different acclimatization

treatments for five days as follows: 1) Control, 2)mist, 3) humidity tent, and 4) test tubeacclimatization by uncapping. Each treatmenthad at least 42 plants. Control plants weretransferred directly to an open bench in thegreenhouse. The initial temperature and relativehumidity were 25oC and 63% respectively. Highand low temperatures averaging 26 and 16oCrespectively with corresponding relativehumidities of 54% and 94%. During winter, theaverage greenhouse relative humidity was 40 5%, and the temperature was 22 3oC, and lightintensities of approximately 400 mol m-2 S-1.The plants subjected to the second treatment

were placed under an automatic misting systemset for six seconds at eight minutes intervals. Togradually reduce the humidity, the mistinginterval was increased to 16 minutes after thefirst two days for the remaining three days ofacclimatization. The third treatment placed plantsin a locally constructed plastic humidity chamberwith the dimension of 127 X 92 X 62 cm. Ahumidifier was used to provide an initial relativehumidity of 98% with a temperature of 25oC.Humidity was gradually reduced after the secondday by partially opening the flaps of the chamber.On third, fourth and fifth days, the relativehumidity was lowered to 96 and 94 and 92%

respectively. The fourth treatment was conductedin culture vessels by partial uncapping. Thecultures were placed on a bench in the sameenvironment as the control plants. Caps wereloosely opened for the first two days, and thentotally removed for the remaining three days ofacclimatization to expose the plants to thenatural environment while in the test tubes. Afterfive days of acclimatization in the test tubes, theplants were taken out, washed and transplantedinto the same soilless mix. All plants weretransferred to an open bench and grown understandard greenhouse conditions. The number ofstomates in tissue culture-derived and

conventionally-propagated plants was examinedto ascertain stomatal function and influencerelative to plant survival upon transfer from invitro to natural conditions. To count stomates,leaf impressions were made using a thin film oftransparent fingernail polish. This was applied toperipheral sections on either side of the midriband on both abaxial and adaxial surfaces of thelamina. After the dryness of the fingernail polish,the epidermal cell layer was peeled with a


4/15

transparent adhesive tape. The ithen placed on microscopeobservations. Also, the epicuticulaon leaves from tissue cultureconventionally propagated plants w

2.4 Data Analysis

Experiments were laid out as a cdesign. All data were subjected tovariance using unequal replicatcontamination. Treatment means wby Tukeys Multiple Range Test atsignificance [37].

3. RESULTS

3.1 Regenerated Plantlets Cha

Tissue culture regenerated cocoyanot show any obvious deviationmorphologically similar to theirpropagated counterparts (Fig. 1).retained the characteristic sagitaconventionally-propagated planmodifications of color or shape.

3.2 Stomata in Tissue Cultand Conventionally-Plants

Fig. 1. A morphological comparimonths in greenho

Sama et al.; AJEA, 5(2): 94-108, 2015; Article

97

mprints wereslides for

r wax content-derived andas compared.

mplete blockan analysis ofions due toere separateda 5% level of

racteristics

plantlets dids, and wereonventionallyThe plantletste leaves ofts without

ure-DerivedPropagated

3.2.1 Stomatal frequency (SF)

Cocoyam leaves from allamphistomatic. Almost twice aswere found on the abaxialsurf

Analysis of variance indicatednumber of stomates per mm2) wgreater in the non-micropropagatethan in the acclimatized gree(Table 1 and Fig. 3). There wadifference in the average SFacclimatized and conventionplants (Table 1 and Fig. 2A). Theadaxial and abaxial surfaces wererespectively. Epidermal cells (Eleaves from the three sources weirregular with undulate anticlinalHowever, the anticlinal walls wereECs of cultured plantlets. The c

varied in size and shape.greenhouse plants were ellipticawhile those of in vitro plants wereand raised, but below the level of(Fig. 3). Stomates from all leafscattered and at unequal distananother. However, ECs and stoveins were smaller and alignedmanner. Abaxial and adaxialsimilar to one another, with varyinsides.

on between conventionally-propagated and tissse) cocoyam plants growing in the greenhouse

o. AJEA.2015.011

ources wereany stomatesce (Fig. 2A).

that SF (theas significantlydcontrol plantshouse plants

s a significantmong control,lly-propagatedaverage SF of17.4 and 30.5) of cocoyame polygonal orwalls (Fig. 3).less distinct in

ells were also

Stomates ofl and sunken,more sphericalepidermal cellssources wereces from oneata along thein stream-like

stomata weresizes on both

ue cultured (3.


5/15


98

Table 1. Analysis of variance with mean squares and treatment significance of the effect ofdifferent cocoyam plant source and leaf surface on stomatal frequency and index and the

effect of plant source on epicuticular wax content on cocoyam leaves and the effect ofacclimatization procedures under different durations during summer on leaf damage, leaf

wilting, leaf initiation and leaf shedding of tissue culture-derived cocoyam plants

Source DF Mean squares P-value*Stomatal frequency:Plant source (S) 2 1052.3 0.001Leaf surface (F) 1 935.0 < 0.0001S x F 2 2251.0 < 0.0001Rep 41 10222.0 0.35Stomatal index:Plant source (S) 2 152.3 0.003Leaf surface (F) 1 235.0 < 0.0001S x F 2 241.0 < 0.0001Rep 41 952.0 0.44Cuticular wax:Plant source 2 4321.0 < 0.0001Rep 41 789.0 0.70Leaf damage:

Acclimatization treatment (T) 3 1252.3 0.005Duration (D) 2 1335.0 < 0.0001T x D 6 3251.0 < 0.0001Rep 41 15222.0 0.14Leaf wilting:

Acclimatization treatment (T) 3 252.3 0.001Duration (D) 2 335.0 < 0.0001T x D 6 351.0 < 0.0001Rep 41 1522.0 0.54Leaf initiation:

Acclimatization treatment (T) 3 211.0 0.01Duration (D) 2 195.0 < 0.0001T x D 6 466.0 < 0.0001Rep 41 952.0 0.24Leaf shedding:

Acclimatization treatment (T) 3 122.0 0.015Duration (D) 2 95.0 < 0.0001T x D 6 166.0 < 0.0001Rep 41 789.0 0.90

*Significant at P 0.05

3.2.2 Stomatal index (SI)

Analysis of variance indicated no significantdifferences in stomatal index calculated as thenumber of stomata / number of epidermal cellsand stomata x 100 mm2among various cocoyamplantlet sources (Table 1 and Fig. 2B). Valuesranged from 8.0 for in vitro propagated plants to8.6 for control plants on the abaxial surface while

it ranged from 4.6 for in vitro plants to 5.3 forcontrol plants on the adaxial surface.

3.3 EpicuticularWax Content on Leavesof Culture-Derived andConventionally-Propagated Plants

Analysis of variance indicated a significantdifference among different plant sources inepicuticular wax (EW) formation on cocoyam

leaves (Table 1). Cocoyam plantlets cultured invitro were found to have greater deposits of EW.Gravimetric determination showed that in vitroleaves had an average of 88.6 g/cm2, ascompared to 50.1 g/cm2 for plantletstransferred and grown in the greenhouse (Fig. 4).

3.4 Ttissue Culture-Derived PlantletsBehavior and Adaptation to DifferentEnvironmental Factors

3.4.1 Summer acclimatization

3.4.1.1 Effects on leaves

Analysis of variance indicated a significantdifference among acclimatization procedures onleaf damage and leaf wilting (Table 1). Cocoyamplantlets showed no significant visual desiccation


6/15


99

one hour after transfer to the open bench. After24 hours, differences were observed in leafwilting among different treatments. Plantletsacclimatized by uncapping the culture tubeshowed 14.9% leaf injury after 24 hours of

acclimatization compared to only 0.6% inplantlets acclimatized under mist (Fig. 5A). Onthe other hand, wilting assessment indicated thatplantlets acclimated under mist suffered lesswilting after 24 hours (Fig. 5B). One week afteracclimatization, more wilting was observed asthe percentage of damaged leaves increasedsignificantly for open tube acclimatization (43.7%) as compared to that from the humidity tentwhich had a damage percentage of 15.9%.Wilting assessment of 2.8 and 3.5 wasassociated with the previous leaf damagepercentages respectively (Fig. 5A and B).

3.4.1.2 Effects on growth habit

Analysis of variance indicated a significantdifference among acclimatization procedures onleaf initiation and leaf shedding (Table 1). Afteracclimatization, plants continued to grow activelyin the greenhouse with normal leaf and wholeplant morphology. Significant differences amongthe different acclimatization treatments werefound in the number of new leaves formed inplants (Fig. 5C). Two weeks after acclimatization,an average of 1.2 leaves were produced fromplants acclimatized in the humidity tent, ascompared to only 0.6 leaves in mist-acclimatized

and control plants. An average rate of one newleaf per plant was produced every two weeks.Mist-acclimatized plants produced fewer leavesthan the other treatments after four and sixweeks and was significantly different from thoseacclimatized in a humidity tent and uncappedtubes.

The number of leaves shed per plant pertreatment was used as indication of the reverseof leaf production. After two weeks ofacclimatization, humidity tent plants shed only anaverage of 0.8 leaves as compared to 1.3 for thecontrol plants (Fig. 5D). A significant differenceamong treatments was observed after two weeksand disappeared at four and six weeks.

3.5 Winter Acclimatization

3.5.1 Effects on leaves

Plantlet leaves wilted slightly duringtransplantation, but those acclimatized under

mist and humidity tent were able to regainturgidity. Plantlets transferred directlyfromculture to the open bench were morestressed after 24 hours compared when to othertreatments. Plantlets acclimatized under mist

and in the humidity tent had wilted leaves onlyafter one week of acclimatization, which weregradually reduced in subsequent weeks. Thecritical period for leaf injury was the first weekafter acclimatization. All plants survived in alltreatments but less wilting and leave injury wereassociated with mist or humidity tent ascompared with the control.

3.5.2 Effects on growth habit

The growth and development of plants was notaffected by the method of acclimatization duringshoot elongation. New leaves were produced by

the second week after acclimatization and moregrew after four weeks. The rate of leaf formationwas low as compared to summer acclimatizedplants except for the mist treatment (Fig. 5C).

4. DISCUSSION

The decrease in stomatal index and increase inepidermal cell size may affect plant growthphysiology. Therefore, altered leaf structuresmight be associated with poor field performanceand increased disease susceptibility [38,39]. Insimilar findings, Donnelly and Vidaver, [40]reported almost twice as many stomates on the

abaxial surface in tissue cultured plantlets ofRubus idaeus. The reduced SF in leaves ofacclimatized plants may have been due to theirenlargement. Blanke and Belcher [41] noticed adrastic decrease in SF of transferred appleplants, which was attributed to leaf expansion. Instrawberry plants, the increase in size ofpersistent leaves was mainly the result of cellenlargement, rather than the increase in cellnumber [42]. On the other hand, Brainerd et al.[43] reported significantly reduced cell length inthe upper epidermis of transferred Pixy plumplants as compared to those aseptically and fieldgrown. Comparable findings, where SF was

greater with in vitro plantlets than those that hadbeen removed from culture, were observed inLinquidambar styraciflua [44,45], and apple [41].


7/15


100

Plant source

In Vitro Acclimatized Conventional

Stom

atalindex

0

2

4

6

8

10

Stomatalfrequency

0

10

20

30

40

50Abaxial surface

Adaxial surface

ab

a

A

B

a

ab

b

ab

a

aa

aa

Fig. 2.Stomatal frequencies (A) and indices (B) of the leaves of in vitro, acclimatized and

conventionally propagated cocoyam. Columns labeled with the same letter are notsignificantly different at P= 0.05 using Multiple Range Test for plant source comparison at

abaxial and adaxial surfaces. Vertical bars at the top represent standard errors


8/15

A

B

C

Sama et al.; AJEA, 5(2): 94-108, 2015; Article

101

o. AJEA.2015.011


9/15


102

Fig. 3. Stomatal frequency on the abaxial surface of different sources of cocoyam plant asindicated by photomicrographs of the leave imprints (X 600). A. in vitro plants, B. acclimatized

plants and C. conventionally-propagated plants

Plant source

In Vitro Acclimatized Conventional

Epicuticularwax(

gcm-2)

0

20

40

60

80

100

Fig. 13.

a

b

ab

Fig. 4. Effect of plant source on epicuticular wax formation on cocoyam leaves. Columns

labeled with the same letter are not significantly different at P= 0.05 using Multiple Range Testfor plant source comparison. Vertical bars at the top represent standard errors

The average SF of 17.4 and 30.5 for adaxial andabaxial surfaces of cocoyam, respectively, arerelatively low, when compared to 27.5 and 150

for Rubus idaeus [40], 184.5 (adaxial only) forVitis sp. Valiant [46]. This low SF may havecontributed to low transpiration rates, whichresulted in less wilting and high survival rates ofcocoyam plantlets after transplantation. Incontrast, Brainerd and Fuchigami [47], suggestedthat the high SF of apple micropropagatedplantlets was responsible for the higher waterloss observed. The rapid water loss could bedue to stomatal malfunction [47] or the size of thestomates [45]. Wetzstein and Sommer [45] foundthat stomata were also larger in vitro plantlets ofsweet gum, in addition to their greater densities.As indicated by Brainerd and Fuchigami [47] and

[48], stomates have a greater part in water lossof plantlets than epicuticular wax.

No significant differences in stomatal indexamong various cocoyam plantlets sources werefound. These results corroborate previousfindings that were reported for Solanumlaciniatum [48], Rosa multiflora [49], and Vitis sp.Valiant [46] in comparisons made between invitro and field grown plants. Dami [46] found

significantly greater stomatal densities in leavesof greenhouse-grown plants than in in vitrocultured leaves but found none when SI

comparisons were made. These results agreewith the idea that SI is a better estimate than SFin comparisons involving leaves with stomates ofdifferent sizes [48, 40, 46]. However, Zhao et al.[38] found that micropropagatedregenerants hadproduced a significantly lower stomatal index, butlarger epidermal cell size than conventionalplants when they investigated the alterations inleaf trichomes, stomatal characteristics andepidermal cellular features in micropropagatedrhubarb (Rheum rhaponticum L.).

Quantitative variation is frequently found amongregenerants derived from tissue culture and often

indicates alteration of numerous loci [50].Quantitative variation has been described formany phenotypes including plant growth habitand agronomic performance [50-52]. The causesof somaclonal variations are believed to resultfrom a range of genetic events during planttissue culture, but it is difficult to interpretsomaclonal variation in a genetic mode [53-55].In recent years the genetic analysis of plantsregenerated from tissue culture has revealed that


10/15


103

extensive genetic changes apparently occurduring tissue culture. The majority ofmorphological variants observed in tissuecultured plants were due to numerical andstructural chromosome changes induced during

culture [56,57].

Cocoyam plantlets cultured in vitro were found tohave greater deposits of EW. Apparently, therewas lees wax deposits per unit area aftertransplantation. Sutter [58] found a similarphenomenon with apple plants, with more EW invitro and less after acclimatization. It wassuggested that the decrease may be related totwo possible causes: leaf enlargement thatexceeded the synthesis of additional wax tocover the additional surface area; and waxmetabolism during acclimatization, sinceprevious studies have shown that wax

biosynthesis and degradation is a continual and

dynamic process [58,59]. These results are incontrast to reports where more extensive waxdeposits were observed in greenhouse and fieldplants than observed in vitro. Examples includecauliflower [60], carnation [61], cabbage [61],

strawberry [42], chrysanthemum [58], and grape[46]. Wax deposition after planlet transplantationoccurs with time. Wax formed after 10-14 days inBrassica oleracea [62,63] and 17/18 days incarnation [61]. Fabri et al. [42] observed anincrease in EW deposits of transferredstrawberry plantlets during the first 20 days,while similar findings were observed in Solanumlaciniatumacclimatized plants after a month [48].The results obtained in this study showed adecrease in wax content per unit area intransferred plantlets at 9 and 12 weeks fromtransplantation. The previous results indicate thatwax deposition and breakdown are species-

dependent.

Numberofinitiatedleaves

0

1

2

3

4

52 weeks

4 weeks

6 weeks

Acclimatization treatment

Ten

t

M

ist

Tub

e

Control

Leaveswilting(0=dead,4=intact)

0

1

2

3

4

5

24 hours

24 hours

4 weeks

Damagedleaves(%)

0

10

20

30

40

50

24 hours

2 weeks

4 weeks

a

ab

b

a

b

A

B

a

ab

ab

b

bb

d

a

aa

a a

aab

a

bc

c

c

c

b

b

b

cb

abb

b

Ten

t

M

ist

Tub

e

Control

Numberofshedleaves

0

1

2

3

2 weeks

4 weeks

6 weeks

D

C

c c

a

a

a

ab ab

b

aab abb

abab

b

Fig. 5. Effect of acclimatization procedures during summer on leaf damage (A), leaf wilting (B),leaf initiation (C) and leaf shedding (D) of tissue culture-derived cocoyam plants after differentdurations. Columns labeled with the same letter are not significantly different at P= 0.05 usingMultiple Range Test for treatment effect comparison at different durations. Vertical bars at the

top represent standard errors


11/15


104

Sutter and Langhans [61] and Wezstein andSommer [45] indicated that the environment inwhich a plant grows determines its morphologyand chemical composition. The in vitro conditionsin which cocoyam plantlets were grown seemed

favorably for EW formation. This high EWcontent may have contributed to plantlet survivalupon transfer ex vitro. On the other hand,Brainerd and Fuchigami [47] and Conner andConner [48] showed that EW was less importantthan stomates in determining the amount ofwater loss in plants. The sunken and ellipsoidalstomata of cocoyam leaves in vitro, in addition totheir high EW content, may have been invaluablein conferring plantlet survival. The low waxcontent of acclimatized plants may have beencaused principally by the rapid leaf expansionthat supressed wax formation. Theenvironmental conditions were not optimum

[45,61], but did favor wax formation in vitro.Another possible cause for the high amounts ofEW observed in vitro may have been thedissolution of internal lipids from open stomata ofin vitro plants to close upon removal from culture[45,47,48]. This could be true for cocoyam. Itcould also relate to the fact that cocoyamtypically grows in high humidity, and thus mayhave wax production even under high humidities.

The relatively poor growth performance ofplantlets acclimatized by mist system may beattributed to the wet conditions they weresubjected to. Griffis et al. [64] reported that

nutrients are leached under a misting system,and that the wetness creates an environmentfavorable for microorganism growth. Cocoyam,unlike taro, cannot withstand water-logging undernatural conditions [8,65,66]. Continuous mistingfor a period of five days, in addition to the highhumidity, may have been too wet to ensurenormal growth. However, the overall trend wasthat more leaves were produced than shed.Reduction in growth upon transplanting of tissueculture plantlets has been frequently reported inthe literature [62,67,68].

The number of leaves shed was comparatively

lower than that encountered from non-tissueculture derived plants under field conditions [69].This could be due to the use of growth regulatorswhile in culture. Spence [69] observed that fieldgrown cocoyam plants were wasteful in themanner in which they produced and maintainedtheir leaves, and suggested the use of growthregulators to alleviate the shedding. Thecontinuous turnover of large leaves reducedphotosynthetic productivity of the plants.

The ability to successfully transfer cocoyamplantlets from culture at a relatively low cost withminimal loss is important to the micropropagationtechnique, especially at the commercial scale. Ingeneral, many tissue culture regenerated plants

are lost during transfer to normal growthconditions. These losses are associated withrapid water loss and desiccation during theacclimatization phase. Mist systems and humiditychambers are most commonly utilized in anattempt to mitigate plant loss [44]. Short et al.[70] evaluated the success of a micropropagationsystem by the percentage of plants that aresuccessfully transferred from culture to naturalsoil conditions.

In this study, all cocoyam plants transferred fromculture to in vivo conditions survived, evenwithout acclimatization. Onokpise et al. [71-73],

also obtained 100 % survival with differentacclimatization studies. Staritsky et al. [74]reported that rootless cocoyam shoots could beeasily rooted and would rapidly develop intoplantlets when transferred into soil.Acclimatization procedures may be eitherunnecessary or just advantageous for a shortperiod, especially in areas such as the humidtropics with relatively high humidities. Otherwise,a humidity tent or cheaper method of maintaininga moderately high humidity is recommended,rather than an expensive misting system, inareas with low relative humidities.

The lag in growth in the case of winteracclimatization could be related to the lowtemperatures, humidity, and lower lightintensities in winter conditions within thegreenhouse. This probably slowed conversionfrom heterotrophic to autotrophic nutrition.Tsafack et al. [75] mentioned that thetuberization rate, the number and weight ofmicrotubers and the leaf weight were affected byday length and temperature. Omokolo et al. [76]obtained the highest tuberization rate (83%) ofthe white cocoyam cultivar with an inductivemedium containing 6-benzylaminopurine (BAP)under Short day regime. Tsafack et al [75]

confirmed the findings of Gopal et al. [77] andTsafack et al. [78] who reported that tubers couldbe induced in vitro without the use of plantgrowth regulators (PGRs). The use of mediawithout PGRs was important to judge the innatecapacity of genotypes to produce microtubersand to avoid the possibility of any undesirablecarry-over effect of PGRs on morphogenesis andsprouting.


12/15


105

5. CONCLUSION

Evaluation of stomatal number showed thatcocoyam leaves have few stomates on bothabaxial and adaxial surfaces with fewer on theadaxial surface. High levels of epicuticular waxfound in vitro may have contributed to reducedtranspiration rates. The reduced amounts of EWon acclimatized plants could be attributed to therapid cell enlargement in expanding leaves, morerapid than the rate of wax formation. Erlenmeyerflasks and test tubes did not prove to be the bestculture vessels. A wider-mouthed culture vesselsshould be used so that the mass of proliferatedtissue can be removed easily. The culturederived plants should be grown in the field undernormal conditions to evaluate trueness-to-type.This study provides additional evidence ofsomaclonal variation in these regenerants.Further investigations on physiologicalparameters will be beneficial to understand theeffect of altered leaf structure on plant growthand abnormal plants. A relatively high humidity(60-80%) is required for approximately twoweeks to prevent leaf injury resulting from wiltingand desiccation. Evaluation of stomatal numbershowed that cocoyam leaves have few stomateson both abaxial and adaxial surfaces.

COMPETING INTERESTS

Authors have declared that no competinginterests exist.

REFERENCES

1. Purseglove JW. Tropical Crops:Monocotyledons. Longmans, London.1992;97-117.

2. Onwueme IC, Charles WB. Cultivation ofcocoyam. In: Tropical root and tuber crops.Production, perspectives and futureprospects. FAO Plant Production andProtection Paper 126, Rome. 1994;139-161.

3. Ndoumou DO, Tsala GN, Kanmegne G,

Balange AP. In vitro induction of multipleshoots, plant generation and tuberizationfrom shoot tips of cocoyam. C. R. Acd. Sci.Paris, Sciences de la vie/Life Sciences.1995;318:773-778.

4. Nyochembeng L, Garton S. Plantregeneration from cocoyam callus derivedfrom shoot tips and petioles. Plant Cell,Tissue and Organ Culture. 1998;53:127-134.

5. Sefa-Dedeh S, Agyir-Sackey KE. Chemicalcomposition and effect of processing onoxalate content of cocoyam Xanthosomasagittifolium and Colocasia esculentacormels. Food Chem. 2004;85:479487.

6. Tambong JT, Ndzana X, Wutoh JG,Dadson R. Variability and germplasm lossin the Cameroon national collection ofcocoyam (Xanthosoma sagittifoliumSchott(L.)). Plant Genetic Resources Newletters.1997;112:49-54.

7. FAO. Food and agriculture organizationstatistical database: world productionoffruitsand vegetables; 2006. Available:http://www.ers.usda.gov/publications/vgs/tables/world.pdf.

8. Onwueme IC. The tropical tuber crops:Yams, cassava, sweet potato, cocoyams.John Wileys and sons Ltd, U. K. 1978;234.

9. Pacumbaba RP, Wutoh JG, Sama AE,Tambong JT, Nyochembeng LM. Isolationand pathogenicity of rhizosphere fungi ofcocoyam in relation to the cocoyam root rotdisease. J. Phytopath. 1992;135:265273.

10. Chen J, Adams MJ. Molecularcharacterization of an isolate of Dasheenmosaic virus from Zantedeschia aethiopicain China and comparisons in the genusPotyvirus. Archives of Virology.2001;146:1821-1829.

11. Debergh PC, Read PE. Micropropagation.In: Debergh PC, Zimmerman RH. (eds.),Micropropagation, Technology and

Application, Kluwer Academic Publishers,Netherlands. 1991;1-13.12. Murashige T, Skoog F. A revised medium

for rapid growth and bioassays withtobacco tissue culture. Plant Physiol.1962;15:473497.

13. Omokolo ND, Tsala NG, Kanmegne G,Balange AP. Production of multiple shoots,callus, plant regeneration and tuberizationin Xanthosoma sagittifolium cultured invitro. C R Acad Sci. 1995;318:773778.

14. Choi JH, Sung ZR. Two dimensional gelanalysis of carrot somatic embryogenesisproteins. Plant Mol. Biol. Rep. 1984;2:19

25.15. Stirn S, Jacobsen HJ. Marker proteins forembryogenic differentiation patterns in peacallus. Plant Cell Rep. 1987;6:5054.

16. Ananthakrishnan G, Xia X, Elman C,Singer S, Paris HS, Gal-On A, Gaba V.Shoot production in squash (Cucurbitapepo) by in vitro organogenesis. Plant CellRep. 2003;21:739-46.


13/15


106

17. Pal SP, Alam I, Anisuzzaman M, SarkerKK, Sharmin SA, Alam MF. Indirectorganogenesis in summer squash(Cucurbita pepo L.). Turk. J. Agric. For.2007;31:63-70.

18. Lee YK, Chung W, Ezura H. Efficient plantregeneration via organogenesis in wintersquash (Cucurbita maxima Duch.). PlantScience. 2003;164:413-418.

19. Joyner EY, Boykin LS, Lodhi MA. Callusinduction and organogenesis in soybean[Glycine max (L.) Merr.] cv. Pyramid frommature cotyledons and embryos. TheOpen Plant Science Journal.2010;4:18-21.

20. Verma VM, Cho JJ. Plantlet developmentthrough somatic embryogenesis andorganogenesis in plant cell cultures ofColocasia esculenta (L.) Schott. AsPac J.Mol. Biol. Biotechnol. 2010;18:167-170.

21. Krug MGZ, Stipp LCL, Rodriguez APM,Mendes BMJ. In vitro organogenesis inwatermelon cotyledons. Pesq. agropec.bras., Braslia. 2005;40:861-865.

22. Alam AKMM, Khaleque MA. In vitroresponse of different explants on callusdevelopment and plant regeneration ingroundnut (Arachis hypogeae L.). Int. J.Expt. Agric. 2010;1:1-4.

23. Sarabi B, Almasi K. Indirect organogenesisis useful for propagation of Iranian ediblewild asparagus (Asparagus officinalisL.).Asian Journal of Agricultural Sciences.2010;2:47-50.

24. Sujatha R, Babu LC, Nazeem PA.Histology of organogenesis from calluscultures of black pepper (Piper nigrum L.).Journal of Tropical Agriculture.2010;41:16-19.

25. Kamal GB, Illich KG, Asadollah A. Effectsof genotype, explant type and nutrientmedium components on canola (Brassicanapus L.) shoot in vitro organogenesis.African Journal of Biotechnology.2007;6:861-867.

26. Ozyigit II. Phenolic changes during in vitroorganogenesis of cotton (Gossypiumhirsutum L.) shoot tips. African Journal of

Biotechnology. 2008;7:1145-1150.27. Khierallah HSM, Bader SM.Micropropagation of date palm (Phoenixdactylifera L.) var. Maktoom through directorganogenesis. ActaHort. 2007;736:213-224.

28. Khawar KM, Sancak C, Uranbey S, ZcanS. Effect of thidiazuron on shootregeneration from different explants oflentil (Lens culinaris Medik.) via

organogenesis. Turk J Bot. 2004;28:421-426.

29. Andrs M, Gatica Arias AMG, ValverdeJM, Fonseca PR, Melara MV. In vitro plantregeneration system for common bean

(Phaseolus vulgaris): effect of N6-benzylaminopurine and adenine sulphate.Electronic Journal of Biotechnology2010;13:1-8.

30. Mayor ML, Nestares G, Zorzoli R, PicardiL. Analysis for combining ability insunflower organogenesis-related traits.Australian Journal of AgriculturalResearch. 2006;57:11231129.

31. An YR, Li XG, Su HY, Zhang XS. Pistilinduction by hormones from callus ofOryza sativa in vitro. Plant Cell Rep.2004;23:448452.

32. Banerjee N. De Langhe E. A tissue culture

technique for rapid clonal propagation andstorage under minimal growth conditions ofMusa (Banana and plantain). Plant CellRep. 1985;4:351354.

33. Hazarika BN. Acclimatization of tissue-cultured plants. Current Science.2003;85:12- 25.

34. Watanabe KZ. Challenges inbiotechnology for abiotic stress toleranceon root and tubers. JIRCAS WorkingReports. 2002;75-83.

35. Goenaga R, Chardon U. Growth, yield andnutrient uptake of taro grown under uplandconditions. Journal of Plant Nutrition.

1995;18(5):1037-1048.36. Gamborg OL, Miller RA, Ojima K. Nutrientrequirements of suspension cultures ofsoybean root cells. Exp. Cell Res.1968;50:151-158.

37. SAS Institute. SAS/STAT users guide.SAS Institute, Cary, N.C; 2006.

38. Zhao Y, Grout BWW, Crisp P. Inadvertentselection for unwanted morphologicalforms during micropropagation adverselyaffects field performance of Europeanrhubarb (Rheum rhaponticum L.). ActaHort. 2003;616:301308.

39. Zhao Y, Grout BWW, Crisp P. Unexpected

susceptibility of novel breeding lines ofEuropean rhubarb (Rheum rhaponticumL.)to leaf and petiole spot disease. Acta Hort.2004;637:139144.

40. Donnelly DJ, Vidaver WE. Leaf anatomy ofred raspberry transferred from culture tosoil. J. Am. Soc. Hort. Sci., 1984;109:172-176.


14/15


107

41. Blanke MM, Belcher AR. Stomata of appleleaves cultured in vitro. Plant Cell Tiss.Org. Cult. 1989;19:8589.

42. Fabbri A, Sutter E, Dunston SJ.Anatomical changes in persistent leaves of

tissue-cultured strawberry plants afterremoval from culture. Scientia Hort.1986;28:331-337.

43. Brainered KE, Fuchigami LH, KwiatkowskiS, Clark CS. Leaf anatomy and waterstress os aseptically cultured pixy plumgrown under different environments. HortSci. 1981;16:173-175.

44. Wardle K, Dobbs EB, Short KC. In vitroacclimatization of aseptically culturedplantlets to humidity. J. Amer. Soc. Hort.Sci. 1983;108:386-389.

45. Wetzstein HY, Sommer HE. Scanningelectron microscopy of in vitro-culturedLiquidambar styraciflua plantlets duringacclimatization. J. Amer. Soc. Hort. Sci.1983;108:475-480.

46. Dami I. In vitro acclimatization of tissuecultured grape (Vitis sp. Valiant) plantlets.M.S. Thesis, Colorado State University;1991.

47. Brainered KE, Fuchigami LH.Acclimatization of aseptically culturedapple plants to low relative humidity. J.Am. Soc. Hort. Sci., 1981;106:515-518.

48. Conner LN, Conner AJ. Comparative waterloss from leaves of Solanum laciniatumplants cultured in vitro and in vivo. Plant

Sci. Lett. 1984;36:241-246.49. Capellades M, Fontarnau R, Carulla C,Debergh P. Environment influencesanatomy of stomata and epidermal cells intissue cultured Rosa multiflora. J. Amer.Soc. Hort. Sci. 1990;115(1):141145.

50. Kaeppler SM, Kaeppler HF, Rhee Y.Epigenetic aspects of somaclonal variationin plants. PlantMol. Biol. 2000;43:179188.

51. Anu A, Babu KN, Peter KV. Variationsamong somaclones and its seedlingprogeny in Capsicum annum. Plant Cell,Tissue Organ Cult. 2004;76:261267.

52. Zhao Y, Grout BWW, Crisp P. Variation in

morphology and disease susceptibility ofmicropropagated rhubarb (Rheumrhaponticum) PC49, compared toconventional plants. Plant Cell, TissueOrgan Cult. 2005;82:357361.

53. Scowcroft WR. Somaclonal variation: Themyth of clonal uniformity. In: Hohn B andE.S. Dennis (eds.) Plant Gene Research:genetic flux in plants. Springer-Verlag,New York. 1985;215245.

54. Larkin PJ, Banks PM, Bhati R, Berttel RIS,Davies PA, Ruan SA, Scowcroft WR,Spindler LH, Tanner GJ. From somaticvariation to variant plant: mechanisms andapplication. Genome. 1989;31:705711.

55. De Klerk GJ, TerBrugge J, Bouman H. Anassay to measure the extent of variation inmicropropagated plants of Begoniahiemalis. Acta Bot. Neerl. 1990;39:145151.

56. DAmato F. Cytogenetics of plant cell andtissue cultures and their regenerates. CRCCrit. Rev. Plant Sci. 1985;3:73112.

57. Duncan RR. Tissue culture-inducedvariation and crop improvement. Adv.Agron. 1997;58:201240.

58. Sutter E. Stomatal and cuticular water lossfrom apple, cherry, and sweet gum plantsafter removal from in vitroculture. J. Amer.

Soc. Hort. Sci. 1988;113:234-238.59. Cassagne C, Lessire R. Studies on alkane

biosynthesis in the epidermis of AlliumporrumL. leaves. Arch. Biochem. Biophys.1974;165:274-280.

60. Grout BWW. Wax development of leafsurfaces of Brassica oleracea var.currawong regenerated from meristemculture. Plant Sci. Lett. 1975;5:401-405.

61. Sutter E, Langhans RW. Epicuticular waxformation on carnation plantletsregenerated from shoot tip culture. J. Am.Soc. Hort. Sci. 1979;104:493-496.

62. Grout BWW, Aston MJ. Transplanting of

cauliflower plants regenerated frommeristem culture. I. Water loss and watertransfer related to changes in leaf wax andto xylem regeneration. Hort. Res.1977;17:1-7.

63. Wardle K, Quinlan A, Simpkins I. Abscisicacid and the regulation of water loss inplantlets of Brassica oleracea L. var.botrytis regenerated through apicalmeristem culture. Ann. Bot. 1979;43:745-752.

64. Griffis Jr JL, Hennen G, Oglesby RP.Establishing tissue cultured plants in soil.Comb. Proc. Intl. Plant Prop. Soc.

1983;33:618-622.65. Caveness FE, Hahn SK, Alvarez MN.Sweet potato, yam, and cocoyamproduction. In: J. Cock (ed.), GlobalWorkshop on Root and Tuber CropsPropagation. Proceedings of a regionalworkshop held in California, 13-16September, 1983. CIAT, Cali, Colombia.1986;22-31.


15/15


108

66. FAO. Root and tuber crops, Plantains andbananas in developing countries:Challenges and opportunities. FAO plantproduction and protection paper 87, Rome,Italy; 1988.

67. Grout BWW, Aston MJ. Modified leafanatomy of cauliflower plantletsregenerated from meristem culture. Ann.Bot. 1978;42:993-995.

68. Grout BWW, Millam S. Photosyntheticdevelopment of micropropagatedstrawberry plantlets following transplanting,Ann. Bot. 1985;55:129131.

69. Spence JA. Growth and development oftannia (Xanthosoma sp.). In: Tropical rootand tubers crops tomorrow 2. Proceedingsof the 2nd International Symposium onTropical Root and Tuber Crops, Honolulu,Hawaii. 1970;47-52.

70. Short KC, Warburton J, Roberts AV. Invitro hardening of cultured cauliflower andchrysanthemum plantlets to humidity. ActaHort. 1987;212:329-334.

71. Onokpise OU, Tambong JT,Nyochembeng L, Wutoh JG.Acclimatization and flower induction oftissue culture derived cocoyam(Xanthosoma sagittifolium Schott) plants.Agronomie. 1992;12:193-199.

72. Onokpise OU, Meboka MM, Eyango AS.Germplasm collection of macabococoyamsin Cameroon. African Tech. Forum.1993;6:2831.

73. Onokpise OU, Wutoh JG, Ndzana X,Tambong JT, Mebeka MM, Sama AE,

Nyochembeng L, Agueguia A, NzietchuengS, Wilson JG, Borns M. Evaluation ofmacabo cocoyam germplasm inCameroon. In: Janick J (ed) Perspectiveson news crops and news uses. Ashs

Press, Alexandra VA USA. 1999;394396.74. Staritsky G, Dekkers AJ, Louwaars NP,

Zandvoort EA. In vitro conservation ofaroid germplasm at reduced temperaturesand under osmotic stress. In: L. A. Withersand P. G. Alderson (eds.). Plant tissueculture and its agricultural applications.1986;277-283. Butterworths, London.

75. Tsafack TJJ, Gilbert PC, Hourmant A,Omokolo ND, Branchard M. Effect ofphotoperiod and thermoperiod onmicrotuberization and carbohydrate levelsin Cocoyam (Xanthosoma sagittifolium L.Schott). Plant Cell Tiss Organ Cult.

2009;96:151-159.76. Omokolo ND, Boudjeko T, Tsafack TJJ. In

vitro tuberization of Xanthosomasagittifolium (L.) Schott: Effects ofphytohormones, sucrose, nitrogen andphotoperiod. Sci Horti. 2003;98:337345.DOI:10.1016/s0304-4238(03)00066-9.

77. Gopal J, Minocha JL, Dhaliwal HS.Microtuberization in potato (Solanumtuberosum L.). Plant Cell Rep.1998;17:794798.

78. Tsafack TJJ, Boudjeko T, Mbouobda HD,Omokolo ND. Effect of nitrogen nutrition onin vitro tuberization of Xanthosoma

sagittifolium L Schott (Cocoyam). J CamAcad Sci. 2004;4:337344._________________________________________________________________________________ 2015 Sama et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.

Peer-review history:The peer review history for this paper can be accessed here:

http://www.sciencedomain.org/review-history.php?iid=665&id=2&aid=6075

Documents

Comparative Growth Analysis and Acclimatization of Tissue Culture Derived Cocoyam (Xanthosoma Sagittifolium L. Schott) Plantlets