2004 Microparticle Bombardment as a Tool in Plant Science an-小麦转基因

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

Effects of Growth Regulators on In Vitro Plant Regeneration in Durum Wheat

V.V.Satyavathi,P.P.Jauhar,*E.M.Elias,and M.B.Rao ABSTRACT

techniques has shown considerable potential for genetic enrichment of cereal crops,including hexaploid wheat Work on improvement of durum wheat (Triticum turgidum L.)(Vasil et al.,1993;Weeks et al.,1993;Nehra et al.,1994;using tools of biotechnology is limited.Development of a reliable in vitro plant regeneration procedure for this important cereal is a Blechl and Anderson,1996;Clausen et al.,2000;Li et prerequisite for its improvement by genetic transformation.Here,al.,2003).Extensive reviews on this subject are also we report the effects of three growth regulators (GRs),2,4-D (2,4-available (Dahleen et al.,2001;Patnaik and Khurana,dichlorophenoxyacetic acid),picloram (4-amino-3,5,6-trichloropicoli-2001;Rakszegi et al.,2001;Repellin et al.,2001;Janaki-nic acid),and dicamba (3,6-dichloro-o-anisic acid),on callus induction raman et al.,2002).

and plant regeneration from scutellum cultures of four commercial A reliable in vitro plant regeneration protocol is a durum cultivars:Ben,Maier,Munich,and Lebsock.Callus induction prerequisite for the application of biotechnological meth-was obtained from isolated scutella cultured on modified Murashige ods in crop improvement.Although significant progress and Skoog (MS)basal medium.After 4wk of callus induction,all calli has been made in the transformation of cereals including were plated on MS basal medium for regeneration.The regenerated bread wheat,similar research in durum wheat is still plantlets were fertile,maintained the normal chromosome number (2n ?4x ?28)and structure as revealed by fluorescent genomic in limited.A major obstacle to genetic transformation of situ hybridization (fl-GISH),and showed no apparent somaclonal durum was the lack of an efficient in vitro regeneration variation.Genotype and callus induction medium played a dominant system.Bommineni and Jauhar (1996)standardized a role in plantlet regeneration.Dicamba proved the best GR for induc-regeneration protocol for four durum cultivars and sub-ing compact callus and also gave the highest proportion (0.16)of sequently,using this protocol,they produced transgenic regenerated plants across the four cultivars.Overall,Maier gave the durum wheat (Bommineni et al.,1997).Since this first highest proportion (0.27)of plantlet regeneration when dicamba at report of genetic transformation of durum wheat there 2.0mg L ?1concentration was used for initial callus induction.These have been several reports on production of transgenic results will facilitate genetic transformation work with durum wheat.

durum (He et al.,1999;Pellegrineschi et al.,2002).It would be advisable to use current commercial durum cul-tivars for genetic transformation to introduce new traits D

urum wheat (2n ?4x ?28;AABB)is an impor-so that the transformants are directly useful.Hence,tant cereal crop used for human consumption an efficient regeneration protocol applicable to current worldwide.Because of its high protein content and glu-commercial cultivars is highly desirable.This report ten strength,it is the wheat of choice for preparing pasta deals with the effects of three GRs,2,4-D,picloram,products.It is an important crop of the Northern Great and dicamba,on callus induction,callus regeneration Plains of the USA.Of the total durum wheat grown in capacity,and plant regeneration from scutellum cultures the USA in the year 2000,71.3%was in the state of of four commercial durum cultivars:Ben,Maier,Mu-North Dakota (USDA-NASS,2001).Durum is also nich,and Lebsock.

grown in several European countries including Italy,France,Turkey,Romania,and Ukraine,and in Canada.Chromosome-mediated gene transfers,involving sex-MATERIALS AND METHODS ual hybridization coupled with manipulation of pairing Plant Materials:Preparing for Culture

among chromosomes of parental cultivars or species,have resulted in genetic improvement of both bread Four agronomically important durum cultivars (Ben,Maier,Munich,and Lebsock)were selected to standardize the in wheat (Triticum aestivum L.)and durum wheat (Jauhar,vitro culture protocol.These cultivars were grown in the field 1993;Friebe et al.,1996;Jauhar and Chibbar,1999;under uniform conditions at the Casselton Seed Farm,North Jauhar,2003).However,this sexual technique of germ-Dakota State University Experiment Station.Spikes from all plasm enhancement is time-consuming and has its own four cultivars were collected approximately 2wk post anthesis limitations (Jauhar,2001;Repellin et al.,2001).In recent (summer season,2002),labeled,and kept in a refrigerator at years,direct introduction of foreign DNA by modern

4?C until the scutella were extracted for culture.Immature caryopses from spikes of all cultivars were removed at the same time for each replication.Thus,spikes of all cultivars in V.V.Satyavathi and E.M.Elias,Dep.of Plant Sciences,and M.B.Rao,Dep.of Statistics,North Dakota State Univ.,Fargo,ND 58105;an experiment were in the refrigerator for the same amount P.P.Jauhar,USDA-ARS,Northern Crop Science Lab.,Fargo,ND of time.Immature caryopses were surface sterilized with 70%58105.This paper embodies Satyavathi’s postdoctoral research done ethyl alcohol for 10min,followed by 15%commercial bleach in Dr.Jauhar’s lab.Mention of a trademark or proprietary product (active ingredient 5.25%sodium hypochlorite)with 0.2%poly-does not constitute a guarantee or warranty of the product by the oxyethylene sorbitan monolaurate for 30min.Explants were USDA or imply approval to the exclusion of other products that then rinsed with sterile,distilled water three times (each wash also may be suitable.Received 18Sept.2003.*Corresponding author for about 5-min duration)and used for culturing.

(prem.jauhar@https://www.wendangku.net/doc/838603172.html,).

Published in Crop Sci.44:1839–1846(2004). Crop Science Society of America

Abbreviations:fl-GISH,fluorescent genomic in situ hybridization;GR,growth regulator;MS,Murashige and Skoog.

677S.Segoe Rd.,Madison,WI 53711USA

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traditional methods of linear regression and ANOVA are not Callus Induction and Plant Regeneration

applicable for analyzing these data because the assumptions Immature embryos were excised from the caryopses under of analyses were not met.This model (Hosmer and Lemeshow,aseptic conditions.The scutella were isolated carefully by re-1989)posits a nonlinear model for the probability of the trait moving embryonic axes and any 20scutella (derived and and can flexibly incorporate categorical or continuous pre-pooled from different spikes of each cultivar)were cultured dictors.The Hosmer and Lemeshow test was used to test the per GR concentration in two Petri dishes,10in each.The adequacy of the logistic regression model.Chi-square tests scutella were placed (with cut surface of the scutellum in have been traditionally used to detect significant differences contact with the medium)in 15-mm high by 100-mm-diam.among the levels of each factor.In the present study,the Petri dishes containing MS (Murashige and Skoog,1962)me-logistic model posited the probability p ijk of an explant showing dium supplemented with 30g L ?1sucrose,100mg L ?1casein callus (Case i)in terms of three predictors (covariates),which hydrolysate,and 100mg L ?1myo-inositol.Four different con-were all categorical:i ?cultivar at Levels 1,2,3,and 4(Ben,centrations,0.5,1.0,2.0,and 2.5mg L ?1,of each of the GRs,Lebsock,Maier,and Munich);j ?GR at Levels 1,2,and 32,4-D (2.26,4.52,9.05,and 11.31?M ),picloram (2.07,4.14,(2,4-D,picloram,and dicamba);k ?concentration of GRs at 8.28,and 10.35?M ),and dicamba (2.26,4.52,9.05,and 11.31Levels 1,2,3,and 4(0.5,1.0,2.0,and 2.5mg L ?1).Theoretically,?M ),were used in different media.The medium was solidified the logistic regression model fitted was as follows:

with 0.8%purified agar (Sigma Chemical Co.,St.Louis,MO)and autoclaved at 120?C for 20min.Dicamba,being heat ln[p ijk /(1?p ijk )]????i ??j ??k ?(??)i ,j ?

sensitive,was filter-sterilized and added to autoclaved me-(??)i ,k ?(??)j ,k ?(???)i ,j ,k ,

dium.However,2,4-D and picloram are not affected by heat and were therefore coautoclaved with the media (Sigma Bio-where ln ?natural logarithm;??general effect;?i ?effect sciences,1996).The cultures were incubated in the dark at 25?of i th cultivar;?j ?effect of j th GR;?k ?effect of k th concen-2?C for 4wk for callus induction.

tration;(??)i ,j ?interaction between i th cultivar and j th GR;After 4wk,the callus was transferred to hormone-free MS (??)i ,k ?interaction between i th cultivar and k th concentra-medium.This regeneration medium was the same as the callus-tion;(??)j ,k ?interaction between j th GR and k th concentra-induction medium but without hormones.The callus obtained tion;and (???)i ,j ,k ?interaction between i th cultivar,j th GR,from each explant was maintained separately.The cultures and k th concentration.

were placed in an incubation room at 25?C,illuminated with For the analysis of the data on calli showing shoot buds two warm white and two cool white,automatically timed fluo-(Case ii),the model posited probability p ijk of calli showing rescent lights (3.1–5.5?mol m ?2s ?1)with a 16-h photoperiod.shoot buds in terms of three predictors.The model was similar Four weeks later,healthy plants with a well developed root to the one presented above.

and shoot were transferred to peat pellets and kept under the Data on the number of plantlets regenerated (Case iii)same growth-room conditions to harden before transplanting were analyzed with a Poisson Regression Model,where the in a greenhouse.After 1wk,the plants were transferred to response variable Y ijk was a count.(Counts are traditionally 13-cm-diam.pots (filled with Sunshine Mix No.1,Sun Gro modeled by a Poisson distribution.)In this case,the model Horticulture,Bellevue,WA)in a greenhouse.When estab-posited the expected or average count E Y ijk of the number of lished,the plantlets were transferred to bigger pots and grown plantlets obtainable and the offset variable was the number to maturity.

of explants cultured.This model had three categorical vari-ables:i ?cultivar,j ?GR,and k ?concentration,and was represented as follows:

Experimental Design

A replicated (four cultivars ?three GRs ?four concentra-ln(E Y ijk )??0??1(2,4-D)??2(dicamba)??3(Ben)?tions)experiment was designed to study the effect of three ?4(Lebsock)??5(Maier)??6(0.05)??7(1.0)?GRs (2,4-D,picloram,and dicamba)on callus induction in four durum cultivars.Twenty scutella were cultured per GR ?8(2.0)?ln(explants).

concentration,with 10explants per Petri dish.The experiment SAS (SAS Institute,2001)procedures were used for statisti-was repeated three times.Data were pooled from about 60cal analyses.

scutella per concentration of each GR.The number of scutella callusing (callus induction rate)was scored 3wk after culture.The number of calli with somatic embryos (that were differ-Cytological Studies

entiating into green shoots)was scored 1wk after transfer For cytological observations,root tips from 85regenerated to regeneration medium.Three weeks later,the number of plants picked at random at the four-leaf stage were collected regenerated plantlets longer than 3.0cm and with well-devel-in chilled distilled water,kept at 4?C for 24h,and then fixed oped shoot and root system were scored before transfer to in acetic alcohol (3:1,95%ethanol to glacial acetic acid).The soil pellets.

fixed root tips of 80regenerants were squashed and stained with carbol fuchsin according to the method described by Statistical Analyses

Jauhar et al.(1999).To discern the details of chromosome complement,somatic spreads of selected regenerants were There were three data sets for which we performed three statistical analyses to determine (i)whether or not each ex-studied using both conventional staining and fl-GISH tech-niques standardized earlier (Jauhar et al.,1999,2000).Ten plant produced calli;(ii)whether or not each explant with calli produced shoot buds,and (iii)how many plantlets each regenerants,selected at random,were studied by fl-GISH.Somatic chromosome spreads of durum regenerants were hy-explant with shoot buds regenerated.The data on the number of explants callusing and the number of calli showing shoot bridized with Triticum urartu Tumanian ex Gandilyan genomic DNA [labeled with biotin-14-dATP (Gibco BRL,Gaithers-buds (Cases i and ii)were analyzed as a 4by 3by 4factorial design using a logistic regression model,where the response burg,MD)100ng per slide],blocking the B genome with Aegilops speltoides Tausch genomic DNA (500ng per slide).variable is presence or absence of some outcome event,and therefore considered binary.It must be emphasized that the

The chromosome preparations were counterstained with pro-

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

SATYAVATHI ET AL.:REGULATORS AND REGENERATION IN DURUM WHEAT

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pidium iodide and the labeled DNA was detected with fluores-hormone-free MS medium.When exposed to light,the cein isothiocyanate.

somatic embryos differentiated into green shoot buds (Fig.1B).On picloram-and dicamba-containing media,shoot-like structures developed even during the dark RESULTS

induction period (Fig.1C).The shoot buds grew into All four durum cultivars (Ben,Maier,Munich,and plantlets with well-developed root systems after 3to 4Lebsock)produced calli from isolated scutellum cul-wk of culture on MS hormone-free medium (Fig.1D).tures.However,they differed in their abilities to pro-The overall effects of the cultivar,GR,and its concen-duce calli on various media.The stages of callus and tration on proportion of explants callusing,calli showing embryo formation and plant regeneration are repre-shoot buds,and plantlets regenerated were studied by sented in Fig.1.Swelling of the explant was observed taking one parameter and grouping the other two and within 2to 3d after culture,and initiation of callus was are presented in the tables and Fig.2.For callus induc-apparent as a white translucent tissue on the surface of tion,all the second-order interactions were significant the scutellar region within 3to 7d,depending on the and the relevant statistical analyses are presented in cultivar and medium.The appearance of dense,translu-Tables 1through 4.For calli showing shoot buds,no cent tissue was an indication of the cell division activity,interactions were significant,and only the main effects resulting in tissue clusters within 2wk of incubation,as were significant and the relevant statistical analyses are shown earlier by Bommineni and Jauhar (1996).Some presented in Tables 5through 8.Finally,for plantlet of these tissue clusters gradually converted into compact regeneration,only the main effects were significant,for white or yellow embryogenic calli (Fig.1A).The per-which the relevant statistical analysis is given in Table 9.centage of explants callusing on average varied from Other summary statistics are presented in Fig.2.

13to 93%,with Lebsock showing the highest callus induction rate on 2.0mg L ?1dicamba medium,and Callus Induction

Munich showing the least on 2.5mg L ?1picloram.Callus was white,friable/watery on the 2,4-D medium,white Main Effects

and friable on picloram medium,and highly compact/If we focus on the main effects,the four cultivars friable,slightly yellow on the dicamba medium.

showed significant differences in callus production Generally,the embryogenic calli differentiated into (Table 1).Lebsock and Maier were clearly the two best

somatic embryos within 3to 4wk of culture on auxin-containing medium.After 4wk of callus induction,the callus was carefully subdivided and transferred to

fresh

Fig.1.Callus formation and plantlet induction in durum wheat

culti-Fig.3.Somatic metaphase chromosomes from root-tip cells and fluo-vars.(A)Scutella showing calli from cultivar Lebsock,3wk after culture on modified Murashige and Skoog (MS)medium containing rescent genomic in situ hybridization of Maier regenerants from callus obtained on 2.0mg L ?1dicamba.(A)28somatic chromo-2.0mg L ?1dicamba.(B)Callus showing shoot buds of cultivar Maier,4wk after culture on medium containing 2.0mg L ?12,4-D somes;(B)A 28-chromosome cell counterstained with propidium iodide (PI);and (C)Same cell as (B)hybridized with total genomic (green shoot-like structures developed after exposure to light),and (C)2.0mg L ?1dicamba (green shoots developed in the dark.(D)DNA of Triticum urartu and detected with fluorescein isothiocya-nate (FITC).Brightly lit chromosomes belong to the A genome,Plantlet regeneration from cultivar Maier on MS basal medium from callus initiated on 2.0mg L ?1dicamba.

while the faded chromosomes are from the B genome.

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

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Table 1.Analysis of effects for callus induction using the logistic regression model.?

Effect

df ?2value p value Cultivar

3135.8034?0.0001Growth regulator (GR)252.5776?0.0001Concentration 3120.4631?0.0001Cultivar ?GR

631.2348?0.0001Cultivar ?concentration 921.60790.0102GR ?concentration

6

26.5882

0.0002

?Hosmer and Lemeshow (1989)?2value test of goodness-of-fit of the model 2.9496at 8df,p ?0.9375.

Table 2.Percentages of callus induction for cultivar ?growth regulator (GR)interaction.?

Cultivar

GR

Ben

Maier

Munich

Lebsock Mean SE?%

2,4-D 42.0(224)§60.8(227)49.3(229)63.3(240)54.00.04Picloram 45.6(226)56.7(238)24.2(240)61.6(232)46.90.04Dicamba 53.4(234)66.3(240)52.1(240)79.6(220)62.5

0.03

Mean 47.161.341.767.9SE?0.050.040.05

0.04

?Chi-squared statistics for comparing 12combinations of cultivar ?GR interaction was 199.0525at 11df (p ?0.0001)with sample size 2790.Chi-squared statistics for comparing Lebsock ?dicamba and Maier ?dicamba was 10.2019at 1df (p ?0.0014)with sample size 460.?SE is the standard error of the relevant mean percentage.

§Number in parentheses represents the total number of explants used with a particular combination of GR and cultivar.

Table 3.Percentages of callus induction for growth regulator (GR)?concentration interaction.?

Concentration,mg L ?1

GR 0.5 1.0

2.0

2.5

%

2,4-D 42.9(240)?46.1(219)63.0(224)64.1(237)Picloram 36.6(232)42.9(238)63.3(226)45.4(240)Dicamba 40.9(230)63.0(235)72.9(240)72.9(229)Mean 40.250.766.560.6SE§

0.05

0.07

0.07

0.04

?Chi-squared statistics for comparing 12combinations of GR ?concen-tration interaction was 180.5799at 11df (p ?0.0001)with sample size 2790.Chi-squared statistics for comparing dicamba ?2.0mg L ?1,2.5mg L ?1and 2,4-D ?2.5mg L ?1was 5.7843at 2df (p ?0.0555)with sample size 706.

?Number in parentheses represents the total number of explants used with a particular combination of GR and concentration.§SE is the standard error of the relevant mean percentage.

test statistic value of 2.9496at 8df,p ?0.9375).How-ever,third-order interactions were not significant and are therefore not shown in Table 1.The growth regula-tor and its concentration in the medium had a

pro-nounced effect on callus production by the cultivars.Fig.2.Effects of (A)the cultivar,(B)growth regulator,and (C)the Thus,all second-order interactions were also significant concentration of the growth regulator on proportion of plants re-generated.

and were further analyzed to determine the best combi-nation.producers of callus.Lebsock produced the highest num-ber of calli (Table 2)and it was significantly different Interactions

from Maier (p ?0.006).Among the three GRs,regard-All 12combinations of the cultivar ?GR interactions less of all other factors,dicamba proved to be the best were significantly different as shown in Table 2(?2value for callus induction (p ?0.0001,Table 2).Of the four of 199.0525at 11df,p ?0.0001).The combinations concentrations of GRs,2.0mg L ?1was found to be Lebsock ?dicamba,and Maier ?dicamba showed the better than other concentrations (p ?0.022,Table 3).best response with probabilities of 79.6and 66.3%,re-When a logistic regression model with main effects spectively (Table 2).When these two combinations were and second-order interactions was applied to the data,the fit was good (Table 1,Hosmer and Lemeshow ?2

further compared,the combination Lebsock ?dicamba

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

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Table 7.Percentages of calli with shoot buds for the effect of Table 4.Percentages of callus induction for cultivar ?concen-tration.?

growth regulator (GR).?

GR

Concentration,mg L ?1

2,4-D

Picloram Dicamba Cultivar 0.5 1.0

2.0

2.5

%

%Ben 37.5(176)?44.7(170)56.3(160)50.6(178)Mean percentage 21.324.417.5SE?

0.08

0.09

0.06

Maier 48.9(178)53.7(175)72.4(172)70.2(178)Munich 30.6(180)38.5(169)51.7(180)46.1(180)?Chi-squared statistics for comparing among the growth regulators was Lebsock

44.1

(168)

65.2

(178)

85.2

(176)

76.5

(170)

7.4432at 2df (p ?0.0242)with sample size 1520.Chi-squared statistics for between 2,4-D and picloram was 1.2301at 1df (p ?0.2674)with ?Chi-squared statistics for comparing 16combinations of cultivar ?con-centration interaction was 251.4756at 15df (p ?0.0001)with sample sample size 936.

?SE is the standard error of the relevant mean percentage.

size 2790.Chi-squared statistics for comparing Lebsock ?2.0,2.5mg L ?1and Maier ?2.0mg L ?1was 8.7735at 2df (p ?0.0124)with sample size 520.

Table 8.Percentages of calli with shoot buds for the effect of ?Number in parentheses represents the total number of explants used concentration of the growth regulator.?

with a particular combination of cultivar and concentration.

Concentration

Table 5.Analysis of effects for the number of calli with shoot 0.5

1.0

2.0

2.5

buds using the logistic regression model.?

%

Effect

df ?2value p value Mean percentage 3.28.642.718.7Cultivar

355.1856?0.0001SE?

0.06

0.08

0.11

0.09

Growth regulator 28.24300.0162?Chi-squared statistics for comparing among concentrations was 220.4641Concentration

3

179.5779

?0.0001

at 3df (p ?0.0001)with sample size 1520.Chi-squared statistics for ?Hosmer and Lemeshow (1989)?2value test of goodness-of-fit of the comparing between 2.0and 2.5mg L ?1was 59.5678at 1df (p ?0.0001)model 16.2551at 8df,p ?0.0389.

with sample size 887.

?SE is the standard error of the relevant mean percentage.

Table 6.Percentages of calli with shoot buds for the effect of cultivar.?

indication of somatic embryo formation.When a logistic regression model with main effects was applied to the Cultivar

data,the fit was good (Hosmer and Lemeshow ?2test Ben

Lebsock

Maier

Munich

statistic value of 16.2551at 8df,p ?0.0389).Thus,the %

main effects were significant (Table 5).However,for Mean percentage 19.914.331.316.6data on calli showing number of shoot buds,both sec-SE?

0.11

0.07

0.11

0.13

ond-and third-order interactions were not significant.?Chi-squared statistics for comparing among cultivars was 44.3786at 3df (p ?0.0001)with sample size 1520.Chi-squared statistics for comparing Effect of Cultivar

between Ben and Maier was 12.2859at 1df (p ?0.0005)with sample size 754.

All four cultivars were significantly different in their ?SE is the standard error of the relevant mean percentage.

ability to produce calli with shoot buds,as shown in Ta-proved to be the best statistically (?2value of 10.2019ble 6(?2value 44.3786at 3df,p ?0.0001).Maier was at 1df,p ?0.0014).

the best in producing calli with shoot buds (Table 6).The 12combinations of the GR ?concentration in-Figure 1B and C shows calli with shoot buds of cultivar teractions (Table 3)were also significantly different for Maier induced on 2.0mg L ?12,4-D and 2.0mg L ?1callus induction (?2value of 180.5799at 11df,p ?dicamba,respectively.0.0001).Dicamba at 2.0mg L ?1and 2.5mg L ?1,and 2,4-D at 2.5mg L ?1were found to be more effective Effect of Growth Regulator

compared with others (Table 3).However,responses As shown in Table 7,all the three GRs were also to dicamba (2.0,2.5mg L ?1)and 2,4-D (2.5mg L ?1)significantly different (?2value of 7.4432at 2df,p ?were not statistically different from each other (?2value 0.0242).Of the three hormones,2,4-D and picloram of 5.7843at 2df,p ?0.0555).

showed the best response (Table 7).On further compar-All 16combinations of the cultivar ?concentration ison between the two,both 2,4-D and picloram were interactions were significantly different as shown in found to be equally effective in inducing calli with shoot Table 4(?2value of 251.4756at 15df,p ?0.0001).buds (?2value of 1.2301at 1df,p ?0.2674).However,for callus induction,Lebsock at 2.0mg L ?1and 2.5mg L ?1and Maier at 2.0mg L ?1proved to be Effect of Concentration of Growth Regulator better combinations than others.When these combina-tions were compared among themselves,Lebsock at The four concentrations were significantly different from each other (?2value of 220.4641at 3df,p ?0.0001);2.0mg L ?1concentration proved to be the best combina-tion irrespective of the GR used (Table 4,?2value of 2.0mg L ?1being the best in inducing calli with shoot buds (Table 8).

8.7735at 2df,p ?0.0124).

Somatic Embryo Formation

Plantlet Regeneration

Poisson regression analysis of the data on plantlet The cultivar,GR,and concentration of GR had signif-icant effects on number of calli showing shoot buds,an

regeneration revealed significant differences among cul-

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Table 9.Analysis of effects for the number of plantlets regener-picloram (Wernicke and Milkowitz,1987),which vary ated using the Poisson Regression Model.

in their efficacy according to plant species used.There-Source

df ?2value p value fore,we studied the effects of these GRs on in vitro plant regeneration in four commercial durum cultivars:Cultivar

3222.71?0.0001Growth regulator 234.62?0.0001Ben,Maier,Munich,and Lebsock.

Concentration

3

291.32

?0.0001

The statistical analysis on callus production and plant regeneration showed significant differences among the four cultivars,the three GRs,and their concentrations.tivars,GRs,and their concentration for regeneration rates The study of the effects of genotype and culture me-with no significant interaction among them (Table 9).dium,as well as any interactions between them using For the purpose of comparison,we calculated the pro-linear regression and ANOVA were considered inap-portion of calli with shoot buds regenerating plantlets.propriate (Onay et al.,2000)because the fitted probabil-Among the four cultivars,Maier showed higher propor-ities from such an analysis could be negative or ?1.tion of plant regeneration compared with the other three Moreover,the variances of their proportions were not cultivars in the order Maier (0.27)?Ben (0.13)?Leb-constant,but depended on the corresponding probabili-sock (0.07)?Munich (0.03)(Fig.2A).For plantlet ties.Therefore,use of logistic regression analysis in pre-regeneration,the effectiveness of GRs was dicamba dicting the probabilities was considered more appro-(0.16)?2,4-D (0.13)?picloram (0.08),irrespective of priate in analyzing data related to probabilities.

the cultivar used (Fig.2B).The order of the most effec-There were clear differences among the four durum tive concentration for regeneration of plantlets was:cultivars in their capacity to produce callus or regenerate 2.0mg L ?1(0.31)?2.5mg L ?1(0.11)?1.0mg L ?1plants (Tables 1and 9).Lebsock and Maier were the (0.06)?0.5mg L ?1(0.01)for the three GRs (Fig.2C).best producers of callus,and Maier produced the most Figure 1D,for example,shows regenerated plantlets of regenerants.The presence of such genotypic differences cultivar Maier from callus induced on 2.0mg L ?1is common in cereal crops.There are several reports of dicamba.

differences among cultivars of bread wheat (Caswell et al.,2000;Przetakiewicz et al.,2003)and durum wheat General Morphology of the Regenerants

(Bommineni and Jauhar,1996;Benkirane et al.,2000;The regenerants (10per treatment)were studied mor-Gonz ′alez et al.,2001).

phologically with regard to general appearance,size,We found dicamba to be more effective for callus and leaf and spike characteristics.They all looked nor-induction and subsequent plant regeneration compared mal and similar to the maternal cultivar they were de-with the other two GRs,2,4-D and picloram.These rived from.There was no evidence of apparent somaclo-results are consistent with those of Mendoza and Kaep-nal variation.

pler (2002),who studied effects of four GRs,2,4-D,dicamba,picloram,and 2-MCPP [2-(2-methyl-4-chloro-Cytological Studies

phenoxy)propionic acid]on callus induction and plant regeneration from mature embryos of wheat cv.Bob-All 80regenerants studied showed 28apparently normal white.They found dicamba to result in a two-fold in-somatic chromosomes (Fig.3A).To determine whether crease in the number of plants regenerated per embryo,the chromosome complement was intact,we did fl-GISH and the amount of time required for plant regeneration analysis on somatic chromosomes of 10randomly picked was reduced by 3to 4wk.Previous reports on durum regenerants obtained from callus on 2.0mg L ?1dicamba.wheat tissue culture used 2,4-D at a concentration of We found precisely 14A-and 14B-genome chromo- 1.0–5.0mg L ?1for callus induction (Borrelli et al.,1991;somes (Fig.3B and 3C),showing absence of aneuploidy Bommineni and Jauhar,1996).

or any chromosomal imbalance.There was no evidence In our study,the number of calli showing shoot buds of chromosomal aberrations.

was not significantly different on media containing 2.0mg L ?1picloram or 2,4-D (Table 7).He and Lazzeri DISCUSSION

(2001)studied the effect of two auxins,2,4-D and piclo-ram,on scutellum culture response.They found that Genetic transformation of a commercial cultivar would addition of auxin to media did not have a significant facilitate direct introduction of genes of interest into that effect on embryogenesis,but it clearly affected regener-cultivar and make it directly usable,thereby speeding up ation,with cultures induced on picloram-containing me-the breeding process.However,an efficient and reliable dia showing higher regeneration frequencies than those in vitro regeneration procedure is the first important induced on 2,4-D.However,these workers did not com-step in any transformation protocol.In many cereal pare the effect of dicamba in their studies.On the other crops,including wheat and barley (Hordeum vulgare hand,Hassan et al.(1999)found that picloram strongly L.),immature scutellum has been the tissue of choice inhibited somatic embryogenesis from mature embryo for in vitro plant regeneration and,hence,for genetic and hypocotyl cultures of oat.We also found that the transformation (Barcelo and Lazzeri,1995;Bommineni proportion of plants regenerated from callus induced and Jauhar,1996;Li et al.,2003).Auxins play an impor-on picloram to be less than that on the dicamba me-tant role in somatic embryogenesis.The auxins com-dium (Fig.2B).

monly used are 2,4-D (Ahloowalia,1982;Maddock et al.,1983),dicamba (Papenfuss and Carman,1987),and

Although Lebsock ?dicamba and Maier ?dicamba

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

SATYAVATHI ET AL.:REGULATORS AND REGENERATION IN DURUM WHEAT

1845

were found to be a better choice for callus production,14A-and 14B-genome chromosomes (Fig.3B and 3C)with no evidence of any chromosomal imbalance Maier gave the highest response with respect to plantlet regeneration when dicamba at 2.0mg L ?1concentration or abnormality.

was used for initial callus induction.Such an indepen-dent nature of callus induction rate and plantlet regener-CONCLUSIONS

ation capacity support the suggestion of the presence of different genetic components controlling these traits We studied the effects of three GRs on callus induc-as reported earlier in bread wheat (Chowdhury et al.,

tion and subsequent plant regeneration in four current 1991;O

¨zgen et al.,1998)and durum wheat (Bohorova commercial durum wheat cultivars.Overall,the results et al.,2001).

revealed dicamba as the most suitable auxin for callus The use of medium with low auxin concentration or formation and subsequent plantlet regeneration across without auxin and/or addition of certain cytokinins seem all four cultivars.Maier had the highest percentage of to influence plantlet regeneration.In the present study,scutellum-regenerating plants,and the highest number only MS medium without GRs was used for regenera-of plantlets regenerated per scutellum with dicamba at tion of plantlets.The percentage of plant regeneration 2.0mg L ?1concentration and thus is a choice cultivar varied from 0to 31%with a mean value of 1.2plantlets for use in genetic transformation research.

per scutellum,which seems to be lower than those re-ported by earlier authors.This might be because of the ACKNOWLEDGMENTS

use of field-grown material,as the source of our ex-We thank Mr.Terrance Peterson for technical help.We are plants,or the use of medium devoid of hormones for also grateful to R.N.Chibbar,Plant Biotechnology Institute,plantlet regeneration.Borrelli et al.(1991)reported re-National Research Council of Canada,Saskatoon,Canada;generation frequencies varying from 0to 7.2%in durum John Carman,Utah State University,Logan,Utah;Lynn Dah-wheat,and Bommineni and Jauhar (1996)showed best leen,USDA-ARS,Northern Crop Science Laboratory,Fargo,values of 62and 100%scutella-regenerating plants for ND;and C.Akula,University of Wisconsin,Madison,WI,four durum cultivars,with an average of 16plantlets for critically reading the manuscript and for giving several per scutellum cultured on RM2(Regeneration Medium suggestions for its improvement.

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