belarmino manuscript

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Belarmino and GonzalesAnnals of Tropical Research 30[2]:22-33(2008)

VSU, Leyte, Philippines

Somatic embryogenesis and plant regeneration in

purple food yam (Dioscorea alata L.)

Marilyn M. Belarmino1 and Jocelyn R. Gonzales2

1Department of Horticulture, College of Agriculture, Visayas State University, Visca,

Baybay, Leyte 6521-A, Philippines;2Laboratory of Vegetable Science, Faculty of Horticulture, Chiba University, 648

Matsudo, Matsudo City, Chiba 271-8510, Japan

AbstrAct

A udy wa ondued o ealih a eliale poedue fo omai emyogenei

and plan egeneaion fom allu ulue of puple food yam (Dioscorea alata L.). the

poedue involved hee ep; (1) ulue of nodal em egmen fom geenhoue-

gown plan o geneae in vitro planle; (2) induion of allu fom he leaf, peiole

and nodal em iue; and (3) iniiaion of omai emyo fom allu. reul howed

that the agar-solidied Murashige ad Skoog (MS) medium cotaiig 30 gl-1 uga,0.1 gl-1-cysteie , 10 mgl-1 calcium patotheic acid, 2.0 mgl-1 asparagie, 2.0 mgl-1

argiie, 80.0 mgl-1 adenine ulfae (AdsO4) ad 0.1 mgl-1 naphhalene aei aid (NAA)

effectively broke dormacy of lateral buds of odal stem cultures from both VU-2

and Kinampayvaieie. Poduion of muliple adveniiou hoo oued afe

trasfer of i vitro odal pieces to the same medium added with 1.0 mgl-1 enzylamino

purie (BAP) or, MSA medium. Callus was effectively iduced from the vegetative

tissues i MS medium added with 1.0 mgl-1 2,4-Dihloophenoxy aei aid (2,4-D)

o, wih piloam. Among he hee ype of explan, he nodal em wa he mo

uiale whih podued puplih nodula emyogeni allu. A highe peenage of

odal stem-derived calli produced globular embryos i MS medium cotaiig 1.0 mgl-12,4-D ad 0.5 mgl-1 BAP, or i 1.0 mgl-1 picloram ad 0.5 mgl-1 bAP han, in he plan

gowh egulao-fee medium (onol). the mauaion of emyo wa failiaed y

oe-moth culture i MS medium cotaiig 0.1 mgl-1 ABA ad 100 mgl-1 gluamine.

thi ep impoved he geminaion of omai emyo in one-half engh PGr-

free MS medium cotaiig 100 mgl-1 gluamine (egeneaion medium). All omai

emyo-deived planle wee mophologially nomal and ealihed well in oil.

Keywod: allu, muliple adveniiou hoo, nodal em explan, planle, omai

emyo

Correspondence: M. M. Belarmio Address: AVRDC-The World Vegetable Ceter, Regioal

Ceter for Africa, P.O.Box 10, Duluti, Arusha, Tazaia.E-mail:mailyn.elamino@woldveg.

og Tel No. +255 27 2553093/2553102Fax No. +255 27 2553125


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Somatic embryogenesis in purple food yam (Dioscorea alata L.) 23

InTRODUCTIOnAomai puple vaieie of geae yam (Dioscorea alata L.) ae

among he impoan food yam ha ae highly ough eaue of hei

ulinay value. Depie hei eonomi impoane, howeve, poduion

is still low due to limited supply of platig materials ad lack of

eiane o vial and fungal dieae (saleil et al. 1990). Although

in vio mulipliaion ofDioscorea peie ha een pefomed uing

explats sice 1980s (cited by Shu et al. 2005), this techique has ot bee

ouinely applied in puple yam due o iue oxidaion and neoi.so fa, epo of de novo egeneaion inD. alata wee limied o

allu deived fom peiole explan (Faue et al. 1985) ad somatic

embryos derived from root cells (Twyford ad Matell 1996) of white

yams. Likewise, somatic embryogeesis has bee reported i several

Dioscorea species (Osifo, 1988; Twyford ad Matell 1996; Shu et al.

2005) but these did ot iclude the purple yams. To date, the techical

kow how o the productio of ulimited umber of somatic embryos,

whih ae unifom in ize and hape and eain he apaiy o give ieto ormal plats, is ot available for ay species (Petrivica, 2004).

In hi pape, we epo he poduion of plan fom puple vaieie

ofD. alata hough he induion of allu and omai emyo uing

exied nodal em of in vio plan. the effe of plan gowh

egulao on adveniiou hoo fomaion, allu induion, omai

emyo fomaion and plan egeneaion ae deied.

MATERIALS AnD METHODS

Preparation of explants

Geenhoue-gown plan (2 monh-old) fom wo puple vaieie

ofD. alata, `VU-2` ad `Kinampay(Type D, LA-609) were used as

oue of nodal em explan fo iniial in vio ulue. Nodal explan

wee pepaed fom vine ha wee hooughly wahed wih ap waead the soaked i 0.1% (v/v) of fugicide solutio (Belate; Dupot,


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Belarmino and Gonzales24

Wilmigto, Delaware, USA) for 10 mi followed by risig i sterile

diilled wae fou ime. Finally, he explan wee ufae eilized

i a commercial chlorie bleach solutio (cotaiig 4.5% chlorie) for

20 miutes ad the, rised four times i a sterile distilled water prior to

cuttig ito sigle ode stem pieces about 10-15 mm log.

Culture medium and incubation condition

The MS basal medium (Murashige ad Skoog 1960) cotaiig 30

gl-1 sugar (commercial white table sugar), 0.1 gl-1-cysteie , 10 mgl-1

calcium patotheic acid, 2.0 mgl-1 asparagie, 2.0 mgl-1 argiie ad 6gl-1 agar (Proadisa, Madrid, Spai) was used throughout this study. The

pH of the medium was adjusted to 5.8 prior to autoclavig at 1.1 kg cm-2

(121 C) for 20 miutes. newly-ioculated explats were icubated i

dark for oe week followed by exposure to 16 hours of daylight (supplied

by two pieces of 40-W uorescet tubes) at 26 1 C.

Nodal stem culture of purple D. alata and adventitious shoot

formation

To break dormacy of lateral bud, odal stems were ioculated

idividually i glass vial (25 mm 95 mm) cotaiig 10 ml of medium.

The medium was supplemeted with 1.0 mgl-1 BAP ad 0.1 mgl-1 NAA

(t1); 2.0 mgl-1 BAP ad 0.1 mgl-1 NAA (t

2); 80 mgl-1 adenine ulfae

(AdsO4) ad 0.1 mgl-1 NAA (t

3); 100 mgl-1 AdsO

4ad 0.1 mgl-1 NAA

(t4). The medium lackig plat growth regulators (PGR) served as

onol (t0

). Twety-ve explats were ioculated for each treatmet

ad replicated four times. All odal cultures were kept i dark for oe

week ad the exposed to 16 h photoperiod for further eight weeks. The

explats were trasferred to fresh medium at oe week iterval util

lateral bud break ad shoot emergece.

Induction of multiple adventitious shoots

Nodal em-deived hoo onaining a lea fou node wee u

ino ingle node em piee and inoulaed veially in one-half engh


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MS medium to iduce multiple advetitious shoot formatio. The rst

medium, MSA was supplemeted with 1.0 mgl-1 BAP, 80 mgl-1 AdSO4ad 0.1 mgl-1 nAA ad the other, MSB was added with 1.0 mgl-1 bAP

ad 0.2 mgl-1 nAA. The MS medium without plat growth regulator

served as cotrol (MSC). Five sigle ode stem pieces were ioculated

per culture bottle (60 mm 120 mm) cotaiig 30 ml of medium. Fifty

nodal em piee wee inoulaed fo eah eamen and epliaed fou

times. All odal cultures were kept uder 16 h photoperiod at 251 C. The

peenage of nodal explan poduing muliple hoo and he aveage

nume of hoo podued pe nodal explan wee deemined afe fouweeks of culture. The odal stem-derived shoots were maitaied by

anfeing hem o feh medium a one monh ineval.

Induction of callus from vegetative tissues

Stem (5 mm log), leaf (5 mm 5 mm), ad petiole (5 mm log)

piee oained fom one monh old in vio planle ofKinampayand

VU-2 were evaluated for callus formatio usig 1.0 mgl-1 of 2,4-D,

nAA, ad picloram that were added idividually ito the MS medium.

the medium wihou PGr eved a onol. the mo uiale ype

of explan (i.e., nodal em) and PGr (i.e., 2,4-D and piloam) wa

deemined aed on he highe peenage of allu fomaion. to

improve the quality of callus, the experimet was repeated usig 0.2,

0.5, 1.0 ad 2.0 mgl-1 of 2,4-D ad picloram. The quality of the callus

wa evaluaed afe wo monh. Fo oh expeimen, he explan wee

ioculated i glass vial (25 mm 95 mm) cotaiig 15 ml of treatmet

medium. Twety-ve explats were cultured per treatmet medium adreplicated four times. The explats were icubated i dark at 251 C for

one monh and uulued o feh medium wo ime.

Induction of somatic embryogenesis and regeneration of plants

to iniiae omai emyo fomaion, he emyogeni alli

derived from odal stem explats were trasferred ito agar-solidied

MS medium composed of macroelemets with reduced ammoiumitrate (800 mgl-1), mioelemen and viamin a full onenaion,

30 gl-1 sugar, ad 0.10 gl-1-yeine (iniiaion medium). the iniiaion


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medium was added with combiatios of 1.0 mgl-1 2,4-D ad 0.5 mg/L

bAP (s1), or 1.0 mgl-1 picloram ad 0.5 mg/L BAP (S2). the mediumwihou PGr eved a onol (s

0). Twety calli (0.5 g fresh weight per

callus) was ioculated for each treatmet medium ad icubated uder 16

h photoperiod at 251 c. Afe wo monh, he alli onaining gloula

somatic embryos were divided ito small pieces (0.2 g fresh weight per

piece) ad the, subcultured idividually to agar-solidied MS medium

cotaiig 0.1 mgl-1 of lter sterilized abscisic acid (ABA) ad 100 mgl-1

gluamine (mauaion medium). Afe one monh, he peenage of alli

piee onaining geminaing emyo wa evaluaed. the geminaingembryos were trasferred to oe-half stregth PGR-free MS medium

cotaiig 100 mgl-1 gluamine (egeneaion medium) o ineae he

geminaion ae. the omai emyo-deived planle wee allowed

o gow in hi medium unil he poduion of a lea hee leave and

ve roots. The platlets were acclimatized at room temperature for oe

wk ad the, trasplated i a mixture of garde soil ad river sad

(1:1, v/v) ad maitaied i the greehouse. Percet survival of potted

planle wa deemined one monh afe anplaning. All expeimenwee ondued following a ompleely andomized deign. the analyi

of ample fom eah eamen wa evaluaed uing analyi of vaiane

(AnOVA,p


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Somatic embryogenesis in purple food yam (Dioscorea alata L.)

media and he onol (tale 1). t3

alo indued he highe peenage

of nodal explan wih emeging laeal ud. Ineaing he onenaionof AdsO

4to 100 mgl-1 (t

4) howeve, did no haen ud emegene.

Moreover, the medium cotaiig 1.0 mgl-1 BAP + 0.1 mgl-1 NAA (t1)

iduced bud emergece but this took loger compared to the medium

added wih ominaion of AdsO4

and NAA o, he onol. Ineaing

the cocetratio of BAP to 2.0 mgl-1 (t2) euled o he welling of he

laeal ud u did no haen hoo emegene. Adenine ulfae elong

to the cytokii group ad kow to iduce shoot formatio from excised

plat tissues (George ad Sherigto, 1984). Likewise, nAA is a auxiha i epoed o effeively indue hoo emegene when omined

with a adeie derivative (Torres, 1989).

The three media: MSA, MSB ad MSC (cotrol) iduced advetitious

hoo fomaion fom in vitro-deived nodal em explan ofKinampay

ad VU-2. MSA medium was the most effective for both varieties sice

it iitiated rapid lateral bud emergece (14.0 days i Kinampayand 12.4

days i VU-2), produced the highest percetage of shoot formatio (60%

i Kiampay ad 75% i VU-2) ad the highest average umber of

hoo pe nodal em explan (4.3 hoo in Kinampayad 5.0 shoots

i VU-2) (Table 2). The multiple advetitious shoot formatio was

meaued afe wo monh of ulue.

tale 1. Fomaion of hoo fom exied ingle nodal em explan ofD. alata vaieie

VU-1 ad Kinampay(KI) i agar-solidied Murashige ad Skoog mediumonaining ominaion of NAA and bAP, and adenine ulfae (AdsO

4) and

NAA.

teamen Ave. no. of Peen hoo Ave. no. of

(mgl-1) days to bud emergece shoots/odal

emegene explan

VU-2 KI VU-2 KI VU-2 KI

t0; 0.0 52.0a 60.0b 30.0b 0.0 1.0 0.0t

1; 1.0 BAP+0.1 nAA 57.0a 65.0a 15.0c 0.0 1.0 0.0

t2; 2.0 BAP +0.1 nAA d* d 0.0 0.0 0.0 0.0

t3; 80.0 AdSO

4+0.1 nAA 30.0c 51.0cd 50.0a 30.0 2.0 1.0

t4;100.0AdSO

4+0.1 nAA 37.0b 55.0c 45.0a 30.0 1.0 1.0

*o data

27


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Table2.Multipleadvetitiou

sshootformatioofodalstemexplatsderivedfrominv

itroplanlesofD.alatavar.

KinampayadVU

-2after2mothsofculture.

treamens

Ave.no.ofd

ays

Percenadv.

Ave.no.ofshoos

(mgl-1)

toadv.shoot

shootformatio

/odalexplat

formaion

D.a

latavar.Kinampay

MSA;1.0BAP+80AdSO4+0

.1nAA

14.01.2

60.41.7

4.30.2

MSB;1.0BAP+0.2nAA

21.21.5

30.70.8

3.20.1

MSC;cotrol

35.00.6

20.11.2

1.00.0

D.a

latavar.VU-2

MSA;1.0BAP+80AdSO4+0

.1nAA

12.41.3

75.51.1

5.00.2

MSB;1.0BAP+0.2nAA

20.41.0

60.51.0

4.10.2

MSC;cotrol

30.00.8

40.91.3

2.00.1


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Figue 1. Peen allu fomaion fom leaf, peiole and nodal em explan ofD.

alata L. vaiey Kinampayafter two moths of culture i agar-solidied

MS medium cotaiig 1.0 mgl-1 of 2,4-D, NAA and piloam

Figue 2. Peen allu fomaion fom leaf, peiole and nodal em explan ofD.

alata L. variety VU-2 after two moths of culture i agar-solidied MS

medium cotaiig 1.0 mgl-1 of 2,4-D, NAA and piloam.

29


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Induction of callus and somatic embryogenesis

2,4-D, nAA ad picloram at 1.0 mgl-1 iduced callus formatio from

leaf, peiole and nodal em iue ofKinampay(Fig. 1) ad VU-2

(Fig. 2) after six weeks of culture i the dark. However, these explats

did no fom allu in he medium wihou PGr (onol). the medium

added with 1.0 mgl-1 2,4-D o piloam indued highe peenage of

explats that produced callus compared to 1.0 mgl-1NAA. Geneally,

VU-2 exhibited higher percetage of explats that produced callus

ompaed o Kinampay. callu aed foming aound he edge of he

u aea of explan iue. Among he hee ype of explan, he nodal

em podued he highe peenage of puplih nodula emyogeni

allu (Fig. 3A). In ona, he leaf and peiole explan podued of

e o-embryogeic calli that were highly vacuolated. More tha 50%

of he leaf and peiole explan eame oally own and neoi due o

phenoli uane ha wee exuded fom he u poion (thoma and

Ravidra 1997). Tissue browig ihibits the productio of callus ad

i severe cases led to the death of tissue (Waez, 1987; Taji ad Williams

1996).

Induction of somatic embryogenesis and regeneration of plants

the emyogeni nodal-deived alli iniiaed omai emyo

(Fig. 3b) ha appeaed a puplih whie gloula uue afe eigh

weeks of culture i the treatmet media (S1

and s2) and onol (s

0). A

highe peenage of alli podued gloula emyo in he medium

cotaiig 1.0 mgl-1

2,4-D ad 0.5 mgl-1

bAP (s1), or 1.0 mgl-1

piloamad 0.5 mgl-1 bAP (s

2) han in he onol (s

0). the gloula emyo

uderwet sequetial stages of developmet ad the germiated oe

moth after trasfer i PGR-free MS medium (Fig. 3C). Embryo

germiatio, however, was low (12-20%) due to slow embryo maturatio.

Hece, the embryos were cultured i maturatio medium cotaiig 0.1

mgl-1 ABA ad 100 mgl-1 gluamine pio o anfe in he egeneaion

medium. thi ep impoved he geminaion of omai emyo. AbA

effeively nomalized he developmen of emyo and foeed nomalmaturatio (Ammirato, 1983). Forty-five ad sixty-two percet of


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Figue 3. Induion of allu and omai emyogenei in puple D. alata L. (A)

nodal em-deived alli, (b) omai emyo, (c) geminaion of planle,

(D) rootig of platlets, (E) pottig of platlets, ad (F) establishmet of

plan in oil. (bar size; A=5mm; B=1mm; C, D & E=1 cm; F= 0.5 m)

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AbA-eaed omai emyo ofKinampayad VU-2, respectively,

were coverted to platlets after three weeks of culture i regeeratiomedium cotaiig 100 mgl-1 glutamie (Fig. 3D). The beecial effects

of gluamine fo plan egeneaion wee alo epoed in he explan of

Dioscoreacomposita Hemsl. adD. cayenensis Lam.,D. composita and

D. cayensis (Viaa ad Matell 1988). The regeerated platlets were

trasplated i soil (Fig. 3E), established well i the greehouse (Fig.

3F)) ad produced oe to two tubers setts at 20-40 g per tuber sett.

the peen udy, heefoe, peen a mehod fo plan egeneaion

via he induion of omai emyo fom exied nodal em. theailiy o onol omai emyogenei in puple yam i a neeay

requisite for the developmet of a efciet biotechological tool with

appliaion in apid ma lonal popagaion, genei anfomaion,

articial seed productio ad cryopreservatio.

LITERATURE CITED

FAUTHERET, A., P. DUBLIn ad P. CHAGVARDIEFF. 1985, Callus formatio adneofomaion wih wo edileDioscorea peie:D. alata andD. trifda (pp 624-

632).Proc. 7th Symp. Intl. Soc. Trop. Root Crops, INRA, Guadaloupe.

GEORGE, E.F. ad P.D. SHERInGTOn. 1984.Plant propagation by tissue culture.

Readig, Berks Easter Press.

MURASHIGE, T. ad F. SKOOG 1962. A revised medium for rapid growth ad

ioaay wih oao iue ulue.Physiol. Plant. 15: 473-492

OSIFO, E. O. 1988. Somatic embryogeesis i Dioscorea. Plant Physiol.133:378-

380

PETREVICA, L. 2004. Micropropagation in horticultural and tropical plant production.

Pure State Horticultural Research Statio Tukuma r.; LV-3124; LATVIA. IITA

cop and Faming syem

SALEIL, V., L. DEGRAS ad R. JOnARD. 1990. Obtetio de plates idemes

du virus de la mosaque de ligame (YMV) par culture in vitro de apex hez

ligame americaieDioscorea trifda L.Agronomie, 10: 605-615.

SHU, Y., Y. YInG-CAI ad L. HOnG-HUI. 2005. Plat regeeratio through somatic

emyogenei fom allu ulue ofDioscorea zingiberensis.Plant Cell Tiss.

Org. Cult. 80(2): 157-161(5)


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TAJI, A. ad R. WILLIAMS. 1996. Tissue culture of Australian plants. Amidale,

nSW: Uiversity of Eglad

THOMAS, P. ad M. B. RAVInDRA. 1997. Effects of pruig or removal ofin vitro

fomed oo on ex vio egeneaion and gowh in miopopagaed gape.

Plant Cell Tiss. Org. Cult., Dordrecht, 51(3): 177-180

TORRES, K.C. 1989. Tissue culture techniques for horticultural crops. AVI Books,

new. York

TWYFORD, C. T. ad S. H. MAnTELL. 1996. Productio of somatic embryos ad

platlets from root cells of the Greater Yam.Plant Cell Tiss. Org. Cult. 46: 17-

26

VIAnA, A. M. ad S. H. MAnTELL. 1989. Callus iductio ad plat regeeratio

fom exied zygoi emyo of he eed-popagaed yamDioscorea composita

Hemsl. adD. cayenensis Lam.Plant Cell, Tiss. Org. Cult.6(2):113-122

WAEZ ,V. J. 1987. The effect of activated charcoal o in vitro popagaion of eeen

Europea orchids.Acta. Horticulture.1: 131-138.

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