Thidiazuron Promotes in vitro Regeneration of Wheat and Barley

Author(s): Xueyan Shan, Desen Li and Rongda Qu

Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 36, No. 3 (May - Jun., 2000), pp.
207-210
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/4293338
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In Vitro Cell. Dev. Biol.-Plant 36:207-210, May-June 2000
? 2000 Society for In Vitro Biology
1054-5476/00 $10.00+0.00

THIDIAZURON PROMOTES IN VITRO REGENERATION OF WHEAT AND BARLEY

XUEYANSHANt*,DESENLIt? AND RONGDAQU*?

Department of Plant, Soil & Environmental Sciences, Montana State University, Bozeman, Montana 59717-0312

(Received 3 August 1999; accepted 23 November1999; editor D. D. Songstad)

SUMMARY

Thidiazuron (TDZ) is a substituted phenylurea which has been shown to be an efficacious regulator of in vitro
morphogenesis of many dicot plant species. However, information regarding the effect of TDZ on in vitro regeneration of
monocot species is limited. We investigated the effects of TDZ on in vitro regeneration of barley (Hordeum vulgare L.) and
wheat (Triticum aestivum L.) and found that it promoted shoot regeneration from callus in these two important cereal
species. Plant regeneration from calluses derived from immature embryo culture of barley and wheat was observed in
regeneration media with a wide range of TDZ concentrations (0.045-45 p[M). Shoot regeneration from barley calluses was
the highest (38.3% for cv. Golden Promise) at 4.5 pM (1 mg 1-1) TDZ, while the optimal TDZ level for wheat regeneration
seemed to be 0.9 piM (0.2 mg 1-1) (87% for cv. Bob White and 49.4% for cv. Hi-Line). Roots developed normally when
the regenerated wheat and barley shoots from TDZ-containing media were transferred to the rooting medium. Comparison
with other plant growth regulators commonly used in wheat and barley regeneration media suggested that TDZ was among
the best for in vitro regeneration of wheat and barley.

Key words: cereal; cytokinin; monocot; thidiazuron; tissue culture.

INTRODUCTION MATERIALS AND METHODS

Thidiazuron (TDZ) is a substituted phenylurea which was first
registered as a cotton defoliant (Arndt et al., 1976). It was later
found that TDZ showed high cytokinin activity in promoting growth
of cytokinin-dependent callus cultures (Mok et al., 1982). TDZ may
stimulate conversion of cytokinin nucleotides to more biologically
active nucleosides (Capelle et al., 1983) and/or stimulate
accumulation of endogenous purine cytokinins (Thomas and
Katterman, 1986). TDZ has received more attention in recent
years because of its ability to promote in vitro regeneration in many
dicot species, particularly woody species recalcitrant to in vitro
regeneration. Thidiazuron is capable of inducing not only
adventitious and/or axillary shoot production through organogen-
esis, but also somatic embryogenesis in many dicot plant species
(for reviews, see Lu, 1993; Murthy et al., 1998). To date, most
studies of the effects of TDZ in plant in vitro culture have been on
dicot plant species. Little information regarding the effects of TDZ
on in vitro culture of cereals and other monocots is available. In this
paper, we report that TDZ promotes shoot regeneration from callus
induced from immature embryos in wheat and barley.
*Authorto whomcorrespondenceis to be addressed:Email rongda_qu@
ncsu.edu
tBoth authorscontributedequally.
*Presentaddress:Departmentof Horticulture,OregonState University,
Corvallis,Oregon97331-7304.
tPermanentaddress:College of Life Sciences, Nan Kai University,
Tianjin,People's Republic of China.
$Presentaddress:Departmentof CropScience, NorthCarolinaState
University,Raleigh, NorthCarolina,27695-7620.
Plant material. Seeds of barley variety Golden Promise and spring
wheat varietiesBob White and Hi-Linewere providedby Drs. P. Bregitzer,
T. Weeks and L. Talbert,respectively.Barley and wheat plantswere grown
in potted soil (Sunshine Mix #1, Fisons, Canada) in the greenhouse.
Greenhouseday/night temperatureswere 25 ? 2oC/19oC and the plants
were grown for a 16-h photoperiodwith supplementarylights providing
150 p[molm-2 s-1 light intensity.Caryopseswere collected approximately
2 wk after pollination,surface-sterilizedfor 20 min with a solutionof 20%
(v/v) commercialbleach (Clorox,containing 5.25% sodium hypochlorite)
and 0.1% (v/v) Tween 20, and rinsed briefly five times with sterile water.
Immatureembryos 1-2 mm in size were dissected from caryopses and
placed scutellum-sideup on callus inductionmedia.
Culture conditions. Immaturebarley embryos were cultured on the
callus inductionmediumdescribedby Wan and Lemaux(1994), except that
maltosewas substitutedwith sucrose (30 g 1-1). Embryoswere culturedfor
two 2-wk periods on inductionmediumat 25 ? 20C in the dark. Immature
wheat embryos were cultured in the same way on the callus induction
mediumdescribedby Weeks et al. (1993) supplementedwith 100 mg 1-1 of
myo-inositol.
After 4 wk on callus induction medium, calluses were transferredto
regeneration medium containing various levels of TDZ. The basal
regeneration medium was MS medium (Murashige and Skoog, 1962)
supplemented with 30 g 1-1 sucrose and solidified with 2.5 g 1-1 Phytagel.
The TDZ concentrationstested for barley regenerationrangedfrom 0.01-
10 mg 1-1 (0.045-45 pM) while the levels tested for wheat were from0.1-
10 mg 1-1 (0.45-45 LMl).The effects of optimalTDZlevels on barley and
wheat regeneration were compared with those of other plant growth
regulatorscommonlyused in the barley and wheat regenerationmedia. In
barley, TDZ (1 mg 1-', 4.5 pM) was compared with a combination of
1 mg 1-' (4.5 pIM)2,4-dichlorophenoxyaceticacid (2,4-D) and 1 mg 1-I
(4.4 plM)6-benzylaminopurine(BAP) (Jaihneet al., 1991), and a combina-
tion of 1 mg 1-I (5.7 pM) indole-3-acetic acid (IAA) and 0.2 mg 1-1
(0.93 LIM)kinetin (Wan and Lemaux, 1994). In wheat, TDZ (0.2 mg 1-',
0.9 pM) was compared with 0.01 mg 1-1 (0.045 pAM)2,4-D (Sears and
Deckerd, 1982; He et al., 1988), 0.5 mg 1-1 (2.3 pM) dicamba(Hunsinger

207

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208 SHAN ET AL.

TABLE 1

EFFECTSOF TDZ CONCENTRATION

ON PERCENTREGENERATIONFROMCALLUSDERIVEDFROMIMMATUREEMBRYOSOF BARLEY
VARIETYGOLDENPROMISE

TDZ concentration(mg 1-1) 0.01 0.1 0.5 1 5 10

Regeneration(%) 21.7 ? 4.7 32.6 ? 9.9 30.0 ? 11.3 38.3 ? 16.6 35.1 ? 0.4 35.5 ? 10.5

and Schauz, 1987; Weeks et al., 1993), and a combinationof 1 mg 1-1
(4.6 ?pM)kinetin + 1 mg 1-' (5.4 RVM) at-naphthaleneaceticacid (NAA)
(Ouyang et al., 1983). Most chemicals used in the experiments were
purchasedfrom Sigma Co. (St. Louis, MO) while TDZ was from Crescent
Chemical Co. (Hauppauge,NY). Dicambawas kindly providedby Sandoz
Agro Inc. (Des Plaines, IL). Regeneratingcultures were incubated in an
illuminated growthchamber at 250C for a 16-h photoperiodwith a light
intensity of 75 p.molm-2 s-1
Regeneratedshoots were transferredto rootingmediumwhen the leaves
were - 1 cm in length. Rooting medium consisted of half-strengthMS
mediumcomponentswith 30 g 1-1 sucrose and 2.5 g 1-1 Phytagel.
Data analysis. For each experiment, 25 immature embryos were
cultured in a petri dish as an experimentalunit. Each treatmentincluded
four replicates. The callus induction rate was 90-100% for all varieties
tested (data not shown). Plantlet regenerationwas recorded 4 wk after
transferto regenerationmedium. Percent regenerationwas determinedby
dividing number of calluses with shoot(s) by the number of calluses
transferred to the regeneration medium. Calluses were considered
'regenerated'if at least one visible shoot with at least one extended leaf
was present. Calluses with only 'leafy' structuresor green color were not
considered to be regenerated as, from our observations, they rarely
developed into plantlets even with extended culture. A completely
randomizeddesign was used for all the experimentsperformed.Analysis
of variance (ANOVA)was carried out for each set of experiments.The
ANOVAor GLMproceduresof the SAS program(SAS Institute,Cary,NC)
were used for statistical analysis of the data. Least significance difference
test (LSD) (P = 0.05) was used for the multiple comparisons. In the
experimentin which the effect of TDZ concentrationon wheat regeneration
was studied,the statisticalanalysiswas performedseparatelyfor each of the
two varieties.
RESULTSANDDIscUSSION
Barley regeneration. The barley variety Golden Promise has
been shown to be highly culturable among many varieties tested
(Luhrs and Lorz, 1987; Bregitzer, 1991), and is often used as a
model system for tissue culture and transformation studies, and
was thus used in this study. Various TDZ concentrations,from
0.01 to 10 mg 1-1 (0.045-45 tRM),were tested for their effects on
barleyregeneration.Plantregenerationwas observedat all levels of
TDZ tested (Table 1). Approximately three-quarters of the
regeneratedshoots were observed 2 wk after transferringto the
TDZ-containingmedia, while the rest formedduringthe additional
2-wk cultureperiod.The highest mean percentregeneration(38%)
resulted from the medium containing 1 mg 1-1 (4.5 [LM) TDZ.
However, no significant difference (a = 0.05) between the
concentrationsof TDZused in the experimentwas found,indicating
that a wide range of TDZ concentrationscan be applied to barley
regenerationmedium. This is similar to the results observed in
peanut (Kanyandet al., 1994).
In a subsequentexperiment,a regenerationmedium containing
1 mg 1-' of TDZ was comparedwith media containing 1 mg 1-1
2,4-D and 1 mg 1-1 BAP (Jihne et al., 1991) or 1 mg 1-1 IAA and
0.2 mg 1-' kinetin (Wanand Lemaux,1994) which are often used
for barley regeneration.Among the three treatments,the mean
percentregenerationfromthe TDZ-containingmedium(40.0%)was
the highest (Table2). The LSDtest indicatedthat use of TDZin the
regenerationmediumproducedsignificantly higher mean percent
regenerationthan the combinationof IAA and kinetin (P < 0.05).
The difference between the media containing TDZ and the
combinationof 2,4-D and BAP was not significant at the a =
0.05 level but was significantat the a = 0.1 level.
Wheatregeneration. Springwheat varieties Bob White and Hi-
Line (Lanninget al., 1992) were used to examinethe effects of TDZ
concentrationon wheat regeneration(Table 3). Bob White is a
variety with good regenerationability and is commonlyused in
wheat transformation experiments(Weeks et al., 1993; Vasil et al.,
1993), while the tissue culture response of Hi-Line has not been
previouslyexamined.In bothvarieties,0.2 mg 1-1 (0.9 p.M)of TDZ

TABLE2

(a) EFFECTSOF PLANTGROWTHREGULATORS

ON PERCENTBARLEYREGENERATION

1 mg 1' 2,4-D 1 mg I' IAA + 1 mg 1-1

Variety 1 mg 1-1 BAP 0.2 mg 1-1 kinetin TDZ
Golden Promise 25.0 yvz 14.0 z 40.0 y

aMeanswith the same letter are not significantlydifferentbased on LSD grouping.

(b) ANOVASUMMARYTABLEb
Source Degree of freedom Sum of squares Mean square F-value Pr > F
Model 2 1362.67 681.33 7.41 0.013
Treatment 2 1362.67 681.33 7.41 0.013
Error 9 828.00 92.00
Correctedtotal 11 2190.67

boa= 0.05.

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 OF WHEATAND BARLEY
THIDIAZURONPROMOTESIN VITROREGENERATION 209

TABLE3

EFFECTSOF TDZ CONCENTRATION OF CALLUSDERIVEDFROMIMMATUREEMBRYOS

ON PERCENTWHEATREGENERATION

TDZ concentration(mg 1-1)

Variety 0.1 0.2 0.5 1 5 10
Bob White 60.0 ? 29.6 87.0 + 15.1 78.0 ? 9.5 81.0 ? 2.0 n.t.a n.t.
Hi-Line 40.1 ? 18.8 49.4 ? 22.4 40.0 ?15.3 42.0 ? 19.7 27.0 ? 8.9 31.0 + 15.1

n.t., not tested.

resulted in the highest mean percent regeneration.Similar to the
results of the barley experiment, no significant difference (a =
0.05) in regenerationwas foundbetweenthe concentrationsof TDZ
used in the wheat experiment.
The effects of the TDZ level (0.2 mg 1-1) on wheat regeneration
of the two varieties were comparedto those of other plant growth
regulatorsand combinationscommonlyused for wheatregeneration.
The plant growth regulators and combinations used in the
experiments included 0.01 mg 1-1 2,4-D (Sears and Deckerd,
1982; He et al., 1988), 0.5 mg 1-1 dicamba(Hunsingerand Schauz,
1987; Weeks et al., 1993) and 1 mg 1-1 kinetin + 1 mg 1-1 NAA
(Ouyanget al., 1983). In both varieties,TDZresultedin the highest
mean percentageregeneration(87% for Bob White and 49.4% for
Hi-Line).ANOVAanalysis and LSD groupingindicatedsignificant
differences between the treatments (Table 4) as well as the
interactionbetweenvarietyand treatment.The combinationof Bob
White and TDZ is superiorto any other combinationused in the
experiment. In Hi-Line, no significant difference was found
between TDZ, 2,4-D and kinetin plus NAA treatmentswhereas
all these treatmentsperformedsignificantlybetter than dicamba.
It has been reportedthat browningor necrosis of the tissues,
hyperhydricityor morphologicalabnormalityof leaves were some-
times associatedwith the employmentof TDZin culturemedia (Lu,
1993). In barley, including TDZ in the regeneration medium
sometimescaused browningand necrosis of calluses, althoughthe
overall regenerationrate was still high. No other abnormalitiesor
hyperhydricitywere observed. In wheat, no detrimentaleffects of
TDZ on calluses or regenerated plantlets were observed. Few
albinos were observed among the regenerated plantlets of both
species. All regenerated barley and wheat plantlets from TDZ
treatment,as well as those from other treatments,developed good
roots after transferringto the rooting medium. No difference was
observed in terms of plant morphologyand seed set between the
plants regenerated by TDZ and those from other plant growth
regulatortreatmentsafter they were transplantedto the soil. TDZ
has been routinelyused in the regenerationmediain ourwheatand
barley transformationexperiments.
CONCLUSION
We have demonstratedthat TDZ is capable of promotingcallus
regenerationin both barley and wheat. Percent regenerationon
mediumcontainingTDZwas as good as, or better than, that of the
widely used plant growthregulatorsin wheat and barley regenera-
tion. A wide range of TDZ concentrationstested promotedin vitro
regenerationin both cereal species, while the optimalconcentration
seemed to be 0.2 mg 1-1 (0.9 piM) for wheat and 1 mg 1-1
(4.5 plM) for barley. Although TDZ is applied to in vitro
regenerationof many dicot species, little informationhas been
availableregardingits effects on in vitroregenerationof cereal and
other monocotspecies. Tian et al. (1994) reportedthat inclusion of
TDZ in the callus subculture medium, together with 2,4-D,
enhanced indica rice regenerationlater when the calluses were
transferredto a regenerationmediumcontainingBAP as a cytokinin
source.Guptaand Conger(1998) observedin vitrodifferentiationof
multiple shoot clumps from intact seedlings in switchgrasswhen
TDZ was used together with 2,4-D. Our work investigated the
effects of TDZ on in vitro shoot regenerationfrom callus in two

TABLE4

(a) COMPARISON RATE (%)

OF PLANTGROWTHREGULATORSON WHEATREGENERATION

Variety 0.01 mg 1-1 2,4-D 0.5 mg 1-1 Dicamba 1 mg 1-1 Kinetin + 1 mg 1-1 NAA 0.2 mg 1-1 TDZ
Bob White 33.5 xyza 25.0 yz 30.0 xyz 87.0 w
Hi-Line 37.0 xy 15.8 z 46.7 x 49.4 x

aMeanswith the same letter are not significantlydifferentbased on LSD grouping.

(b)ANOVAANALYSISb
Source Degreeof freedom Sumof squares Meansquare F-value Pr > F
Model 7 13 1978.714 1885.388 10.06 <0.0001
Variety 1 355.111 355.111 1.90 0.181
Treatment 3 9619.126 3206.375 17.11 <0.0001
VarXTrt 3 3223.476 1074.492 5.73 0.004
Error 24 4497.285 187.387
Corrected
total 31 17 694.999

boL= 0.05.

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210 SHANETAL.
major cereal crop species. The results indicate that TDZ, by itself,
is an efficient regulator for in vitro regeneration of wheat and barley.
The results also implied that TDZ could have potential for
enhancing the regeneration of other cereal and grass species.
ACKNOW1LEDGMENTS
We are gratefulto Dr. William Dyer for helpful discussions and critical
readingof the manuscript,and Joy Smith and Yuyu Bai for help with SAS
statistical analysis. This work was supported by a NSF-EPSCoRgrant
awardedto MontanaState University.
REFERENCES
Arndt,F.; Rusxh,R.; Stilfried,H. V. SN 49537, a new cottondefoliant.Plant
Physiol. 57:99; 1976.
Bregitzer,P. Plantregenerationand callus type in barley:effects of genotype
and culture medium.CropSci. 32:1108-1112; 1991.
Capelle, S. C.; Mok, D. W. S.; Kirchner,S. C.; Mok, M. C. Effects of
thidiazuronon cytokinin autonomyand the metabolismof N6-(A2-
isopentenyl)[8-14C]adenosine in callus tissues of Phaseoluslunatus
L. Plant Physiol. 73:796-802; 1983.
Gupta,S. D.; Conger,B. V. In vitrodifferentiationof multipleshoot clumps
from intact seedlings of switchgrass.In Vitro Cell. Dev. Biol.-
Plant. 34:196-202; 1998.
He, D. G.; Yang, Y. M.; Scott, K. J. A comparisonof scutellum callus and
epiblast callus inductionin wheat:the effect of genotype,embryoage
and medium.Plant Sci. 57:225-233; 1988.
Hunsinger, H.; Schauz, K. The influence of dicamba on somatic
embryogenesisand frequency of plant regenerationfrom cultured
embryosof wheat (TriticumaestivumL.). Plant Breed. 98:119-123;
1987.
Jiihne, A.; Lazzeri, P. A.; Lorz, H. Regenerationof fertile plants from
protoplastsderived from embryogeniccell suspensions of barley
(HordeumvulgareL.). Plant Cell Rep. 10:1-6; 1991.
This content downloaded from 129.82.28.144 on Fri, 08 Jan 2016 09:58:33 UTC
All use subject to JSTOR Terms and Conditions
Kanyand, M.; Dessai, A. P.; Prakash, C. S. Thidiazuronpromotes high
frequencyregenerationof peanut(Arachishypogaea)plants in vitro.
Plant Cell Rep. 14:1-5; 1994.
Lanning,S. P.; Talbert,L. E.; McNeal, F. H.; Alexander,W. L.; McGuire,
C. F.; Bowman,H.; Carlson,G.; Jackson,G.; Eckhoff,J.; Kushnak,
G.; Stewart,V.; Stallknecht, G. Registrationof "Hi-Line"wheat.
CropSci. 32:283-284; 1992.
Lu, C.-Y. The use of thidiazuronin tissue culture.In VitroCell. Dev. Biol.
29P:92-96; 1993.
Luhrs,R.; Lorz,H. Plantregenerationin vitrofromembryogenicculturesof
spring- and winter-typebarley (Hordeumvulgare L.). Theor. Appl.
Genet. 82:74-80; 1987.
Mok,M. C.; Mok,D. W. S.; Armstrong,D. J.; Shudo,K.; Isogai,Y.; Okamoto,
T. Cytokinin activity of N-phenyl-N'-1,2,3-thiadiazol-5-ylurea
(thidiazuron).Phytochemistry.21:1509-1511; 1982.
Murashige,T.; Skoog, F. A revised mediumfor rapid growthand bioassays
with tobacco tissue cultures. Physiol. Plant. 15:473-497; 1962.
Murthy,B. N. S.; Murch,S. J.; Saxena,P. K. Thidiazuron:a potentregulator
of in vitro plant morphogenesis.In Vitro Cell. Dev. Biol.-Plant.
34:267-275; 1998.
Ouyang,J. W.; Zhou, S. M.; Jia, S. E. The response of anotherculture to
temperaturein Triticumaestivum.Theor.Appl. Genet. 66:101-109;
1983.
Sears, R. G.; Deckerd, E. L. Tissue culture variabilityin wheat: callus
inductionand plant regeneration.Crop.Sci. 22:546-550; 1982.
Thomas,J. C.; Katterman,F. R. Cytokininactivity induced by thidiazuron.
Plant Physiol. 81:681-683; 1986.
Tian, W.; Rance, I.; Sivamani,E.; Fauquet,C.; Beachy, R. N. Improvement
of plantregenerationfrequencyin vitroin indica rice. Chin.J. Genet.
21:1-9; 1994.
Vasil, V.; Srivastava,V.; Castillo,A. M.; Fromm,M. E.; Vasil, I. K. Rapid
productionof transgenic wheat plants by direct bombardmentof
culturedimmatureembryos.Bio/Technology.11:1553-1558; 1993.
Wan, Y.; Lemaux,P. G. Generationof large numbers of independently
transformedfertile barley plants. Plant Physiol. 104:37-48; 1994.
Weeks, J. T.; Anderson,O. D.; Blechl, A. E. Rapid productionof multiple
independent lines of fertile transgenic wheat (Triticumaestivum).
Plant Physiol. 102:1077-1084; 1993.