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Ill/I W H l 3 1293 01421 $953 This is to certify that the dissertation entitled Vernalization Response and its Implication in Wheat (Triticum Aestivum L.) presented by ShiYing Wang has been accepted towards fulfillment of the requirements for Doctoral Crop & Soil Sciences degree in W Major professor MK MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 Date LIBRARY Mlchlgan State University PLACE ll RETURN BOX to remove this checkout from your record. TO AVOID FINES Mum on or before date duo. DATE DUE DATE DUE DATE DUE MSU I. An Affirmative Action/Equal Opportunity Institulon WM! VERNALIZATION RESPONSE AND ITS IMPLICATION IN WHEAT (TRITICUM AESTIVUM L.) BY ShiYing Wang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1 995 ABSTRACT VERNALIZATION RESPONSE AND ITS IMPLICATION IN WHEAT (TRITICUM AESTIVUM L.) BY ShiYing Wang Vemalization response in wheat so far has been characterized poorly, and less well quantified. The discrepancies and inconsistencies in the literature regarding terminology, measure of response, classification of response types, operative temperatures, etc. stem in part from the lack of a general conceptual model of vemalization phenomena. A fundamental technique in wheat vemalization study is to count the number of leaves emerged before, during, and after vemalization treatment, rather than to calculate calendar days or thermal time after the end of vemalization treatment. Final leaf number on the main stem for vemalization-sensitive cultivars in general decreased until reaching a plateau as days of vemalization treatment increased. There is not an absolute “vemalization requirement" for wheat. Accumulated plant age, expressed as leaf stage, enables attainment of vemalization insensitivity, independent of, or in combination with vemalization treatment. There is an interchangeability between plant age and the duration of vemalization treatment. After the onset of vemalization insensitivity, a plant will emerge six more leaves under long photoperiod conditions. The quantitative features of this vemalization response, up to the point of insensitivity, were characterized with a linear regression: (F. - 6) = a - BTW where F. is the number of leaves observed for a particular vemalization treatment, TV is the time in days of that vemalization treatment, and a and B are the Y-intercept and the slope of the regression, respectively. The parameters a and B varied among cultivars, and are useful for quantifying vemalization response in wheat. The implication of each parameter can be interpreted biologically: a is the “changeable number of leaves”, i.e., how many leaves can be potentially decreased by vemalization treatment; and [3 represents the “exchange rate” between leaf numbers and vemalization days, i.e., how many leaves can be reduced by one day of vemalization treatment. A novel conceptual framework was proposed for characterizing and quantifying wheat vemalization response. Copyright by SHIYING WANG 1 995 Affectionately Dedicated to my Loving Father, Zhan-ao Wang and Mother, Zheng-ai Shen ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all those individuals that in one way or another helped me to complete my doctoral program: Dr. Richard W. Ward, my advisor, Dr. Joe T. Ritchie, my co-advisor, Drs. Royal D. Heins and James A. Flore, my guidance committee member, for all their guidance, encouragement, constructive criticism, and constant friendship. Dr. Ralph A. Fischer, director of wheat program in lntemational Maize and Wheat Improvement Center (CIMMYT) in Mexico, for his fundamental role in making my Ph. D. dream come true: finding financial resource and institution for starting my program, and frequent guidance throughout my research project; for his special trip coming to Michigan State for my oral examination. The MSU wheat group: Mr. Dave Glenn, Ms. Erica Jenkins, and other graduate and undergraduate students, for their help in the experiments and routine work, for their friendship. The MSU Homer Nowlin Chair group: Drs. Urs Schulthess, Jun Wei, and other visiting scholars and postdoctoral fellows, e.g., Drs. D.B. Fowler, I.R. Brooking, and M.J. Robertson, for thoughtful discussion; Mr. Brian Baer, for his assistance in solving the computer-related problems. Dr. Larry Copeland, for serving on my extension program instructor. vi My wife, Bin Liu, and my daughter, Xin Wang, for their love and support. Gratitude is extended to Dr. R.S. Loomis, editor-in-chief, and other unnamed reviewers of the ‘Field Crops Research”, for their constructive criticism and valuable comments on the first two sections; to Professor Guo-Yuan Miao and Dr. E.J.M. Kirby, for their reviews and valuable comments on the third section. The financial support from the CIMMYT Fellowship, the MSU wheat program, and the MSU Homer Nowlin Chair is greatly appreciated. vii TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................ viii LIST OF FIGURES .............................................................................................. ix GENERAL INTRODUCTION ................................................................................ 1 References ..................................................................................................... 3 SECTION I. Vemalization in wheat. I. A model based on the interchangeability of plant age and vemalization duration ........................................................ 5 Abstract ......................................................................................................... 6 1. Introduction ............................................................................................... 6 2. Materials and methods ............................................................................. 8 3. Results ...................................................................................................... 8 4. Discussion .............................................................................................. 1 1 Acknowledgements ..................................................................................... 14 References ................................................................................................. 14 SECTION II. Vemalization in wheat. ll. Genetic variability for the interchangeability of plant age and vemalization duration ......................... 16 Abstract ....................................................................................................... 17 1. Introduction ............................................................................................. 17 2. Materials and methods ........................................................................... 18 3. Results and discussion ........................................................................... 19 Acknowledgements ..................................................................................... 22 References ................................................................................................. 22 SECTION III. A novel conceptual framework for wheat vemalization ................ 23 Abstract ....................................................................................................... 24 1. Introduction ............................................................................................. 26 2. Terminology ............................................................................................ 26 3. Measure of response .............................................................................. 29 4. Response types ...................................................................................... 33 5. Operative temperatures .......................................................................... 37 6. Relationship between leaf number and vemalization responsiveness ...40 7. Interchangeability between plant age and vemalization duration ........... 44 8. A novel conceptual framework ................................................................ 46 9. Concluding remarks ................................................................................ 53 Acknowledgements ..................................................................................... 54 References ................................................................................................. 54 viii Table 1. LIST OF TABLES Page Section I Effect of vemalization duration and leaf tip stage (LTS) at the onset of vemalization treatment on the total number of leaves on the main shoot at heading (final leaf number, FLN), for cultivar Pioneer 2548 .......................................................................................... 9 Section II Table 1 wheat lines used in the experiments with their countries and Table 2. Table 3. latitude of origin ................................................................................... 18 Mean F LN in various lines at different vemalization treatments .......... 20 The Y-intercept (a) and the slope ([3) derived from linear regression analysis of F. minus six and the days of vemalization treatment for a range of wheat lines, and resultant estimates of substituted days of vemalization treatment by one leaf’s growth, 1/3. The r’ is coefficient of determination of the linear regression. F0 is the mean FLN of unvemalized plants. ..................... 21 Fig. 1. Fig. 2. Fig. 1. Fig. 2. Fig. 1. Fig. 2. LIST OF FIGURES Page Section I Relationship between FLN minus six and days of vemalization duration for Pioneer 2548. The data include all age treatments except those with fewer than six leaves emerging after vemalization. The linear regression line is fitted as: (FLN - 6) = 15.7 - 0.244%... The r2 is 0.89. ........................................ 10 Relationship between FLN minus six and days of vemalization duration for Augusta. The data include all age treatments except those with fewer than six leaves emerging after vemalization. The linear regression line is fitted as: (FLN - 6) = 17.0 - O.236‘V...,.. The r2 is 0.85. ......................................................................................... 10 Section II Vemalization response for wheat lines with a similar Y-intercept (a) but different slopes (B). The parameters of the lines are listed in Table 3. The symbols represent the observed values after discarding points in the region of vemalization insensitivity ................... 21 Vemalization response for wheat lines with a similar slope (B) but different Y-intercepts (a). The parameters of the lines are listed in Table 3. The symbols represent the observed values after discarding points in the region of vemalization insensitivity ................... 21 Section III Days from the end of vemalization treatment to heading in response to weeks of vemalization treatment. Adapted from Ahrens and Loomis (1963), imbibed seeds of winter wheat Minter were vemalized at 1°C for varying periods, then transferred to a warm greenhouse (24°C, 18/6-h), and observed for heading date. ........ 31 Final leaf number on the main stem in response to weeks of vemalization treatment. Adapted from Wang et al. (1995a), seedlings of winter wheat Pioneer 2548, aged at the third leaf tip visible, were vemalized at the growth chamber (513°C, 8/16-h) for varying periods, then transferred to a warm greenhouse (20°C, 20I4-h), and observed for final leaf number on the main stem. .............. 34 Fig. 3. Fig. 4. Fig. 5. Fig. 6. The effectiveness of temperature (°C) on vemalization response in the literature. Adapted from (a) Kirby (1992, solid line) and Maas and Arkin (1980, dashed line), (b) Hansel (1953, solid line) and Reinink at al. (1986 dashed line), (c) Ritchie (1991, solid line) and Weir et al. (1984, dashed line). ......................................................... Schematic representation of the effects of vemalization and photoperiod on the number of leaves in wheat. The capital Roman numerals represent the ordinal leaf number from the flag leaf. .................................................................................................... Vemalization response quantified with a linear regression: (F. - 6) = a - [3Tv for different cultivars. The parameters are as follows: 01 = 5.4, B = 0.1174 for Pitic 62; a = 5.4, B = 0.0750 for FL 303; 01 = 15.1, B = 0.2240 for Pioneer 2548; and 01 = 17.4, B = 0.1880 for NY 73116-4w. Data taken from Wang et al. (1 995b) .............................................................................................. Model of relation between leaf number and vemalization responsiveness. The vemalization response line (line AD) used as an example is: (F. - 6) = 17.4 - 0.188T.,, i.e., NY 73116-4w in Fig. 5. Point A, B, C or D represents vemalization insensitivity under different temperature regimes. The abbreviation “v.” stands for “vemalization” and “i." for “insensitivity“ ............................................ xi Page ..... 38 ..... 43 ..... 49 ..... 51 GENERAL INTRODUCTION Wheat originated in the so-called Fertile Crescent of southwestern Iran, northeastern Iraq, and southeastern Turkey some 8 to 10 thousand years ago, and spread to India, China, and even England by about 5000 8.0. (Harlan and Zohary, 1966; Bell, 1987; Tahir and Valkoun, 1994). Today, it is grown across a wide range of agrogeographical regions from the equator to greater than 60° latitude (Briggle and Curtis, 1987). Among all the world’s major food crops, wheat is ranked number one based on either the harvest area (about 221.7 million hectares in 1993), or total production (about 564.5 million tonnes in 1993, 95% being bread wheat) (FAO, 1994). Wheat represents almost 30% of world’s grain production, with nearly 40% of the population utilizing it as a staple (Hancock, 1992). The native home of wheat is basically characterized by long, hot, dry summers and short, mild, wet winters. Wheat is planted soon after the first fall rains and it undergoes vegetative growth in winter. It switches to reproductive growth as temperatures and photoperiods increase in spring, and matures in early summer (Loss and Siddique, 1994). Wheat thus evolved as a cool-season annual. After several thousand years, both natural and human selection have resulted in different wheat genotypes adapted to specific environments and cropping practices. 2 A principal taming objective and a major contribution of the plant breeder to it is to ensure that the life cycles of particular genotypes fit the constraints of the local (or target) environment (Summerfield et al., 1991 ). Timely anthesis and maturity in relation to the growing season available in a particular location are also essential for large potential yields from annual crops (Bunting, 1975). Crop phonological development is a result of interaction between genotype and environmental factors. Marcellos and Single (1970) envisaged the length of any developmental period (D) to be a function of a number variables such that D=f(G,V,T,P,, M) where G symbolizes genetic factors, V those for vemalization, T those for temperature P, for photoperiod, and M. miscellaneous factors such as plant nutrition and plant water status. Evidence indicated that vemalization, photoperiod and temperature are the main and almost exclusive determinants for wheat’s phenological development (Pinthus, 1985). Although vemalization response in wheat was studied extensively from 1930s to 19503 (Whyte, 1948; Chouard, 1960), it so far has been characterized poorly, and less well quantified as compared with the photoperiod and general thermal responses (Ellis et al., 1989). There is not consistency in the literature regarding a variety of key issues, such as how to express changes in development induced by vemalization, how to characterize and quantify the genetic variability for vemalization response, which temperatures are the effective temperature for vemalization response, whether vemalization-sensitive 3 wheats have an absolute requirement for vemalization. Those were the primary reasons for initiation of the present studies. The starting point was to investigate the relationship between plant age at the onset of vemalization treatment and the duration of vemalization treatment. The parameters of vemalization responsiveness were determined and quantified in a set of wheat cultivars and lines from diverse geographical origins. The main objective was to outline a novel conceptual framework that integrates both the recent advances in literature and the enlightenment from current studies for wheat vemalization. References Bell, G.D.H., 1987. The evolution of wheat cultivation. In: F.G.H. Lupton (Editor), Wheat Breeding. Its Scientific Basis. Chapman and Hall, London and New York. Briggle, L.W. and Curtis, 80., 1987. Wheat worldwide. In: E.G. Heyne (Editor), Wheat and Wheat Improvement. ASA, CSSA, SSSA Publishers, Madison, pp 1-31. Bunting, A.H., 1975. Time, phenology and the yields of crops. Weather, 30: 312-325. Chouard, P., 1960. Vemalization and its relations to dormancy. Ann. Rev. Plant Physiol., 11: 119-238. Ellis, R.H., Summerfield, R.J., Roberts, SH, and Cooper, JP, 1989. Environmental control flowering in Barley (Hordeum Vulgare). Ill. Analysis of potential vemalization responses, and methods of screening gerrnplasm for sensitivity to photoperiod and temperature. Ann. Bot, 63: 687-704. 4 FAQ, 1994. FAO yearbook, 1993 Production. Vol. 47. UN, Rome. Hancock, J.F., 1992. Plant evolution and the origin of crop species. Prentice- Hall, Inc., New Jersey. Harlan, JR, and Zohary, D., 1966. Distribution of wild wheats and barley. Sci, 1 53: 1 074-1080. Loss. SP, and Siddique, KH.M., 1994. Morphological and physiological traits associated with wheat yield increases in Mediterranean environments. Adv. 'Agron., 52: 229-276. Marcellos, H., and Single, W.V. 1971. Quantitative responses of wheat to photoperiod and temperature in the field. Aust. J. Agric. Res. 22:343-357. Pinthus,'M.J., 1985. Triticum. In: AH. Halevy (Editor), CRC Handbook of Flowering, N. CRC Press, Inc., Boca Raton, Florida, pp. 418-443. Summerfield, R.J., Roberts, E.H., Ellis, RH. and Lawn, R.J. 1991. Towards the reliab!e prediction of time to flowering in six annual crops. l. The development of simple models for fluctuating field environment. Expl. Agric. 27:11-31. Tahir, M., and Valkoun, J., 1994. Genetic diversity in wheat - an international approach in its evaluation and utilization. Wheat Inf. Serv., 78: 1-12. Whyte, R.O., 1948. History of research in vemalization. In: AE. Mumeek and RC. Whyte (Editors), Vemalization and Photoperiodism. Waltham, Mass, pp. 1— 38. Section I Vemalization In Wheat I. A Model Based On The Interchangeability Of Plant Age And Vemalization Duration Field Crops Research ELSEVIER saucepan-m4! (1995) 91.100 Vemalization in wheat I. A model based on the interchangeability of plant age and vemalization duration Shi-Ying w'ahg 1, Richard w. Ward u. Joe '1'. Ritchie -, Ralph Anthony Fischer b, Urs Schulthess ‘ -Wdcmuwmuwpmum.mm.mm¢ USA 'Wheorhogrmn. mmummwrauummw.” Received'lllmehIMueomtad'IFmr-uytfis Abstract VernalizationoeatmentsofOtomdinitiatedwhenOtoBIeafdpsworevisihtewereappIiedtopImofdtewimrwheat (Triricmacuivan)culdvaioneer2548mtdAugusta.Anplmheaded‘ ofdmationofvernalizatioo.Unver- naliaed plants of Pioneer 2548 and Augusta had final leafnumbers (FLN) 01103213 and 21.7tl.0. respectively. WWwfiWMleymmmmmthmm-mt‘mdwm insensitivitywasreached.Esdmofdmmimmumdaysofvunahndmnquuedmruchmndinummumydeuumd inalinearfashionasplantageattheonsetofwmnmmtmmmofleamappeanngaftermeom ofvernalizationinsensitiviryaveraged63105.Mnunussixappemstobeavalidesdrnateinotucxperimenulcmdiuons fordteonsetofver'nalizationinsensitivity.atIeastforphnudtathadsixormueleavesappemingafterdteendofvunalindm treaunentljnearregreasionsofl-INminussixagainmdaysofwrnalizationwereaignificantforbodrcultim(fortreatrnetls widtsixormoreleavesemergingaftervemalization). WY-intaceptsofthefinedmgreasionswerecloaetovaluesobtained bysubtractingsixfromI-‘lfiofunvemalizedplants. Bodainteroeptandslopewerecontrolledgenetically. Accumulatedplam ageexpreuedasleafstage. enablesanainmentofvernalintioninsensitivity. independemot’. orincomhinatioowithvernalimrm treatment Keywords: Leaf number: Modelling; Plat age: Tritium: Ver'rnliution: Wheat 1. Introduction The well-established concept of using thermal unit as a measure of physiological time derives from the fact that duration in calendar days of any phenological phase of plant development is generally increased as temperatures are lowered (Ritchie and NeSmith. ‘ Correspondiagarnhor. Email: 22857mgrOibm.cl.msu.edu:Faa: (517) 353-5114. OWN/95309.50 0 I995 EIaevier Science B.V. All righs tenured 30103784290( 95 )00006-2 I99]).Ontheotherhand.thedtnationofthevegetative phascot'manyspeciesisreducedbyexposuretolow temper-attire: (Pin-iris. 1961; lang. I965). Lynette (I928. seeWhyte. 1948 andChooard. 1960) coined dretennvemalintionflomakespr'inglikchoreferto thisphenomenoninwheot(TriticrnnaesrimnnL).One ofdtegreatchallengestowheatphysiologistsandmod- elerscontinuestobetheaccuratepredictionofwheat‘s developntentalresponsetolowternper'annes.primarily 92 S.-Y. V" «IL I Field Cm M (I (1995) 91-!” because a good conceptttal model of vernalintioo phe- nomena does not exist. Research has shown that several factors influence wheat’s vemalization response. including temperature and duration of low temperature conditions photope- rind. andgenotype (Evansetal.. I975;Pinthus. 1985). ‘l‘hereislittleunityamongresearehersonavarietyof key issues. however. including mearts of expressing vemalization response. whether vemalization is requiredforsomewheatstoflower.andhowgenetic variabilityforvernalizationresponseshorddbechn- acterizetL‘I'hisispartiallyreflectedintherangeof terminology that is used to describe vemalization. beginning with use of the word “vemalization" itself. which is used in reference to both a plant’s physiolog- icalstateandthestateoftheenvironmentinwhichit is grown. Plantsthatno Iongerrespondto vemalization have been described as “fully vemalized". or “ver- nalined", which practice suggests that vemalization refers to a plant’s physiological status. On the other hand.itiscommontorefertotheprocessofsubjecting imbibed seeds or young plants to low temperatures as “vemalization". or to say that plants were “vemali- zed" for a certain number of days. These two usages of forms of the word vemalization lead to problems interpreting a simple statement such as ‘ ‘the plants were not vemalized". because that could mean either that nolowtemperatureconditionswereimposcd.orthat the plants had not yet reached a particularstate of phys- iological development. or both. Here. vemalization is used to describe environmental circumstances rather than a plant's physiological state. and vemalization response is used to refer to a plant’s developmental response to exposure to low. nonfieez- ing temperatures. Consequently. a plant that has been vemalized will not necessarily show any response. while an unvemalized plant is one that was not exposed to vemalizing conditions. Likewise. a vemalization treatment is one that exposes plants to low temperature and will not necessarily elicit vemalization responses from wheat plants. There is not consistency in the literature regarding how to express changes in development induced by vemalization. Some authors use calendar days or ther- mal time as their primary unit of measure. while others use the final number of leaves on main stems (Pugslcy. I971: Levy and Peterson. 1972; Berry et al.. 1980; Flood and Halloran. 1984;Miao et al.. 1992). Neither calendardaynordtermaltimeappnachesarelikelyto revalclearbiologicalprinciplesbwauseinbothcans aplant’sresponseresultsfromthestnnofboththe acceleratingandretardingeffectsoflowtemperannes. Theleafntnnber'appr-oachismoreappropriambecause itdirectlyrefiectsdifferencesinthetimingoftheuan- sitionfromvegetarivetofioralapexdevelopmenttl-lay andKirby. 1991). Another aspect of vermlintion that seems poorly resolved is whether vemalization-responsive wheats haveanabsoluterequirementforvemalintionfienetic linesareofmoassessedfortheir“vernalintionrequi- rements".i.e..howmanydaysoflowtemperattuethey requireinordertoundergofloral initiationandtlow- cring (Krekule. 1987).Ontheotherhand.thereare reports where all tested wheats. even those adapted to autumn planting at high latitudes. will evenntally flower without exposure to low temperatures (Ahrens and Loomis. 1963; Chain. 1966; Martinis. 1973: Catch. 1976;Ledent. 1980;Miaoetal.. I992).Studies involving low-temperature treatments of varying dttra- tion almost always yield quantimive response craves (Ledent. I980; Miao ct al.. 1992). which also seems inconsistent with tlte notion of absolute vemalization mquirernenmatleastinutificialconditionswheresea- sort length is not limiting. Techniques for the characterization of genetic vari- ationinvernalizationresponsearealsonotwelldevel- oped. Various approaches have revealed that each of wheat’sthreegenomeshasoneortwolociwhoseallelic variants influence vemalization response in a qualita- tive fashion, but minor genes are also reported (see Flood and Halloran. I986. for review). This picture of the genetic control of vemalization response logically leads to a cmtinuum of phenotypic classes. and tltat expectation is confirmed by many studies (Flood and Halloran. I986). To this day. however. there is no formal system for classifying wheats beyond use of the terms “spring" and “winter" in combination with modifiers such as “strong" and “weak".‘l'hatsystern fundamentally refers to a genotype's adaptation to spring- or fall-sown systems and does little to charac- terize variability in vemalization response. For instance.itiswellestablishedthatmanyspringwheats exhibitsorneresponsetovernalizationflewand Peterson. 1972; Jedel et al., 1986). Several reports demonstrate that wheat’s vemaliza- tion response is influenced by stage of development S-Y. Wag ad. I Field Cm M 41 (1993) 91-100 93 (e.g., developing embryos. germinating seeds. and growing plants) (Ridden and Gries. 1958; Pugsley. 1971;800an 1984: SharmaandMaseia. 1987: Kato and Yamashita.1991;Whelan and Schaalje. l992).andplantage(Gott. l957;AhrensandLoomis. 1963; Chujo, 1966;.Iedel et al.. 1986). Those reports allconcludedthatwheatgraduallylosesitssensitivity to vemalization as it grows older. However. that phe- nomenonwasnotconsideredinmostofthesmdiesdtat employed more than one vemalization duration. The reports that addressed plant age generally used only onelowternperahneheatrnentoffixeddrnatiomora single vemalization duration plus an unvemalized con- uol. The work of Jedel et al. (1986) addressed both plant age and duration of vemalization treatment with springwheats.‘l'heexperirrtentsreportedherewere designedtoextendtheworkofledeletal.towinter wheats.Aricharrayoftreatrnentcombinationspro- videddatausedinthederivation ofageneralizedcon- ceptual model for wheat vemalization. The applicabilityofthatmodeltowheatsfromawiderange ofadaptationaonesisreportedinacompanionpaper (Wang et al.,1995. in press). 2. Materialsandmethods ‘I'hestudywascarriedoutintwogreenhousesanda growth chamber in 1992 at Michigan State University (42°N). Greenhouse A (2015?) was used for both pre- and post-vemalization growth. Greenhouse B ( 15 i 2°C) was used to acclimate plants to higher tem- perature conditions after vemalization. PhotOperiods in both greenhouses were extended to 20 h with high- pressure sodium lamps providing a photosynthetic pho- ton fiux density of approximately 200 mol rn’2 s“l at pot level. Vemalization treatments were applied in a growth chamber that had a photoperiod of 8 h and a photosynthetic photon flux density of 200 urnol m’2 s ' ' from fluorescent and incandescent lamps. The short photoperiod during vemalization treatment was used in order to ntirnic natural conditions. Temperatures in the growth chamber were 5°C and 2°C during the light and dark periods. respecrively. Two winter wheat cultivars adapted to Michigan. Pioneer 2548 and Augusta. were used in this study. The genotype of these cultivars at known vemalization loci isunknown.Seedssoakedfor24hat20t025°Cwere sowningreenhonseAin lS-cmdiameterplasticpots in a soil mixture of 5 loam:2 pest:3 sand (v/v/v). There were four plants per pot. All pots remained in greenhouseAuntilinitiationofthevernalizationtreat- ments.Plantagetrestrnentswerebasedonthenumber ofleaftipsvisibleonthemainshootattheonsetof vernalintiontreatrnentNineplantages.fromIeaftip stage 0 (LTSO, germinated seed) through LTSS (eighdt true leaftip visible). were evaluated. Forth: L'l‘SOagetreaunenu,potsweretransferredtothever- nalintion chamber immediately upon sowing. The daysfromsowingtotheonsetofvernalizationtreat- meat for LTSO through LT88 were 0. 5. 8. l3. 17. 23. 30. 39.and46d.respectively. Plants were subjectedto vernalizing conditionsinthegrowth chamberfor 7. I4. 21, 28. 35, 42. 49. or ‘70 d. The 49- and 70-d vernali- zationueatrnentswereomittedfortheLTS4through LTSS treatments. The 42-d vemalization treatment was alsoomittedfortheLTS8 treatrnentAftertheendof each vemalization treatment, pots were moved to greenhouseror3dtostabilizevernalizationeffects (Chouard, 1960).Potswerethenretinnedtogreen- house A. Unvernalized cannot plants were grown con- tinuouslyingreenhouseA.1heexperimentwas terminatedafterallplantsreachedmamrity. A completely randomized design with two replica- tions was used. Each replication consisted of one pot of each treatment. Pot positions were randomized weekly to minimize possible position effects. The num- berofernergedleavesattheendofeachvernalization treatment and the final leaf number (FLN. i.e. total number of leaves on the main stems at heading) were determined for the main stems of four plants in each potDatawereanalyzedwiththeGLMpr-ooedmeof SAS ( SAS Institute. 1991). Where appropriate. differ- enoesamongtreatrnentmeanswereexaminedusingdte Duncan r-test. FLN data were transformed to logarith- mic values for means comparison tests. 3. Results 3.]. Pioneer 2548 Plants in all treatments. including unvemalized con- trols, produced flag leaves and headed. Average final leafnurnber(FLN) forcontrols was 20.811.114.14 averagesforthevernaliaedtreatmentswerealways 94 Tdiel S.-1’. Wag etal. [Field Cups M 4111995191400 Effectofveraalintionrhrrauoomdleaftipstage(L13)nmeooaaofvuul'nlionmondmnnalmunbuofhavesonmemainshoa tintingthlleafamnber.flfl).foreuhivar1’ioneer2548 VennLrhuxiontdayI) leafnpstageILTS) 0 I 2 3 4 5 6 7 8 0 20.8a' 20.8a Ma Ma 20.8a 20.8a 20.8a 20.8a 20.8a 1 20.3 ab 20.0a 19.5s 20.8a 203a 20.8a 19.8a 20.0a 21.0a 14 19.8 ab 19.0 a 18.3 b 17.8 b 17.8 b 18.3 b 18.3 b 18.0 b 18.8 b 21 18.8 b 16.3 b 14.3 c 15.0 c 16.0 c 16.0 c 16.3 c 17.0 c 17.0 c 28 17.3 c 13.5 c 13.3 d 12.8 d 15.0 d 15.0 c 15.3 d 16.0d 16.8 c 35 14.5 d 11.3 d 10.8 e 12.0 d 13.3 c 13.8 d 13.5 s 14.8 s 15.0 d 42 10.3 e 10.5 de 10.3 cf 11.. a 12.8 f 12.3 s 13.0 e 14.3 e - 49 9.0 f 9.8 ef 10.0 f 10.8 e 11.8 f 12.0 c 12.8 e 14.0 e — 70 8.0 g 9.0 f 9.8 f 11.0c - ' - - - - wmwmmmumwMaWMWeMmWmemmm trestrnent. -Notreaunentswereapplied. 'VduswiMacdunmnufoflowedbyhnasmeommonmeWydiffuemmmeSQ:levelofprobability. smallerorequaltothemeanFLNforunvemalized controls (Table 1). Average FLNs for the 7-d vernal- ization treatments were not significantly different fiorn the average of unvemalized controls in any of the age treamtents. All vemalization treatrrtents equal to or longer than 14 d reduced FLN relative to unvemalized controls in one or more age treatments. Response to vemalization reached a plateau in the LTSZ to LTS7 treatments. Plants at and after the stage where the vemalization response began to plateau were vemalization insensitive because additional vemaliza- tion did not reduce FLN. Cells in Table l with bold FLN values are the points where a response plateau became evident as vemalization duration was extended within an age treatment. The number of leaves remain- ing to emerge after vemalization was remarkably con- sistent (6.3 i 0.5) among plants from those treatments. The minimum vemalization duration required to reach a stage of vemalization insensitivity decreased as plant age at the onset of vemalization increased. Linear regression of actual leaf stage ( including leaves emerg- ing during vemalization) versus days of vemalization for plants from treaunents that brought the plants to the onsets of the response plateaus was significant (r2 -0.73). Whether that linearity also applied to plants that were not yet vemalization insensitive was answered by assuming. for conditions of nonlimiting photoperiod. that a plant that had flowered became insensitive to vemalization when it had six leaves left toemerge.‘1'hatpointisestimatedforourtreatments byaverageFLNminus6.soaveragevaluesofFLN-6 were plotted versus days of vemalization (Fig. I). Treatments with fewer than six leaves emerging after vemalization were assumed to have passed the point of vemalization insensitivity during treatment and were excluded from the plot. Linear regression (s10pe - -0.244 leaves/day. Y-intercept- 15.7 leaves) of the observed values was significant (1‘2 - 0.89). The Y-intereept of the regression in Fig. l is the predicted age of an unvemalized plant when it reaches a developmental state equivalent to that of plants at the vemalization insensitive points. i.e.. bold values in Table 1. It was expected that plants at that develop- mental stage would have approximately six more leaves appear prior to heading. because that was a com- mon attribute of those that just became vemalization insensitive. In fact. FLN of unvemalized plants was 20.8. which is very close to the predicted value of 21 .7 (derived from Fig. 1's regression equation). The slope of the regression in Fig. 1 can be inter- pretedtomeanthattheleafstageattheonsetofver- nalization insensitivity is reduced by 0.244 leaves for each additional day of vemalization exposure. Put another way, the dtuation of vemalization required to reach vemalization insensitivity is reduced by 4.1 days when plant age is increased by one leaf. This linear relationship implies that accumulated age and vernali- zation days independently contribute to a plant’s attain- S.-1'. Wang ad. lFt'dd Crawlers-eh 41 (1995) 91-100 16 14- 12’ 1O 1C) 009040-50 CD :1, a 5. 4. 2. 0o {b 25 18 16 1 14 12 10 FLN-6 30 50 6O 70 Vdoys Fig.1.MMM-6Nflndmflmmhm3fljhmmmmmmmwith fewathanaixlumewgingaftervunahndonflhehnecmgresaionhnebfiuedu: (FLN-6)-15.7-0.244 wai‘be 330.89. caboaolaa 20 1O ment of vemalization insensitivity. A series of combinations of vemalization days and plant ages can result in vemalization insensitivity. Assuming that the Y-intercept is estimated by the RN of unvemalized plants minus six. then a general- ization of the relationship portrayed in Fig. 1 is: l'i-(FO-6)-m.v oronrearrangement. (Fa-6)-Li+flTv (l) (2) 30 40 50 60 70 Ways Fig. 2. RelationshipbetweenFLN-banddaysofvemalintiondurationforAugustaMdataineludeallagetreatrnentsexceptthoaewith fewerthansix leaves ernergingaftervemalization.‘l'he linearregressionlineisfittedas: (FLN-6)- 17.0-0.236 V.,..The Fis0.85. whereAistheleafstageattheonsetofvernalization insensitivity. F0 is the final leaf number with no ver- nalization. T, is the days of vemalization. and B. which represents the “exchange rate" between leaf numbers and vemalization days. is the absolute value of the slope of the linear regression line in Fig. l. Eq.2indicatesthatap1antbecornesvema1ization insensitivewhenthesurnofU) thecurrentleafstage and (2) the leaf equivalents gained by vemalization days (i.e.. the product ofthe days of vemalization and the leaf number/vemalization days exchange rate) is 11 96 S.-Y. Wag era]. lFr‘eldCrupsRaaarchu (1995191-1w equaltotheFlNofunvernalizedplantsminussix.One shouldbearinrnindthatleavescontinuetogrowand develop even dtuingvemalizationtreatmenLTheleaf emergenceratewas0.029t00.0361eavesperdayin ourgrowth chamber. litherelationshipbetween vemalization days and leafstageatvernalizationinsensitivityislinear.then plantagedoesnotaltertheeffeetofvemalizationas long as the plant is still responsive to vemalization. The wide range of plant ages at the onset of vemalization employed in this study allows confirmation of that assumption. Regressions of vemalization days versus FLN-6wereconductedwithineachofthenineage treatments. That analysis compares the effects of 7 to 70 d of vemalization applied at leaf tip stages ranging from zero to eight. In all age groups. r2 values were greater than 0.91 (data not shown). confirming that plant age during vemalization had little effect on the interrelationships implied in Eq. 2. 3.2. Augusta Average FLN for unvemalized plants was 21.7 :1; 1.0. Trends in vemalization effects were similar to those for Pioneer 2548 except that distinctive pla« teaus were observed only for age treatments LTSB. LTSS and LTS6. The relationship between FLN-6 and vemalization days for treatments which had six or more leaves appearing after vemalization seems non- linear (Frg. 2). The linear regression of the points in Fig. 2. however. was significant ( r: - 0.85). The fitted line had a Y-intercept of 17.0 leaves and slope of - 0.236 leaves/ day. The expected Y-intercept. derived from the FLN of unvemalized plants. was 15.7 leaves. 4. Discussion Vemalization insensitivity. evidenced by plateaus in the response of plants to increased duration of vemal- ization. was observed in a number of other studies (Gun. 1957; Ahrens and Loomis. 1963'. Halloran. 1977: Berry et al.. 1980; Flood and Halloran. 1984: Jedel et al.. 1986: Kato and Yamagata. 1988). The number of days of vemalization required to achieve vemalization insensitivity has sometimes been consid- ered a genotype's “vemalization requirement" (Kato andYamashita. l991).1ialloran ( 1977) referredtothe duration of vermlization needed to attain vernalizationinsensitivityastheammrntofvernaliza- tionrequiredtosatisfyaplant's vemalizationresponae. Om'findingisthatthenurnberofdaysofvernalization neededtoreachinsensitivitychangeswith plantage. expr'essedas leafstage. as well as with genotype. Severalsutdiescanbeinterpretedtoindicatethatthe accumulated thermal time between floral initiation and flowering is constant (Halloran and Pennell. 1982; Flood and Halloran. 1984: Griffiths and Lyndon. 1985). It is reasonable to assume that other phenolog- ical events coupled to floral initiation. such as the pro- posed state of vernalintion insensitivity. would also show consistency as to its timing relative to flowering. The number of leaves remaining to emerge in plants that had just become vemalization insensitive was aboutsixforPioneer2548. Thegood fit ofthe Augusta data to Eq. 2 indirectly confirms that Augusta also exhibits a constant number of leaves emerging after attainment of vemalization insensitivity. Support for this concept can be found through reinterpretation of several other published reports. Hoogendoom ( 1985) reported that the average final leaf number for a range of wheat lines subjected to 8 weeks of imbibed seed vemalization at 5°C was 6 to 10 leaves. Two cultivars classified as “super-winter" wheats had mean FLNs of 8.7 and 9.5. By our model. those plants became vemalization insensitive at about the two- to three-leaf stage. which is a reasonable approximation of the age they would have attained at the end of vemalization at that temperature. Similar analysis of data from Griffiths and Lyndon (1985) and Miao et al. (1992) alsotends to confirm that the number of leaves emerging after vemalization insensitivity under long-day conditions is six. it is likely that photoperiod can influence the number of leaves emerging between vemalization insensitivity andfiowering. In thedataofLevyandPeterson ( 1972). the average FLN of the winter wheat Triumph given a 56 days of vemalization treatment changed from 7.0 to 13.7 when the post-vemalization photoperiod was decreased from 17 to 9 h. The plants with seven leaves were vemalization insensitive. because further reduc- tion in their leaf number was unlikely. so transfer to a shorter daylength must have increased the number of leaves emerging after vemalization insensitivity. This means that vemalization insensitivity is not equivalent to floral initiation. The period between vemalization 12 61-11 Wag eral. [Field Cm m 41 (1995191400 97 insensitivityandfloral initiationis veryprobablystable fora given genotype grown inconstantpost-vemali- utionconditions.however.becausetbatisthemost likelyexplanationoftheconstancyofthenumberof leaves emerging after vemalization insensitivity. The-rate of apical primordia production is usually greater than the rate of leaf appearance during vegeta- tivegrowth (Kirby. 1990). AsarestrlLthenumberof pimordia acropetal to the emerging leaf increases as leafnumberincreases.‘1'hemecbanismbywhicbaplurt fixes the number of leaves emerging after vemalizatim insensitivity must therefore involve a zone of deter- minationwheretbeemergingleafandtbefivetosix primordiaandleaves immediately youngerthan itare committed to becoming leaves. while younger primor- diaarelabileandwillbecomeeitherspikeletsorleaves dependingon when floral initiationtakesplace. IanCt. Griffiths and Lyndon (1985) and Rawson and Ljac (1993) showedtbatprimor'diacanbelabile.'l'hecon- stancy of the number of leaves emerging after vernal- ization insensitivity or floral initiation could be of adaptive significance. because the additional acropetal primordia of plants with more emerged leaves would increase the duration of the period from floral initiation to flowering if all primordia did become leaves. That strategywouldmakethetimefromfloralinitiationto flowering vary with the number of leaves at floral ini- tiation. It is interesting to note that our estimate of number of leaves emerging at vemalization insensitivity under long-day conditions is close to five to seven leaves. which is also the number of leaves either postulated or observed to be the minimum number of leaves possible in wheat ( Purvis. 1934; Aitken. 1966: Miao et al.. 1992). Is this accidental or is there some basic under- lying mechanism linking these phenomena? Minimum leaf number is probably related to the number of inter- nodes that elongate in wheat. This value stays very constant at five (only a few could be four or six). irrespective of the number of leaf nodes actually present ontbestem. lnordertoconstructastem with fourto six internodes. the plant must develop at least that many leaves. It therefore appears that wheat has adopted devel- opmental strategies that enable it to avoid having fewer than four to six primordia committed to becoming leaves. and to maintain a constant number of leaves emerging after floral initiation. Perhaps the postulated aoneofdeterminationfromtheemergingleaftothe5th or 6th younger primordium serves both to prevent initiation. and to prevent primordia within the zone from becoming spikelets. The result would be the obaervedsimilaritybetweenminimumleafnumberand the number of leaves emerging after floral initiation under long photoperiods. How the plant identifies the emergingleaformaintainstbepostulatedzoneofdem- mination is not clear. A similar and possibly related featisaccomplishedwhentbewbeatplantacctuately identifiesthe4thor5thintemodebelowthelowest spikeletasthefirstintemodetoelongate.irrespective ofthetotalnumberofnodesonthestematthattime. Neitheroftbewinterwbeatssmdiedhererequired vemalization in order for flowering to take place. Sim- ilarresultscanbefoundinnumerousreports(Gott. 1957;AhrensandLoomis. 1963;Ledent.1980;David- son et al.. 1985; Masle et al.. 1989; Hay and Kirby. 1991). Final leaf numbers of unvemalized plants grown under long photoperiods were consistent for individuallinesandrangedfrom7t023inasetof springandwinterwheatsadaptedtoawiderangeof conditions (Wang et al.. 1995. in press). Final leaf numbersintherangeonOtoZl havebeenreported for unvemalized plants of otherwinter wheats (Cooper. 1956; Riddell and Gries. 1958; Ahrens and Loomis. 1963; Aitken. 1966). The ability of winter wheats to flower without vemalization is evidence that plant age can substitute for vemalization. A logical corollary of that view is that vemalization can substitute for plant age as a determinant of time of flowering. A state of vemalization insensitivity is also probably reached in unvemalized winter wheats at or prior to the onset of floral initiation. when the plant has only six more leaves left to emerge (under long photoperiods). That means that unvemalized plants would become insensitive to vemalization when they had a leaf number equal to their maximum minus six. That hypothesis was not directly tested. but evidence to support it can be found in the literature. Chujo (1966) presented data that can be interpreted to showthatplants with more laavesthan the FLN for unvemalized plants minus six could not respond to vemalization. while younger plants could. Asimilarinterpretationcanbeappliedtodanfrom Gott (1957). The relative linearity of the relationship between FLN-6 and vemalization days. and the good fit 13 9s s-r. Wag oral. lFieldCropsResaarehfl (1995) 91-100 between the predicted and scars] Y-intercepts of the fitted regression lines for those data indirectly confirm thataboutsixleavesremsintoemergeatthestageof vemalization insensitivity. irrespective of the plant age atwhicbthatstateisattained. Similarly.thenumberof vemalization days experienced by a plant does not influence the number of leaves remaining to emerge oncevemalizationinsensitivityisattainedFigs. land 2 demonstrate that the interchangeability of vemaliza- tiondaysandplantageiseffectiveacrosstherangeof plant ages and vemalization durations. at least within thebomithriessetbymeX-andY-intercepts.Ledent (1980) proposeda linearrelationship betweenageand vemalization days. Expressing the time dimension in days. he concluded that one day of cold exposure of “incompletely vemalized plants". i.e.. plants not yet insensitive to vemalization. would reduce the sowing to anthesis time by 2.6 days. No leaf-numberdata were presented so a direct comparison with our results is not possible. However. a linear relationship between ver- nalization days and plant age is a clear conclusion of Ledent’s work. Halloran (1977) also found an almost linear reduction in leaf number for spring wheats with increased duration of vemalization. The biological implication of Eq. 2 is that accumu- lation ( or depletion) of the products of a process whose rate is tied to the rate of leaf emergence is accelerated by low temperatures. The products of that process even- tually reach a level that enables floral initiation. The fact that vemalization insensitivity is probably not equivalent to floral initiation suggests either that a sec- ond process is initiated at the onset of vemalization insensitivity or that the contributions from vemaliza- tion are suppressed beyond that point. The second proc- ess clearly can involve photoperiod. although photoperiod effects during the period of vemalization response should not be ruled out. Plants of different age and vemalization duration treatments in the present studies had varying photoperiod regimes because of the difference in daylength between the growth chamber and the greenhouses. That probably .did not influence the results greatly. because there was no increase in FLN from additional vemalization days in those cases where response plateaus were evident. As indicated in the introduction. the literature is con- sistent that older plants are less responsive to vemali- zation than younger plants. That may seem a contradiction to our conclusion that the interchangea- bilityofvemalizationandplantageisindependentof theageofplantsdruingvemalization (uptotheonset ofvernalization insensitivity). The apparent conflict is resolved. however. by application of a common vocab- ularytoresultsofthosestudies.1nallthecaseswbere older plants were less responsive. “less responsive“ was used to mean either requiring fewer vemalization daysinm'dertoflowerinapredeterminedtimeperiod. or showing lesstotal reduction in either final leaf num- berordaystoflower.‘1'hatisexactlytherelationship portrayedbyEq. 2.sothereisnoconflictbetweenour viewandthe literatureonplantageand vemalization. Departure fiom the linearity between vemalization daysandFLN-6isapparentforboth Pioneer2548 and Augusta where plants were vemalized for only 7 days. Average FLNs for those treatments were often not significantly different from those for the unvemal- izedcontrolsJedeletal. (1986) alsonotedalagperiod prior to the initial response to vemalization with some spring wheats. Ahrens and Loomis (1963). Halloran (1977). Ledent (1980). Davidson et al. (1985). and Griffiths and Lyndon ( 1985) all found similar results. This lag phase would be most critical in situation where totalvemalizationtirnewasshortorwherethetotal vemalization days were large but individual episodes of vemalization were brief. The key experimental techniques that enabled devel- opment ofthese concepts were use of leaf numbers as the measure of vemalization response and employment of plant age at the onset of vemalization as a treaunent factor. Analysis of our data in terms of calendar days reveals no biologically meaningful trends such as the FLN vemalization response plateaus (and conse- quently vemalization insensitivity). or the remaining leaf number at vemalization insensitivity. Omission of the age component of the treatment combinations would have also obscured what appear to be significant relationships. The concepts developed here have utility in wheat modeling. The key genetic coefficients for vemalizao tion response would be the Y-intercept of Eqs. 1 and 2. and the slope of the line. The onset of vemalization insensitivity during crop simulation can be determined by maintaining a running total for both accumulated vemalization days (7;) and accumulated phyllochrons. When the sum of the phyllochrons and vemalization day leaf equivalents (BL) equals that genotype’s Y- intercept. vemalization insensitivity has been reached. 14 3.4: wmaaL/mu cmmu (1995;91-100 99 Furtherresearcbisneededtocbaracterizetheeffectsof photoperiod and other factors on the duration of the period between vemalization insensitivity and flower- mg. Theconstancyofthe numberofleaves remainingat attainment of vemalization insensitivity within an savi- ronment enables study of apexes as they change from a state of vemalization sensitivity to a state of vemali- zation insensitivity. Comparative biochemical and ultrastructural characterization of vemalization sensi- tive and insensitive apexes with varying numbers of total primordia could lead to a deeper understanding of the underlying processes leading up to floral initiation. Acknowledgements Grateful acknowledgement is made to Dr. R.D. Heins for his helpful discussions and constructive crit- icism of the manuscript. We thank Mr. D. Glenn for his assistance in executing theseexperiments, and Dr. B. Fowler for his comments on an early draft of the manuscript. We acknowledge support from Japanese Government Scholarship funds given to S.-Y. Wang through CIMMYT. References Ahrens. J.F. and Loomis. W.E.. 1963. Floral induction and devel- opment in winter wheat. Crop Sci.. 3: 463-466. Aitken. Y.. 1966. Flower initiation in relation to maturity in crop plants. In. The flowering response of early and late cereal vari- eties to Australian environments. Aust. J. Agric. Res.. 17: 1-15. Berry. GJ.. Salisbury. P.A. and Halloran. GM. 1980. Expression of vemalization genes in near-isogenic wheat lines: duration of vemalization period. Ann. Bot. 46: 235-241. Chouard, P.. 1960. Vemalization and its relations to dormancy. Ann. Rev. Plant Physiol.. 11: 191-238. Chujo. 11.. 1966. Difference in vemalization effect in wheat under various temperatures. Crop Sci. Soc. Jpn. Proc.. 35: 177-186. Cooper. J.P.. 1956. Developmental analysis of population in cereals and herbage grasses. 1. Methods and techniques. J. Agric. Sci.. Cambridge. 47: 262. Davidson. J.L.. Christian. K.R.. Jones. DB and Bremner. P.M.. 1985. Response ofwheat to vemalization and photoperiod. Aust. J. Agric. Res.. 36: 347-359. Evans. LT.. Wardlaw. LF. and Fischer. R.A.. 1975. Wheat. 1n: LT. Evans (Editor). Crop Physiology. Cambridge University Press. Cambridge. pp. 101-149. Flood.R.G.and1-1alloran.G.M.. 1984.Thenanrreandduraticnof geneaetionforvermlintionresporneinwheacAnn.Bot..53: 363-368. FMRG.and1-1alloran.G.M.. 1986.0eneticsusdphysiologyof vemalization response in wheat. Adv. Agrun.. 39: 87-125. Gotob.T.. 1976. Snrdiesonvarietaldifferenminvemalization requirement in wheat. (in Japanese. with English summary.) Jpn. J. Breed. 26: 307-327. 000.143.. 1957. Vernalizationofgreenplantsofwinterwbeat. Nature. 180: 714-715. Griffiths. FEW. and Lyndon. R.F.. 1985. The effects of vemaliza- tiononthegrowthofthewhessshootapex.Ann.Bot..56: 501- 511. . llalloran. GM.. 1977. Developmental basis of maturity differences in spring wheat. Agron. J.. 69: 899—902. Halloran. GM. and Pennell. A.M.. 1982. Duration and rate of devel- opmentpbasesinwheatintwoenvironments.Ann.Bot..49: 115-121. Hay. KK.M. and Kirby. E.J.M.. 1991. Convergence and synchrony: A review of the coordination of development in wheat. Aust. J. Agric. Res.. 42: 661-700. licogendoorn.J.. 1984. Acomparisonof different vemalization tech- niques in wheat (Tr-trim «strewn L). J. Plant Physiol.. 116: 10-20. Hoogendoorn. J.. 1985. The physiology of variation in the time of ear emergence among wheat varieties from different regions of the world. Eupbytica. 34: 599-571. Jedel. P.E.. Evans. LE and Scarth. R.. 1986. Vemalization responses of a selected group of spring wheat (Triricran aesrivrm l...) ctrl- tivars. Can. J. Plant Sci.. 66: 1-9. Kato. K. and Yamagata. 11.. 1988. Method for evaluation of chilling requirement and narrow-sense earliness of wheat cultivars. Jpn. J. Breed. 38: 172-186. Kato. K.and Yamashita.S..1991.Varietalvariationinphotoperiodic response. chilling requirement and narrow-sense earliness and their relation to heading time in wheat (Trin'cam aestivum L). Jpn. J. Breed. 41: 475-484. Kirby. E.J.M.. 1990. Co-ordination of leaf emergence and leaf and spikelet primordium initiation in wheat. Field Crops Res.. 25: 253-264. Krelrule. J.. 1987. Vemalization in wheat. in: J.G. Athenon (Editor). Manipulation of Flowering. Butterworths. London. pp. 159-169. Lang. A.. 1965. Physiology of flower initiation. in: W. Ruhland (Editor). Encyclopedia of Plant Physiology. Vol. XV /1. Springer-Verlag. Berlin. pp. 1380-1536. Ledent. J .F.. 1980. Vemalization and anthesis in a collection of wheat cultivars (Triricrarr aestivunr L.). a quantitative study in con- trolled environment. Bull. Soc. R. Bot. Belg.. 112: 186-192. Levy. J. and Peterson. ML. 1972. Responses of spring wheats to vemalization and photoperiod. Crop Sci.. 12: 487-490. Martinic. 2.. 1973. Vemalization and photoperiodism of common wheatasrelatedtothegeneralandspecificadaptabilityofvari- eties. 1n: R.O. Slatyer (Editor). Plant Response to Climate Fac- tors. Proc. Uppsala Symp. UNESCO. Paris. pp. 153—163. Masle. J.. Doussinault. G. and Sun. B..'1989. Response of wheat genotypestotemperanrreandphotoperiodinnannalconditions. Crop Sci.. 29: 712-721. 15 100 I sac 111-; 1rd. [Field Couple-read (1995) 91-100 Miao. G.-Y.. flung. Y.-T.. Halt. Y.-S.. Yin. J. and Wu. S.-Y.. 1992.811eetsofverulia-fea-dphateperiodaaleat‘aumber «anwunthmmmmim Agron. Sin. 16: 321-330. mm. 1985.1“n'n'crsa.ln:A.l-I.1‘1alevy(Editor).CJlC11-rd- baakofFlowering.Vol.1V.CRCPress.BocaRmn.FLpp. 418-443. Pugsley.A.T..1971.AgenricanalysisoflheWMitof MinwbuAml.Agric.Res..22:21-23. MON. 1934.Anana1ysisoftbe'urfluenceofme (hiring germimion on the subsequent development ofutain winterwbeatcerealsanditsrelationtotbeefiectofleagthof day. An. Bot. 48: 919-955. Purvis.O.N.. 1961.1hepbysiologicalarnlysisofvernalianicndn: W.Rubland (Editor).Eneyclopediaoflethysiology.Vol. XVI. Springer-Venn. Berlin. pp. 76-122. Rawson. 11M. and Zajac. M.. 1993. Effects ofbigberurrpermes. [isotoperiodandseedvernaliznionondevelopneatintwoqling wheats.Aust.J. Plant Pbysiol..20.211-222. Riddell.J.A.andGries.G.A..1958.Developmeruofspringwbeat: III.Terrrperanrreofmatrnationurdageofaeedsasfactusinflu- mumnmmhmm-m. Richie.J.T.udNeSafl.DS~l991.Tw-dcrcpdevel- cpareat.1n:1.T.kisdie-dRJ.11-th(&isun).uadelmg MudeSymASA-CSSA-SSSAMWLppJ- 29. SASW.1991.SASISTATasersguide.Rele-e6.03ad.SAS Mac-Mm. Shea-.11.C.aadMaseia1L. lulvmcrinue enhyosofwimerwbenmfiqirytiea. 36:161-165. Wag. S.-Y.. Wad. KW. Ritchie. J.T.. Fischer. ILA. and Schul— mu. 1995.Vernalifianinwbeat.ll.Wof vanaliauiaarupcueofadivaaesetofgendymfieldow Res..-pres. MEDIAmdScMIjeal. 1992 Vernalianicnoferabry- cumiculhsfiumeemh-yadwmermoopSci. 32:78-80. Wbye.R.O..1948.11istcrycfreaelchinvernalintica.1n:A.E. manowmtumxvmmm paindim-ASynrpcdmmoaicaBa-nanM pp.1-38. Section II Vemalization In Wheat 11. Genetic Variability For The Interchangeability Of Plant Age And Vemalization Duration 16 17 l ,' ELSVIER m Crops ReaearchOO (1995) ooo-ooo ResearCh Vemalization in wheat H. Genetic variability for the interchangeability of plant age and vemalization duration Shi-Ying Wang ‘, Richard W. Ward ""‘, Joe T. Ritchie ‘, Ralph Anthony Fischer b, Urs Schulthess " ' Department of Crop and Soil Sciences. Michigan State University. East lasing. Ml 48824. USA " Wheat Program. International Maize and Wheat Improvement Center (CIMMYT). Mexico. D.I-'.. Mexico Received 15 May 1995; acceptedZOSepterriJer 1995 Abstract Differences in response to vemalization in wheat (Triticron aestivum L.) were quantified through controlled enviromnent experiments with 26 lines with diverse geographical origins. Vemalization treaunents of 0 to 56 d were applied to plants at their first leaf stage. All plants headed irrespective of duration of vemalization treatment. Vemalization response was assessed through the change of final leaf number (FLN) on the main stem at heading. Five lines did not respond to vemalization. FLN for vemalization-sensitive lines generally decreased to a minimum as days of vemalization treatment increased. Plants at and after the stage where additional vemalization did not reduce FLN were vemalization insensitive. The quantitative features of this vemalization response. up to the point of insensitivity. were characterized with a linear regression: (F ,- - 6) - a — 3T... where F, is FLN observed for a particular vemalization treatment. 7‘. is the time in days of that vemalization treatment. and a and B are the Y-intercept and the slope of the regression. respectively. This model fitted the experimental results well. The parameters a and B varied among lines. and are useful for quantifying vemalization response in wheat. The implication of each parameter can be interpreted biologically: a is the “changeable number of leaves". i.e.. how many leaves can be potentially decreased by vemalization treatment. and B represents the “exchange rate” between leaf numbers and vermlization days. i.e.. how many leaves can be reduced by one day of vemalization treatment. Keywords: Leaf number. Modelling: Tn’tt‘cum: Vemalization: Wheat 1. Introduction Wheat (Triticum aestivum L.) is grown across a wide range of agrogeographical regions from the equa- tor to greater than 60° latitude (Briggle and Curtis. 1987). Wheat phenology varies widely depending upon the genotype. location. and date of sowing. I'Corresponding author. Fax: (+1-517) 353-3955; E-mail: 22857mgr@ibmcl.msu.edu 0378-4290/95/50950 O 1995 Elsevier Science B.V. All rights reserved 550/0378-4290( 95 )00076-3 Response to vemalization is one of the most important factors affecting wheat’s environmental adaptation. At least five loci involved in the control of the response to vemalization have been identified (Pugsley. 1971. 1972: Law and Scarth. 1984). Some studies have reported that vemalization response is polygenically controlled (as reviewed by Flood and I-lalloran. 1986) . Despite this rather detailed genetic knowledge. there is no system in general use for the quantification of a 18 2 SJ. WargetaL/FieldCrapsReaearehwfl993)M-M wheat line’s vemalization response. The universally used spring/ winter classification system relates more to the sowing system to which a wheat line is adapted than to the specific nature of a line’s response to ver— nalization. For instance. many wheats that are adapted to spring sowing (so-called spring wheat) can respond to vemalization (Levy and Peterson. 1972; Wall and Cartwright. 1974; Halloran. 1977; Jedel et al.. 1986). and wheats adapted to fall sowing (so-called winter wheat) vary markedly in their response to vemalization ( Gotoh. 1976; Ledent. 1980; Miao et al.. 1992) . More- over. some wheats included in the International Winter Wheat Performance Nursery had little or no response to vemalization (Gotoh. 1975 ) . The lack of a workable system for quantifying wheats for vemalization response stems in part from the lack of a general con- ceptual model of this complex phenomenon. We recently reported that plant age and vemalization duration are related as follows (Wang et al.. 1995): where F o is the final leaf number of unvemalized plants. L. is the leaf stage at the onset of vemalization insen- sitivity. T. is the days of vemalization treatment. and B represents the “exchange rate” between leaf num- bers and vemalization days. Eq. 1 indicates that a wheat plant becomes vemalization insensitive when the sum of the current leaf stage and the leaf equivalents gained by vemalization days ( i.e.. the product of the days of vemalization and the leaf number/vemalization days exchange rate) is equal to the FLN of unvemalized plants minus six. A key premise underlying Eq. I is that plants will emerge six more leaves after the onset of vemalization insensitivity. The leaf stage at which a plant with an emerged flag leaf reached vemalization insensitivity is consequently estimated by FLN minus six. lf leaf stage at the onset of vemalization insensitivity (Y) is plotted against vemalization days (X). then Eq. 1 implies a linear relationship as follows: (Ft-6)‘a-BTV (2) where F ,- is the FLN observed for a particular vemali- zation treatment. a is the Y-intercept and is an estimate of F" minus six. and B and T. are as described for Eq. 1. Eq. 2 applies only up to the point of vemalization insensitivity. as judged retrospectively by the attain- Tablet mmwnummmmum ofcrigin Series Line Country of origin [nitride ('N) Type 1 Seri 82 Mexico 25 Spring (CMIYT) Pitic 62 Mexico 25 Spring (CIMMYT) Yecora Rojo Mexico 25 Spring (CIMMYT) Sonata 64 Mexico 25 Spring (CIMMYT) Gleason Mexico 25 Spring (CIMMYT) FL 303 USA (Florida) 27 Winter Phoenix USA (California) 30 Winter Ana USA (Califania) 30 Spring MO 298 USA (Missouri) 37 Winter WW USA (Virginia) 37 Winter MD 286-21 USA (Maryland) 38 Winter CA 841 Clans (Beijing) 39 Winter Excel USA (Ohio) 39 Winter Cluk USA (Indiana) 40 Winter Pioneer 2548 USA (Indiana) 40 Winter Augmta USA (Michigan) 42 Winter NY 73] 164w USA (New York) 42 Winter Tincher Calida 52 Spring 2 Xingrnai China (Hunan) 28 Winter Shun-i China (Jiangshu) 33 Winter 215953 China (Shandang) 36 Winter Ji 84—5418 China (Hebei) 38 Wilmer CA 8686 China (Beijing) 39 Winter CA 8646 China (Be-iii!!!) 39 Winter ling 411 China (Beijing) 39 Winter Jingnong 86-74 China (Beijing) 39 Winter ment of a minimum in FLN. This report explores the utility of Eq. 2 in development of vemalization response parameters for a set of wheat lines with diverse geographical origins. 2. Material and methods The 26 wheat ( Triticum aestivum L.) lines used in this study are listed in Table 1 along with their country and latitude of origin. Seeds were sown in lO—cm diam- eter clay pots in a greenhouse soil mixture of 5 loam : 2 peat : 3 sand (u/v/v). Plants were grown in a green- 19 SJ. WangetoL/Ft‘eldeRemrrthMS)M-M 3 house except during the vemalization treaunents. whichwereappliedtoseedlingsatthefirstleafstage (7 d after sowing). The greenhouse was maintained at about 20°C and the natural photoperiod was extended to 20 h by high-pressure sodium lamps that delivered a photosynthetic photon flux of approximately 200 mol m ’ ' s " ' at pot level. The vemalization chamber had an 8-h photoperiod with a photon flux of 200 umol rn'I s’l provikd by fluorescent and incandescent lamps. Temperatures were 5°C and 2°C during the light and dark periods. respectively. In experiment 1. seedlings of 18 lines (series 1 in Table 1) were vemalized for 0. 7. 21. 28. or 42 d. In experiment 2. seedlings of eight lines (series 2 in Table l) were vemalized for 0. 16. 28. 42. or 56 d. The experiments were terminated when all plants reached maturity. Five seedlings were kept in each pot afterplant emer- gence. After appearance of the fifth leaf tip. plants were thinned again. and only two well-established seedlings were left in each pot. Measurements were made on the main stems of the plants in each of two pots for each treatment. except for the zero vemalization treatment which had four pots. Final leaf number (FLN) was recorded as the total number of leaves on the main stem at heading. A separate linear regression analysis was performed for each wheat line. Values of FLN minus six were used as dependent variables and days of vemalization treatment were used as independent variables. Plants at and after the stage that additional vemalization did not reduce FLN were vemalization insensitive. The regres- sions were made after discarding points in the region of insensitivity. 3. Results and discussion All 26 wheat lines headed even in the absence of low temperature vemalization. This confirms that there is not an absolute vemalization requirement in wheat's life cycle ( Martinic. 1973; Ledent. 1980; Pinthus. 1985; Miao etal.. 1988; Wang et al.. 1995). Two quan- titative features of the vemalization response were evi- dent in terms of FLN change (Table 2). Firstly. MS of a line generally decreased to a minimum as days of vemalization treatment increased. Secondly. mean FLNs differed markedly among the lines under the samevernaliaationtreatrnentsflhiswasespeciallytrue intheunvernalizedcontrols.wheremeanFLNwasas smallas7.0forYecoraRojo andas largeasZZ.7 for NY 731 l6-4w. a range of 15.7 leaves. 'Ihat range diminishedas vemalization duration increased. Aftera 42-d vemalization treatment. the range of FLNs among all 26 lines became 7.7 leaves. The regression analysis was not performed for five lines whose FLNs changed only about one leaf among all treatments. Three of these lines. Yecora Rojo (Davidson et al.. 1985; Jedel et al.. 1986). Sonora 64 (Levyand Peterson. 1972; WallandCartwright. 1974) andThatcher (McIntosh. 1973; Syme. 1973; Flood and Halloran. 1984; Davidson et al.. 1985; Penrose et al.. 1991) have been studied extensively. and were reported not to respond to vemalization treatment. Our results coincide with the previous conclusions. The other two lines. Ana and Seri 82. were developed in Californiaand Mexico. respectively and both have been described as not responding to vemalization. FLN of Sonora 64 changed little with treatment whereas that of Thatcher increased from 7.3 to 8.8 as days of ver- nalization treatment increased from 0 to 42 d. This may be because Sonora 64 is insensitive to photoperiod (Levy and Peterson. 1972) where” Thatcher is sen- sitive (Halloran and Pennell. 1982; Crofts et al.. 1984). In the current study. photoperiod during the vemali- zation treatment was only 8 h. Mean FLNs of the remaining 21 lines were subjected to separate linear regression analyses as described. The coefficients of determination (r’) of this model were greater than 0.80 for most lines (Table 3). The resul- tant estimates ofthe parameters a and B are also listed in Table 3. Both a and B are useful parameters for quantifying vemalization response in wheat. The implication of each parameter can be interpreted biologically. The slope. B. represents the “exchange rate" between leaf numbers and vemalization days. i.e.. how many leaves can be reduced by one day of vemalization treatment. The meaning of B is clarified by ”B. which represents how many days of vemalization treatment are neces- sary to reduce the leaf number by one. The Y-intercept. a. is the “changeable number of leaves". i.e.. how many leaves can be potentially decreased by vemali- zation treatment. Alpha is also biologically equivalent to the leaf number of an unvemalized plant at the onset of vemalization insensitivity. Accordingly. the mean 20 4 S.«l'. wmuu/chmkcmwumlm Title 2 Mean FIN in various lines at different vemalization cements Line‘ Days of mediation uenrnear 0 7 16 21 28 42 56 l Yecora Rojo 7.0.. 7.0. -‘ 7.3a 7.5a 7.8a - 2 Sonora 64 7.3a 71h - 71h 7.3a 7.3a - 3 Thacher 73c son - 8.0a 81h 8.8a - 4 Ann 8.1]: 8.3b - 8.11: 8.1» 9.0a - 5 Seri 82 8.8a 81b - 8.1!: 8.11) 83d) - 6 Glenruon 10.8a 9.0a - 8.5bc 8.1!: 91!) - 7 Shunni 1 1.3a - 9 (I: - 8.8b 9 (I) 9.3b 8 Pitic 62 11.5a 10.51: - 9.0c 9.0: 8 8c - 9 FL 303 l 1.5a 10.81: - 10.0c 93d 8.5a - lO ' ' 15 3a - 11.1]: - 10.0: 8.8d 9 0d 11 215953 16.0a - 11.0b - 9.8c 9 0c 9 0c 12 CA 8646 17.8a - 15.!!! - 9.5c 8.5d 9.0cd 13 ll 84-5418 18.8a - 18.0a - 11.3b 10.11) 10.0) 14 Phoenix 19.0a 18.5a - 11.515 10.0: 9.011 - 15 CA 8686 192a - 18.0a - 10.8b 9.0c 9 3b: 16 Wakefield 19 2a 185a - 13.5b 12.0c 9.3d - 17 ling 41 I 19.3a - 16.31: - 11.0c 9.8d 10.0d 18 lingnong 86-74 19.4. - 17.8b — 11.5c tons 10.0d 19 Clark 19.7a 18.8b 17.5: 16.011 12.4e - 20 Pioneer 2548 20.3a 19.80 18.“) 14.8c 11.0d - 21 CA 841 20.3a - 19m) 13.8c 11.5d - 22 Augusta 20.8a 205a 18 8b 16.5c 12.5d - 23 Excel 21.0a 19.8ab 18 (be 16.8c 13.8d - 24 MD 286-21 21.3a 20.3a 19 8a 16.9b l 1.8c - 25 MO 298 21.3a 195b 18.5b 15.8c 12.0d - 26 NY 731 l6-4w 22.71: 22.311 20.5b 18.0c 15.0d - Range 15.7 10.7 7.7 'The lines have been arranged in order of their FLNs for unvemalized plants. ”Values within a row not followed by letters in common are significantly different at the 5% level ofprobability in using the Duncan r-test: ‘No treatment is represented by -. FLN minus six for plants in the zero vemalization treat- ments was close to the a value (Table 3). The values of a: and B in general are lower in so- called spring wheats than in so—called winter wheats. Their values can be equal to or close to zero in some wheats such as the five lines identified above. which did not respond to vemalization. However. some spring-sown wheats may have the same a or B values as fall-sown wheats. For instance. a spring wheat. Pitic 62. which was reported to respond to vemalization (Levy and Peterson. 1972; Syme. 1973;Wa11 and Can- wright. 1974; Davidson et a1..‘l985; ledel et al.. 1986) . had the same a value as a winter wheat. FL 303. The B value of Pitic 62 was greater than that ofFL 303. which means that FL 303 needs more days of vemali- zation or accumulated leaves to reach vemalization insensitivity. Some lines had the same a value but dif. ferent B values (Fig. 1). or the same B but different a (Fig.2). Therefore. the vemalization responsiveness of a line is described by both the “changeable number of leaves” due to vemalization. a. and the “exchange rate" between vemalization days and plant age. B. The extrapolated regression lines in Fig. 1 and Fig. 2 indicate that FLN for a particular vemalization neat- ment is also related to temperatures applied in that vemalization treatment. The FLN will increase a tem- 21 S-l’. Wang etal. IFieldCropsReaauehw0995lm-M 5 Twle3 TheY-irlercept(a)andtheslope(fllderivedfrornlinettegression uralysisofl-‘mrinussixandthedaysofvemalintiontleatnentfor arangeofwheatlinesandresultmtestimaesofsubstituteddaysof vemalization treaunentbyoneleaf'sgrowth. llB.Ther’iscoefll- cientofdeterminaionofthelineuregression.FoisthemeanFLN oftmvernaliaedpl-u Line' F0 - 6 a B 1 l B r'1 1 Yecora Rojo" 1.0 2 Sonora 64" 1.3 3 Thatcher" 1.3 4 Anza" 2.0 5 Seri 82" 2.8 6 FL 303 5.5 5.4 0.0750 13.3 0.98 7 Shumai 5.3 5.0 0.0921 10.9 0.88 8 Glennson 4.8 4.3 0.0969 10.3 0.77 9 Pitic 62 5.5 5.4 0.1 174 85 0.99 10 Xiangmai 9.3 8.5 0.1508 6.6 0.91 1 1 215953 10.0 9.0 0.1640 6.1 0.86 12 Clark 13.7 14.1 0.1668 6.0 0.95 13 Excel 15.0 15.1 0.1670 6.0 0.99 14 NY 731 l6-4w 16.7 17.4 0.1880 5.3 0.95 15 Augusta 14.8 15.7 0.1971 5.1 0.93 16 M0 298 15.3 15.5 0.2105 4.8 0.95 17 MD 286-21 15.3 16.2 0.2138 4.7 0.87 18 CA 841 14.3 15.1 0.2181 4.6 0.84 19 Pioneer 2548 14.3 15.1 0.2240 4.5 0.94 20 ii 84-5418 12.8 13.5 0.2346 4.3 0.86 21 CA 8646 11.8 11.8 0.2389 4.2 0.93 22 ling 41 1 13.3 13.3 0.2428 4.1 0.94 23 lingnong 86-74 13.4 14.0 0.2461 4.1 0.91 24 Wakefield 13.2 13.4 0.2529 4.0 0.98 25 CA 8686 13.2 14.0 0.2700 3.7 0.89 26 Phoenix 13.0 12.9 0.2719 3.7 0.90 'The lines have been arranged in order of their B values. 'The regression analysis was nor performed because the FLNs of those lines changed only about one leaf among all treatments. perature of vemalization treatment increases. However. the values of a and B were relatively constant for a given cultivar. For instance. in the present study. Pio- neer 2548 had a and B values of 15.1 and 0.224. respec- tively. In a previous study (Wang et al.. 1995). they were 15.7 and 0.244. respectively. At 20°C and with a 16-h photoperiod. unvemalized plants of Pioneer 2548 had a mean FLN of 19.7 (Fowler et al.. 1995). That FLN is close to 20.3 that was observed in this reported experiment. These results confirm the general linearity of the relationship between plant age and vemalization days as reported by Wang et al. ( 1995). FLN data of plants not yet vemalization insensitive were used here as dependent variables in linear regressions with days of vernalintion as the independent variables. Values of a. indicating the “changeable number of leaves“. exhibited pronounced variation both among and within the lines previously classified as winter or spring types. Beta. or the ‘ ‘exchange rate" between leaf number and vemalization days also varied between and within the winter and spring types. The vemalization response of wheat can be experi- mentally quantified by using the response parameters derived from Eq. 2. Differences among lines for the parameters a and B are presumbly caused by different allelic configurations at genes influencing response to vemalization. Alpha and beta. although loosely related. appear to vary independently. indicating that some of genes conditioning variation in the vemalization days Daysolnmdzflontmdmant Fig.1.Verna1ieation response forwheat lines witha similar Y- interoept (a) but different slopes (B). The parameters ofthe lines arelistedinTable3.1hesymbolsreprewnttheobaervedvaluesalter discudingpointsintheregion of vemalization insensitivity. an N r 3 O v v . v Findlodnumberninuafl Dayaofvomainticnw Fig.2. Vemalizationresponseforwheatlineswithasimilarslope (B) butdifferent Y-intercepts(a).Thepararneter-softhelinesue listedinTable3.Thesymbolsrepresenttheobservedvaluesafier discardingpointsintheregionofvemaliznioninaensitivity. 6 S.-Y. WangetoL/FieldCropsReaearehw09951M-M vs. plant age exchange rate (B) are probably distinct from those influencing the changeable number of leaves (a) . The continuous nature of variation in both a and B indicates a large number of possible genetic states for each of the physiological mechanisms. Genet- ically. that could be caused by allelic variation at a few loci or by allelic variation at several to many loci. Acknowledgements We thank H.L. Wang and F.1-1. Sun of Chinese Acad- emy of Agricultural Sciences. 1. l-leaton of University of California. L. Talbert of Montana State University. and D. Glenn of Michigan State University for provid- ing seed. and Dr. R.D. Heins for comments on the manuscript. We acknowledge support from Japanese Government Scholarship funds given to S.-Y. Wang through CIMMYT. References Briggle. L.W. and Curtis. B.C.. 1987. ereat worldwide. In: E.G. Heyne (Editor). Wheat and Wheat lmproverrrent. Am Soc. Agron.. Madison. WI. pp. 1-31. Crofts. H.l.. Gardner. W.l(. and Velthuis. R.O., 1984. A phenological evaluation of wheat for South-western Victoria. Aust. J. Agric. Res.. 35: 521-528. Davidson. l.L.. Christian. K.R.. lones. DB. and Bremner. P.M.. 1985. Responses of wheat to vemalization ad photoperiod. Aust. J. Agric. Res.. 36: 347-359. Flood. RC. and Halloran. G.M.. 1984. Basic development rue in spring wheat. Agron. l.. 76: 260—264. Flood. RC. and l-lalloran. GM. 1986. Genetics and physiology of vemalization response in wheat. Adv. Agron.. 39: 87-125. Fowler. D.B.. Limin. A.E.. Wang. S.-Y. and Ward. R.W.. 1995. Relationship between low-temperature tolerance and vemaliza- tion requirement in wheat and rye. Can. J. Plant Sci.. in press. Gotoh. '1'.. 1975. Variation in vemalization requirements in winter wheat cultivars. Nebr. Agric. Exp. Stn. Misc. Publ.. 32: 292-297. Gotoh. T.. 1976. Studies on varietal differences in vemalization requirement in wheat. Jpn. J. Breed. 26: 307-327. Halloran. G.M.. 1977. Developmental basis of maturity differences in spring when. Agron. J.. 69: 899-902. MGM-fl MAL.1982.Dur-uionndrneofdevel- opmentphaesinwheatintwoenvironrnenrs.Ann. Bot..49: 115-121. lede1.P.E..Evans.L.E.urdScarth.R.. 1%6.Vemhzaimresponses ofanlxtedgronpofspringm (Trln'crrrnaern'vsrml...) cul- tivars.Can.l. P1urtSci..66: 1-9. Law.C.N.andSarth.R.. 1984.Geneties-1ditspotentialforunder- standingtheactionoflightinllowering. In: D. Vince-Prue (Edi- tor).LightndtheFloweringProcess.Aeadenn’cPress.London. pp. 193-209. mm. 1980. Vernalinionandltthesisinaoollectionofwhel cultivars (Triticanr aesnm 1...). aquantitnive study in corr- trolled environment. Bull. Soc. R. Bot. Belg.. 112: 186-192. Levy..l.md Peterson. ML. 1972. Responsesofspringwheusto vernalinion and photoperiod. Crop Sci..12: 487-490. Martinic. Z... 1973. Vemaliuion and photoperiodism of corrunon whenssrelatedtothepneralandspecificadaptabilityofvari- eties. In: 11.0. Slatyer (Editor). Plant Response to Climue Fac- tors. Prue. Uppsala Syn». UNESCO. Paris. pp. 153-163. McInosh.R.A..1973. Aenalogueofgenesynlrolsforwheat Proc. 4th Int. WluuGenet. Symp.. University ofMissouri. Columbia. pp. 893-937. Miao. G.-Y.. Zhang. Y.-T.. Hou. Y.-S.. Yin. 1.. Wurg. S.-Y. lid 1.i.. 1-1.-2.. 1988. A study on the types ofthe development ofwheat (Triticrrrn aestiurtrn I...) under the difletut cmdititms of tem- perunre and daylength. 1. Beijing Agric. College. 3(2): 8—17 (in Grinese. withEnglish abstract). Miao. G.-Y.. 21mg. Y.-T'.. l-lou. Y..S.. Yin. l. and Wang. S.-Y.. 1992.Eflectsofvernalintionandphotoperiodonleafnumber ofnnin stern in wheat. Acts Agron. Sin“ 16: 321-330 (in Clu- nese. with English abstract). Panose. LDJ.. Marin. 11.11. md Landers. CF. 1991. Measurement ofresponsetovemaliaationinAustr-alianwheatswithwinrer habit. Euphytica. 57: 9-17. Pincers. MJ.. 1985. Trr‘rr‘cunr. 111: All. Halevy (Editor). CRC Hand- book off-Towering. Vol. IV. CRC Press. Boca R1011. FL. pp. 418-443. Pugsley. A.T.. 1971. A poetic analysis of the spring-whiter habit of growth in wheat. Aust. J. Agric. Res.. 22: 21-23. Pugsley. A.T.. 1972. Additional genes inhibiting winter habit of growth in wheat Euphytica. 21: 547—552. Sync. 1.11.. 1973. Quantitruive control of flowering time in wheat cultivars by vemalization and photoperiod sensitivities. Aust. J. Agric. Res.. 24: 657-665.' Wall. P.C. and Cartwright. P.M.. 1974. Effects of photoperiod. tem- perauremdvemaliaationonthephenologyndspikeletnum- bers of spring wheats. Ann. Appl. Biol.. 76: 299—309. Wang. S.-Y.. Ward. R.W.. Ritchie. J.T.. Fischer. RA. and Schul- tires. 8.. 1995. Vernaliaarionin wheat. l. A model based on the interchangeability of plant age and vemalization diction. Field Crops Res.. 41: 91-100. Section III A Novel Conceptual Framework For Wheat Vemalization 23 24 A novel conceptual framework for wheat vemalization" Abstract Vemalization response in wheat so far has been characterized poorly. and less well quantified. The discrepancies and inconsistencies in the literature regarding terrninclogy. measure of response, classification of response types, operative temperatures, etc. stem in part from the lack of a general conceptual model of vemalization phenomena. Low temperature during the early development stage of wheat reduces the number of leaves emerged during that period and results in a lower final leaf number. A plant’s response to temperature during that physiological phase is its vemalization response. Temperature as an unity affects both growth and development. There is not a so-called operative temperature for wheat vemalization. The key technique for measure of vemalization response is to count the number of leaves emerged before. during, and after vemalization treatment. rather than only to calculate * The paper format was adopted for this section in accordance with “Field Crops Research”. Dr. R.A. Fischer of lntemational Maize and Wheat Improvement Center (CIMMYT) in Mexico, Drs. R.W. Ward and J.T. Ritchie of Michigan State University in the United States, Professor G.-Y. Miao of Shanxi Agricultural University in People’s Republic of China, and Dr. E.J.M. Kirby of West Australia University in Australia involved in this study. 25 calendar days or thermal time after the end of vemalization treatment. Plants become vemalization insensitive through accumulating leaf number, the leaf equivalents gained by vemalization days, or both. After vemalization insensitivity, a plant will emerge six more leaves before heading under long photoperiods. Vemalization response of wheat. up to the point of insensitivity, can be quantified experimentally by using the response parameters, or and [3, derived from a linear regression: (F. - 6) = a - BT... where F. is final leaf number observed for a particular vemalization treatment, '1'. is the time in days of that vemalization treatment. Key words: Leaf number; Model; Triticum; Vemalization; Wheat. 26 1. Introduction The three main factors which modulate the development of wheat are photoperiod. temperature and vemalization. Although vemalization in wheat was studied extensively from 1930s to 1950s (Whyte, 1948; Chouard, 1960), vemalization response so far has been characterized poorly. and less well quantified as compared with the photoperiod and the general thermal responses (Ellis et al., 1989). Except the greatest advances in understanding the genetic background (as reviewed by Flood and Halloran, 1986), the progress in this subject has been slow at least in last two decades. Physiological investigations are marked by the accumulation of phenomena (Krekule, 1987). Most of the recent work on wheat vemalization is done in the field of agronomy. Over the years crop physiologists and modellers attempt to make generalizations for wheat’s vemalization response, but they found that it is extremely difficult. This is not only because the results obtained are from a wide range of tests on different cultivars in various environments, but also because there is not a well established conceptual model. The objective here is to outline a novel conceptual framework that integrates both the recent advances and our knowledge on wheat vemalization. 2. Terminology Since Lysenko (1928, see Whyte. 1948) coined the term “vemalization” to describe the phenomena that development of winter wheat is hastened by chilling 27 germinated seeds, this term had been used with various meanings. A furtherrnost derived meaning is that any physiological action stimulating the capacity for flowering, whatever the agent (Chouard. 1960). In this sense, vemalization could be obtained by heat or cold, by long days or short days, by light or dark, by nutrition or chemical. In order to make clarification, Chouard (1960) gave the restricted definition for vemalization as: "the acquisition or acceleration of the ability to flower by a chilling treatment" A similar definition, suggested by Wnce-Pnre (1975), is that vemalization is the specific promotion of flower initiation by a previous cold treatment given to the imbibed seed or young plant. Although this definition has been well accepted, the confusion in terminology of vemalization seems never- ending. Chouard’s definition clearly means that vemalization is the physiological or biochemical processes leading to flowering. In other words, vemalization is plant’s flowering response to a cold environment. The term “vemalization” in the recent literature is, however, used at least in reference to both a plant’s physiological state and the state of the environment in which it is grown (Napp-Zinn, 1987). Plants that no longer respond to vemalization have been described as “fully vemalized”, or “vemalized", which suggests that vemalization refers to a plant’s physiological status. On the other hand, it is common to refer to the process of subjecting imbibed seeds or young plants to low temperatures as “vemalization”. or to say that plants were “vemalized” for a certain number of days. These two meanings of vemalization lead to problems interpreting a simple statement such 28 as 'the plants were not vemalized”, because that could mean either that no low temperature conditions were imposed. or that the plants had not yet reached a particular state of physiological development. or both. Wang et al. (1995a) proposed using vemalization to describe environmental circumstances rather than a plant’s physiological state, and using vemalization response to refer to a plant’s developmental response to exposure to low, nonfreezing temperatures. Therefore, a plant that has been vemalized will not necessarily show any response, while an unvemalized plant is one that was not exposed to vemalizing conditions. Likewise, a vemalization treatment is one that exposes plants to low temperature and will not necessarily elicit vemalization responses from wheat plants. The terminology of vemalization in this paper will follow the definition of Wang et al. (1995a). Another misused term is “short day vemalization”. Without experience of any low temperatures, ear emergence is fastest when long days are preceded by short days (McKinney and Sande, 1935; Cooper, 1960; Krekule, 1964; Davidson et al., 1985). The phenomenon, that short days can substitute for low temperature vemalization to promote flowering in winter cereals, was termed as “short day vemalization” by Purvis and Gregory (1937). However, evidence indicated that the mechanisms of winter wheat plants response to short day and low temperature are different and independent of each other. 1) all winter wheat cultivars respond to vemalization, only some of them respond to short days (Krekule, 1964; Miao et al. 1993); 2) the shoot apices of plants vemalized for eight weeks at 2-3°C reveal no 29 progress towards inflorescence initiation, double ridges are apparent in the apices of plants given short days for eight weeks (Evans, 1987); 3) exposure of the developing grains in ear to short days and low temperatures is less effective than to long days and low temperatures (Evans, 1987); 4) the shoot apical meristem responds to vemalization (lshihara, 1961 ), and the leaves perceive the short days (6011 et al., 1955). Although the effect of short day will not be discussed further in this paper, we suggest use “short day response" instead of “short day vemalization”. 3. Measure of response Vemalization response of wheat is usually evaluated by the degree of acceleration, due to the cold treatment, of floral initiation, stem elongation. flag leaf unfolding, heading, and/or flowering under a long photoperiod (>15-h) and high temperature (>15°C) regime in terms of aalendar days or thermal time from the end of vemalization treatment. The minimum length of cold treatment which maximized vemalization response was defined as the “vemalization requirement” of a cultivar (Martinic, 1973). Gotoh (1976) used a criterion that flag leaf was able to unfold within 34 days after the termination of cold treatment to determine a cultivar's “vemalization requirement”. A similar way, but 40 days for head emergence, was used by Hunt (1979). This criterion is associated with some distortion resulting from the varietal difference in narrow-sense earliness (T akahashi and Yasuda, 1971; also called "intrinsic earliness" by Hoogendoom, 1985; and "flowering tendency” by 30 Wallace, 1985; defined as the time to reach anthesis when vemalization and photoperiod do not constrain development). The results presented in calendar days provide little information about the exact response and has little basis in morphophysiology, and are hardly to be compared among different experiments which different temperatures are applied at post-vemalization growth. When the duration of cold treatment is not long enough to saturate plant’s vemalization response (i.e., smlled partial vemalization), neither calendar day nor thermal time are likely to reveal clear biological principles because in both approaches a plant’s response results from the sum of both the accelerating and retarding effects of low temperatures used for post- vemalization growth. The confusions encountered in explaining vemalization effects for the field data can also be attributed to using calendar day or thermal time as the primary unit of measure. While being cold treated, wheat continues to grow and develop. The higher the temperature used in vemalization treatment, the more growth for treated plant (Chujo, 1966). Therefore, increasing the vemalization duration, even when a plant has become insensitive to vemalization, the calendar days or thermal time from the end of cold treatment to heading has a tendency to be decreased (Fig. 1). In order to measure vemalization response per se, several ingenious vemalization techniques and data-analysis methods have been developed, such as: 1) vemalizing developing grains in the ear (Hoogendoom, 1984); 2) reducing the amount of growth during the vemalization treatment through using lower Fig. 1. 31 8' 5100‘ 8 2140 i- C . 8 ~ . c . 0120 25 g m - g . o 80 . > "6 a °° ' . G 40 r 0 g r ' P O a E 2° ' g o l I l l l l 1 l 1 I, l as 0 1 2 3 4 5 6 7 8 9 17 18 19 0 Weeks of vemalization Days from the end of vemalization treatment to heading in response to weeks of vemalization treatment. Adapted from Ahrens and Loomis (1963), imbibed seeds of winter wheat Minter were vemalized at 1°C for varying periods, then transferred to a warm greenhouse (24°C. 18/6-h), and observed for heading date. 32 temperatures (Riddell and Gries, 1958; Pirasteh and Welsh, 1980), or less moisture (Hoogendoom, 1984); 3) growing control plants at a nonvemalizing temperature to the same size as the vemalized seedlings (McKinney and Sando, 1933; Syme, 1968); 4) estimating growth during vemalization treatment in terms of days of growth at high temperature through the linear regression of primordia (Hoogendoom, 1984), or growth increment (Kate and Yamagata, 1988), or regression on the leaf number at transfer against final leaf number (Halloran, 1975). However, no one of these approaches has been incorporated into a general model. The coleoptile and early leaves are shortened and hair development on the leaf sheath is suppressed in wheat plant due to vemalization treatment (Purvis and Hatcher, 1959). but those are not related to flower initiation and hardly have meaning in evaluation of vemalization response. The morphology of main shoot apices should be a good indiaator which shows the transition from vegetative to reproductive stage. However, in a detailed study for the effects of vemalization on shoot apices, Griffiths et al. (1985) were unable to detect any major changes before the appearance of double ridges. Plant become vemalization insensitive before floral initials are present (Thomas and Vince-Pme, 1984). It is no question that wheat has lost its vemalization responsiveness at the double ridge stage (Halse and Weir, 1970; Weir et al.. 1984; Flood and Halloran, 1986). The question is how long the lag period between the onset of vemalization insensitivity and double ridge stage could be. 33 Many reports showed that the effect of vemalization can be interpreted more clearly from the results of experiments in which final leaf number is recorded (Levy and Peterson, 1972; Berry at al., 1980; Hay and lGrby. 1991). Although final leaf number is an indirect indicator, it does give me unambiguous information about the transition of plant from a state of vemalization sensitivity to vemalization insensitivity. Final leaf number decreases with increase of vemalization duration until reaching a plateau (Fig. 2). Plants at and after the stage where final leaf number begins to plateau is vemalization insensitive because additional vemalization did not reduce final leaf number. In addition. final leaf number is a non-destructive observation and easy to be recorded. As discussed later on, the different effects of temperature on vemalization response and on general growth can be analyzed through counting the leaf number change. Before other more reliable physiological or phenological marker(s) is established, using final leaf number as a measure in vemalization research is suggested. 4. Response types Wheat is a world-wide distribution crop, and characterized by marked variability in its vemalization pattem which is associated with the geographies! origin and the cultivation season of a specific cultivar (Hunt, 1979; Ford et al., 1981; Hoogendoom, 1985). Various approaches have revealed that each of wheat’s three genomes has one or two loci whose allelic variants influence vemalization 181- . 16*- 14- 12- 0 Final leaf number 101' 0 1 2 3 4 5 6 7 8 9 10 Weeks of vemalization Fig. 2. Final leaf number on the main stem in response to weeks of vemalization treatment. Adapted from Wang et al. (1995a). seedlings of winter wheat Pioneer 2548, aged at the third leaf tip visible. were vemalized at the growth chamber (513°C, 8/16-h) for varying periods, then transferred to a warm greenhouse (20°C, 20l4-h), and observed for final leaf number on the main stem. 35 response in a qualitative fashion. but minor genes are also reported (see Flood and Halloran, 1986 for review). This picture of the genetic control of vemalization response logically leads to a continuum of phenotypic classes, and that expectation is confirmed by many studies (Flood and Halloran, 1986). However, the vemalization responsiveness of wheat (including all species) in general is only classified into three types (Pinthus. 1985), i.e., 1) distinct winter wheat (true winter wheat, winter wheat): cultivars of this type require vemalization to reach the stage of spike differentiation within a normal season of growth; 2) intermediate wheat (semi-winter wheat. facultative wheat): cultivars of this type do not require vemalization for normal floral initiation but will respond to it by accelerated progress towards this stage; 3) spring wheat cultivars of this type do not respond at all to vemalization. The terms “spring” and “winter” in combination with modifiers such as “strong" 'and “weak” are also commonly used. Furthermore, the terms “spring’ and “winter“ are held to be synonymous with “early” and “late” in some papers (e.g., Aitken, 1966). In North America the terms "Winter" and "spring" are also used to categorize certain market grades (Pugsley, 1983). The universally used spring/winter classification system relates more to the sewing system to which a wheat cultivar is adapted rather than to the specific nature of a cultivars response to vemalization. For instance. many wheats that are adapted to spring sowing (so-called spring wheat) can respond to vemalization (Levy and Peterson, 1972; Wall and Cartwright. 1974; Halloran, 36 1977; Jedel et al., 1986), and wheats adapted to fall sowing (so-called winter wheat) vary markedly in their response to vemalization (Gotoh. 1976; Ledent, 1980; Miao et al.. 1992). Moreover, some wheats included in the lntemational Winter Wheat Performance Nursery had little or no response to vemalization (Gotoh, 1975). In order to reveal major genotype by site interactions in vemalization response of different cultivars, more groups were divided. For example, Kakizaki and Suzuki (1937. cited by Gotoh, 1976) grouped Japanese cultivars into the classes I (extreme spring habit) to VII (extreme winter habit). Miao et al. (1988) classified 40 cultivars into six groups. i.e., from strong springness cultivar which vernal ization response was zero to ultra-strong wintemess cultivar which responded to over 70 days of vemalization treatment. Those arbitrary groups may work better than only describing cultivar as spring or winter type. This kind of classification was. however, based either on the difference of days from the end of vemalization treatment to heading (or flowering) or on the difference of heading date among tested cultivars at the special vemalization experiment. There was no unambiguous parameter(s) for quantifying vemalization responsiveness of each group. Pugsley (1983) suggested that the classification of cultivars for their vemalization response should be based on genotype. He proposed three genetic types as follows: 1) spring wheats: bearing the major gene Vm1; 2) semi-winter wheats: lacking Vm1, but carrying Vm2, Vm3, Vm4, or a combination of those; 3) winter wheats: bearing the recessive alleles vm1, vrn2, vm3 and vm4. This 37 classification system is hardly to be generally awted. At first, it was only based on four loci (Vm1 to Vm4). The genetic background of vemalization in fact is very complicated and by no means clear (Flood and Halloran, 1986). Except the nuclear genes, there was also some evidence that cytoplasm can influence vemalization response (e.g., Ward, 1983). In addition. it is impossible to know each cultivar's genetic background due to technical reason and economic reason. 5. Operative temperatures There is not consistency in the literature regarding effective vemalization temperatures. Vemalization response is generally believed to take place at 2 to 8°C (Fischer. 1984). Ahrens and Loomis (1963), working with a winter wheat cultivar, found the vemalization effect at 1°C and 3°C, but no effect at -2°C. The effectiveness of the cold treatment in wheat seedlings was maximum at 7°C and decreased very rapidly if temperature was raised to 9°C or lowered to 3°C (T rione and Metzger, 1970). Chujo (1966) demonstrated that vemalization response progressed more rapidly at 4, 8, or 11°C than at 1°C. Many efforts have been made to integrate results of such studies into simulation models. However, the lower limit. the range of optimum, and the upper limit for vemalization temperatures are discrepant among different studies (Fig. 3). Some researchers believed that the operative temperatures for vemalization vary among cultivars. Higher temperatures (e.g., 8 and 11°C) were more favorable for vemalizing semi-winter wheats, but winter wheats were more sensitive to lower Fig. 3. 38 1.0 [ 0.5 i Relative vemalization effectiveness 0.0 ‘ . . . _ -5 0 5 10 15 20 Temperature (centi-degree) The effectiveness of temperature (°C) on vemalization response in the literature. Adapted from (a) Kirby (1992, solid line) and Maas and Arkin (1980, dashed line), (b) Hansel (1953. solid line) and Reinink et al. (1986 dashed line), (c) Ritchie (1991 , solid line) and Weir et al. (1984, dashed line). (continued) 39 — p 0. 1 5 mmocoéuoto c0mo~=nEm> 2553.0 O 15 10 Temperature (centi-degree) .i * 0. O .. mmocoéooto commn..oEo> 253010. -5 Temperature (centi-degree) Fig. 3. -Continued. 40 temperatures (e.g., 4 and 8°C) (Chujo, 1966). Vavilov (1951) showed that the most effective vemalization temperatures for different types of cultivars were as follows: 10 to 12°C for soft-grained spring. 2 to 5°C for hard-grained spring, 5 to 10°C for semi-winter. and 0 to 5°C for winter cultivars. Almost all studies on the operative vemalization temperatures was based on the measure of calendar days or thermal time after the end of vemalization treatment The magnitude of growth under different vemalization temperatures was not taken into account. I.R. Brooking (pers. comm.) suggested to separate the temperature effects on vemalization response and on vegetative growth by using isogenic lines ormonitoring the floral transition through recording the primordia at the end of treatment and final leaf number. Emphasis on final leaf number rather than calendar time should make interpretation more relevant. 6. Relationship between leaf number and vemalization responsiveness Final leaf number on the main stem can be affected by vemalization treatment. To explore the relationship between final leaf number and the number of leaves emerged at the onset of vemalization insensitivity should be of great importance in wheat vemalization study. Wang et al. (1995a) estimated that the number of leaves emerging at vemalization insensitivity under long day conditions is about six leaves. In other words, the leaf stage at which a plant with an emerged flag leaf reached vemalization insensitivity is estimated through 41 final leaf number minus six Evidence to support this hypothesis can be found in the literature. Chujo (1966) presented data that can be interpreted to show that plants with more leaves than final leaf number for unvemalized plants minus six could not respond to vemalization, while younger plants could. A similar interpretation can be applied to Gott’s (1957) data. Data from Hoogendoom (1985). Griffiths and Lyndon (1985), and Miao et al. (1992) also tend to confirm that the number of leaves emerging after vemalization insensitivity under long day conditions is about six. It is interesting to note that after the onset of vemalization insensitivity. plants will emerge six more leaves, which is also the number of leaves either postulated or observed to be the minimum number of leaves possible in wheat (Purvis, 1934; Aitken, 1966; Miao et al., 1992; Brooking et al., 1995), and the number of leaves emerged in spring for most normal fall-sowing wheats in commercial production. Minimum leaf number is probably related to the number of intemodes that elongate in wheat. This value stays very constant at five (only a few could be four or six). irrespective of the number of leaf nodes actually present on the stem. Final leaf number can be reduced by the vemalization treatment. However, in order to construct a stem with four to six intemodes. the plant must develop at least that many leaves. It is no question that photoperiod has an effect on the number of leaves emerging after the onset of vemalization insensitivity (Wang et al.. 1995c; Brooking et al.. 1995). In the data of Levy and Peterson (1972), the average 42 final leaf number of the winter wheat Triumph given a 56 days of vemalization treatment changed from 7.0 to 13.7 when the post-vemalization photoperiod was decreased from 17- to 9—h. The similar results were reported by Miao et al. (1992). Anyhow, the number of leaves emerging between the onset of vemalization insensitivity and flowering is stable for a given cultivar grown in constant post-vemalization conditions. When the photoperiod used in post- vemalization is long enough, the number of leaves emerging after the onset of vemalization sensitivity will be minimized, i.e.. six. The concepts based on the above discussion are presented in Fig. 4. Under long day conditions, the number of leaves below the sixth leaf from the flag leaf is determined by a cultivar’s vemalization responsiveness. That value can be as low as 0 (i.e.. the cultivar which does not respond to vemalization at all and has a final leaf number of six), or as large as more than 15 (i.e., the cultivar which is very sensitive to vemalization treatment and has a final leaf number of great than 21). Although the photoperiod effect will not be discussed in detail in this paper, plant is very probably sensitive to photoperiod until the fourth leaf stage that is counted from the flag leaf, because final leaf number is determined at about the leaf stage of the fourth leaf from the flag leaf (Aitken, 1974; Zhang et al., 1986). 43 Head I l ._ (Flagleaf) 11 -- 111 -- Photopa'iod {V " mfivity :> / (Double ridges) V «1— VI -- Vemalimtron Photoperiod :> i \ CZ! insensitivity scnsitivepbasc l 1. l . V 1' I'm 1'- <2 sensitivephasc I \ Gemination «'- / Fig. 4. Schematic representation of the effects of vemalization and photoperiod on me number of leaves in wheat. The capital Roman numerals represent the ordinal leaf number from the flag leaf. 44 7. Interchangeability between plant age and vemalization duration Wheat does not have an absolute cold requirement for flowering. Numerous reports demonstrated that all tested wheats, including those adapted to fall sowing system at high latitudes, will eventually flower even without exposure to low temperatures (Purvis, 1961; Pauli et al., 1962; Ahrens and Loomis, 1963; Chujo, 1966; Martinic. 1973; Gotoh, 1976; Ledent, 1980; Rahman, 1980; Miao et al., 1992; Wang et al., 1995a, b). It is also clear that there is not a so-called juvenile stage in wheat in terms of vemalization response. The developing grain, even still attached on the mother ear, or embryogenic callus from immature embryos, can respond to vemalization (Purvis, 1961; Pugsley and Warrington, 1979; Hoogendoom, 1 984; Sharma and Mascia, 1987; Whelan and Schaalje, 1992). The vemalization response of the developing grain, anyhow, is unlikely to be of major signifiaance in most commercial wheat growing areas (Hay and Kirby, 1991). In the conventional vemalization studies, imbibed seeds or young seedlings are usually treated with low temperature in refrigerator or growth chamber for a certain number of days, and then transferred to a high temperature environment. Several reports noted that the young seedlings are more sensitive to vemalization treatments (Gotoh, 1976; Salisbury et al., 1979). Most fall-sowing wheats are subject to low temperatures as seedlings during late fall, winter, and even early spring. Therefore, understanding vemalization response of the growing plant is of great importance. Wheat plant loses its sensitivity to vemalization as it grows older (Ahrens and Loomis,1963). The upper age limit for vemalization response varies with cultivars. 45 The cultivar"W1nter Minflor" was responsive to vemalization at any stage from just- genninated seed up to 42 days’ old plant with six to seven leaves on the main shoot (Gott, 1957). Another winter cultivar "Norin No. 27" had no vemalization response in the plants aged 90 days (Chujo, 1966). Spring wl'ieat "Pitic 62" responded to vemalization only at ages less than 14 days (Jedel et al.. 1986). With a rich array of treatment combinations of plant age and vemalization duration, Wang et al. (1995a) demonstrated that the vemalization effect in terms of the change of final leaf number on the main stem decreased linearly as the plant age at the onset of vemalization increased. The age effect of vemalization response was quantified as follows: (F0'6)=LI+BTV (Fo>6) (1) where P.. is final leaf number with no vemalization. L. is the leaf stage at the onset of vemalization insensitivity, T. is the days of vemalization treatment, and B is the absolute value of the slope in the linear regression between average values of final leaf number minus six and days of vemalization. The B represents the “exchange rate” between leaf numbers and vemalization days. Eq. 1 indicates that a plant becomes vemalization insensitivity when the sum of the current leaf stage and the leaf equivalents gained by vemalization days (i.e., the product of the days of vemalization and the leaf number/vemalization days exchange rate) is equal to final leaf number of unvemalized plants minus six. In other words, vemalization can substitute for plant age as a determinant of time of flowering. Chujo (1966) found that the 46 vemalization effect is large and vemalization response is rapid when the growth of plants is possible during the vemalization treatment. The weaker the vemalization responsiveness of a cultivar. the higher the vemalizing temperature necessary for maximum rate of vemalization response (Flood and Halloran, 1986). That is because the spring wheat in general has a small “exchange rate" (Wang et al. 1 995b). 8. A novel conceptual framework It is a well-established concept that the base temperature for wheat, at least at wheat’s early developmental stage, is 0°C (Ritchie. 1991; Kirby, 1992). Any vemalization treatment which is above the base temperature will allow plant to accumulate vemalization response and thermal time simultaneously. The higher the temperature used in vemalization treatment, the higher the rate of primodium initiation and leaf emergence during that period. The interchangeability between plant age and vemalization duration indicates that the effect of vemalization days can be expressed as the leaf equivalents gained by vemalization days. Another important assumption is that plant has a constant number of leaves, which is six, to emerge after the onset of vemalization insensitivity. Therefore, it is possible to estimate the general thermal response and vemalization response through counting the change of leaf number. 47 As mentioned above, plants at and after the stage where additional vemalization does not reduce final leaf number are vemalization insensitive. The quantitative features of vemalization response, up to the point of insensitivity, can be characterized with a linear regression (Wang et al.. 1995b): (Fr-6)=a-BTV (F126) (2) where F. is final leaf number observed for a particular vemalization treatment, a is the Y-intercept and is an estimate of average final leaf number of unvemalized plants (F0) minus six. and B and T. are the same as described for Eq. 1. The fundamental concept essential to generalize this linear regression equation as a novel development model for wheat vemalization response is to consider the number of leaves produced before. during, and after vemalization treatment. rather than only final leaf number, or calendar days (or thermal time) after the end of vemalization treatment. Eq. 2 is further described as: ((Fa+Fa+F.)-6)=a-BTV (Fa+Fu+F.>-6) (3) where F.,, F... and F. is the number of leaves emerged before, during, and after vemalization treatment, respectively. If the same high temperature (e.g., 20°C) is used before and after vemalization treatment, the relationship represented in Eq. 2 or Eq. 3 is not affected by the temperatures applied at vemalization treatment, or by the plant age expressed as leaf stage at the onset of vemalization treatment. Plants perceive leaf number (F.) (via thermal time and phyllochron) and calendar day (TV) (via clock time) as its physiological time. The state of vemalization insensitivity is reached at F.2d, T.,2(otIB), or (F.+BT.)2a. 48 The parameters a and B varied among cultivars (Fig. 5), and are useful for quantifying vemalization response in wheat. The implication of each parameter can be interpreted biologically: a is the “changeable number of leaves", i.e., how many leaves can be potentially decreased by vemalization treatment; and B represents the “exchange rate” between leaf numbers and vemalization days, i.e., how many leaves can be reduced by one day of vemalization treatment. The values of or and B in general are lower in so-called spring wheat than in so-called winter wheat. However. 01 and B appear to vary independently. For instance, the winter wheat FL 303 has the same or value as the spring wheat Pitic 62 (Fig. 5). Eq. 3 reveals that plants can be induced at an early leaf stage because of lower temperatures. A logical corollary of that view is that there are no so-called operative temperatures for wheat vemalization. The ability of unvemalized winter wheats to flower under a high temperature (e.g., 20°C) environment is evidence that plant is able to be induced at high temperature. However, different temperatures result in different final leaf numbers for the same cultivar, and different cultivars have different temperature responses. For instance, Chujo (1966) grew four winter wheats under constant temperature of 15 and 20°C, and all plants headed, but plants from 20°C environment had a greater final leaf number than that from 15°C. Under higher temperature condition in the field, plants which respond to vemalization produced more leaves and had a higher total number of leaves at heading (Ford et al.. 1981; Midmore et al., 1982). 49 18" — NY 73116-4w 18 r . ------- Pioneer 2548 ‘4 l- ‘\‘ .............. FL303 ’ ---— Pitic62 Final leaf number minus six 0 10 20 30 40 50 00 70 80 90 100 Days of vemalization treatment Fig. 5. Vemalization response quantified with a linear regression: (F. - 6) = or - BT. for different cultivars. The parameters are as follows: 01 = 5.4, B = 0.1174 for Pitic 62; o = 5.4, B = 0.0750 for FL 303; o = 15.1, B = 0.2240 for Pioneer 2548; or = 17.4, B = 0.1880 for NY 73116-4w. Data taken from Wang et al. (1995b). 50 Our concept for wheat vemalization is that in wheat life cycle, there is a special physiological phase that low temperature can reduce the number of leaves emerging during that period and result in a low final leaf number. A plant’s response to temperature during that phase is its vemalization response. This concept clarifies that temperature as an unity affects both growth and development. Vemalization response of wheat can be experimentally quantified by using the response parameters, or and B, derived from Eq. 2 or Eq. 3. Two key premises underlying a simple scheme to get the parameters in a comprehensive screening protocol suitable for all wheats are: 1) the same temperature of about 20°C should be used in pre- and post-vemalization growth, because 20°C is the possible high temperature encountered before the onset of vemalization insensitivity in the field for normal fall-sowing wheats; 2) long photoperiods should be applied. A simplified model relating leaf number and vemalization responsiveness for wheat can be constructed as Fig 6. Line AD is a cultivar’s vemalization response curve determined by Eq. 2 or Eq. 3. Point A, B, C or D represents vemalization insensitivity under different temperature regimes. The slope of the dotted line increases as temperature increases. Plant becomes vemalization insensitive after T. 2 (or I B) days at temperature about 0°C (point A in Fig. 6). At that special case, plants of vemalization-sensitive wheats will have a final leaf number of six. 51 ..: I; ‘24 >. . . 2 16 r ‘22 s “i 3m .n L - 'U o 12 .. -1e 1- E. . - s g 10 - ~16 g d) L " c a 8 "14 - s - i 3 3 e - ~12 3 7- ‘ 1 .5 e 4 - 410 LI. 8 ’ i E 2 " I 18 3 " ......... ‘ Z 0 W“ e c 20 30 40 so so .70 so so 100 1; Days after germrnatron 8 >° .0 2 .1! 3 g < E 100 Fig. 6. Model of relation between leaf number and vemalization responsiveness. The vemalization response line (line AD) used as an example is: F. - 6 == 17.4 - 0.188T., i.e., NY 73116-4w in Fig. 5. Point A, B. C or D represents vemalization insensitivity under different temperature regimes. The abbreviation “v.” stands for “vemalization’ and “i.” for “insensitivity”. 52 If either F. or T. can make plant insensitive to vemalization. the calendar days from seed germination to flag leaf unfolding should be very close between vemalized and unvemalized plants if days of vemalization treatment is taken into account. However. many experiments with winter wheats did not come to that conclusion. The winter wheat Pioneer 2548 is used here as an example (unpublished date from authors). The calendar days from seed germination to flag leaf unfolding were 102 days for plants which experienced 70 days cold treatment (5/3°C, 8/16-h) started from seed germination and then transferred to a high temperature condition (20°C, 20I4-h), and 147 days for plants which were grown under the high temperature condition (20°C, 20I4-h) with no pre-treatment of low temperature. The apparent conflict is resolved, however, by taking the difference of phyllochron into account. Although considerable debate exists on how constant the phyllochron is among leaves during a growing season, there does seem to be variation among leaves (McMaster and Wilhelm, 1995). Constant phyllochrons can only occur if the rate of extension of each subsequent leaf increases enoUgh to counterbalance the increasing distance each leaf primordia has to cover from apex, where it is initiated, to the point of emergence. Otherwise, there will be a constant decline in the rate of appearance of subsequent leaves (Miglietta. 1991). The increase of phyllochron for subsequent leaves may be not obvious in the field or in the controlled condition where the vemalized plant is used, so that a constant phyllochron is observed. 53 However, the difference of phyllochron between early emerging and late emerging leaves in a plant of unvemalized winter wheat which has a final leaf number of 20 or more could be significant. In the experiment mentioned above, plants vemalized for 70 days had a final leaf number of eight, and unvemalized plants had 21. That is one of the reasons why flowering of winter wheat canbe promoted by low temperature applied in the vemalization sensitive phase. 9. Concluding remarks The conceptual framework proposed in this paper provides some new clues for understanding vemalization response in wheat. However. some well designed experiments are needed to further develop a general vemalization model. An important element of the present model is the concept that plant will emerge six new leaves after the onset of vemalization insensitivity, but more than six leaves might be emerged under shorter photoperiod conditions. The interactions between temperature and photoperiod before and after vemalization insensitivity need to be clarified. Temperature ranged from 0 to 20°C is used as the effective temperature for wheat development in this paper. A more accurate temperature function should be worked out. It would be interested to look at apex condition in relation to final leaf number minus six Comparative biochemical and ultrastructural characterization of vemalization sensitive and insensitive apexes with varying numbers of total 54 primordia could lead to a deeper understanding of the underlying processes leading up to floral initiation. Acknowledgements Grateful acknowledgement is made to Dr. R. S. Loomis for his encouragement to write this paper. We thank Drs. R.D. Heins and J.A. Flore for their helpful discussion and constructive criticism of the manuscript. We acknowledge support from Japanese GoVemment Scholarship funds given to S.- Y. Wang through CIMMYT. References Ahrens, J.F., and Loomis, W.E., 1963. Floral induction and development in winter wheat. Crop Sci.. 3: 463-466. Aitken, Y., 1966. Flower initiation in relation to maturity in crop plants. Ill. The flowering response of early and late cereal varieties to Australian environments. Aust. J. Agric. Res, 17: 1-15. 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