Coefficient of Variation Can Identify the Most Important Effects of Experimental Treatments

Transcription

1 Aville online: Print ISSN -96X; Electronic 8-9 Not Bot Horti Agroo,, ():87-9. DOI:.8/nh988 Coefficient of Vrition Cn Identify the Most Importnt Effects of Experimentl Tretments José Crlos LORENZO *, Lourdes YABOR, Norm MEDINA, Nicolás QUINTANA, Vness WELLS University of Ciego de Ávil, Bioplnt Centre, Lortory for Plnt Breeding. Ciego de Ávil 69, Cu; (*corresponding uthor); University of Ciego de Ávil, Ciego de Ávil 69, Cu; The University of Sydney, Fculty of Agriculture nd Environment, Plnt Breeding Institute, Austrli; Astrct Most griculturl experiments involve evlution of multiple vriles nd t times it cn e difficult to identify the iologiclly relevnt effects of the experimentl tretments fter performing the trditionl ANOVA, Tukey nd t-tests. The coefficient of vrition formul could e n importnt tool to focus Result nd Discussion sections only on the most importnt chnges produced y the experimentl tretments. This short report is intended to exemplify the use of the coefficient of vrition in three plnt physiology experiments. The first one delt with the effects of common en plntlet exposure to high temperture under controlled conditions (levels: 8 nd C). The second experiment ws relted to common en seed exposure to liquid nitrogen during five different periods of time (levels:, 7,, nd 8 dys). The third experiment ws i-fctoril: fctor ws the type of plnt mteril (levels: pinepple plnts geneticlly trnsformed nd the untrnsformed control); nd fctor ws the time of in vitro-plntlet hrdening (levels:, nd dys). Contents of phenolics, ldehydes, chlorophylls nd proteins were determined. Percentge of seed germintion, electrolyte lekge, peroxidse ctivity, plnt height nd weight were lso mesured. Experiments were monofctoril with two levels, monofctoril with five levels nd ifctoril, respectively, with rndomized design. The coefficient of vrition showed tht the most remrkle effects of high temperture were recorded in free phenolics nd chlorophylls (,, totl). Electrolyte lekge nd chlorophyll concentrtion were the most modified indictors s result of seed exposure to liquid nitrogen. In the third experiment, modifiction in the levels of mlondildehyde nd other ldehydes were the most relevnt chnges resulting from fctors interctions. A similr procedure hs not een pulished, except for our previous pulictions, not focused on the use of the coefficient of vrition, just on the iologicl results. Keywords: iotic stress, Anns comosus, iochemicl mrkers, iosttistics, genetic trnsformtion, OCV, Phseolus vulgris Introduction Most griculturl experiments involve evlution of multiple vriles such s yield, plnt height, iochemicl indictors, etc. After performing, for instnce the trditionl ANOVA, Tukey nd t-tests, it is sometimes difficult to identify the most relevnt effects of the experimentl tretments from the iologicl point of view. We elieve the coefficient of vrition formul could e n importnt tool to focus Result nd Discussion sections only on the most importnt chnges produced during the experiment. The coefficient of vrition is clculted s the rte: (Stndrd devition/averge)*, nd mesures the vriility of series of numers independently of the unit of mesurement used for these numers. In order to do so, the coefficient of vrition elimintes the unit of mesurement of the stndrd devition of series of numers y dividing it y the men of these numers. The coefficient of vrition cn e used to compre distriutions otined with different units, such s, for exmple, cm, mg nd kg/h (Adi, ; Ivnov, 989). The present short report is intended to exemplify the use of the coefficient of vrition formul, in n untrditionl wy, in three plnt physiology experiments. Our method is untrditionl ecuse the originl figures we used to clculte the coefficient of vrition re the verges of ech tretment. In the present pper, we clled this rte s Overll Coefficient of Vrition (OCV). We re not trying to convince sttisticins out ny new method; rther, we would like prcticing scientists to consider in their reserch the coefficient of vrition s descried here. Although we hve Received: 6 Mr. Received in revised form: / Apr. Accepted: My. Pulished online: My.

2 Lorenzo CL et l. / Not Bot Horti Agroo,, (): performed comprehensive literture serch, we hve not found ny procedure similr nd simpler to the one descried in this pper, except for our previous pulictions (Cejs et l., ; Gomez-Pndo et l., 9; González et l., ; González et l., ; Pérez et l., ; Pérez et l., ; Zevllos et l., ; Zevllos et l., ), not focused on the use of sttisticl or mthemticl methods, just on the results. Mterils nd methods The first experiment ws relted to the effects of high temperture on levels of phenolics, ldehydes nd chlorophylls in common en plntlets (cv. Milgro Villclreño ). After hrvesting, seeds were stored t C in the drk, in hermeticlly closed continers. Seeds with % moisture, sed on fresh weight (ISTA, ), were used in this experiment. Seeds were llowed to germinte nd grow without high temperture stress (8 C) during dys, nd then plntlets were exposed to C during 9. hours (% plnt survivl ws previously oserved fter 9. h of het tretment; unpulished dt). A control tretment (8 C) ws estlished. Ech plnt continer ( cm of Ferrlitic red soil) ws irrigted dily with ml wter. Ech tretment involved five plnt continers nd three seeds per continer were sown. At 9. hours of stress, middle-ged leves of ech plnt continer were collected, pooled nd ground in liquid nitrogen to produce lef powder. Three independent smples ( mg powder ech) per tretment were used to perform every iochemicl mesurement. Levels of chlorophylls (,, totl), phenolics (free, cell wll-linked, totl), nd mlondildehyde nd other ldehydes were determined ccording to the methods of Porr (), Gurr et l. (99) nd Heth nd Pcker (968), respectively. Results of this experiment re unpulished. In the second experiment, common en seeds otined s descried ove, were plced in cryo-vils (volume: ml; seeds per cryo-vil) nd immersed in liquid nitrogen for 7,, or 8 dys. Control seeds were set to germinte or nlyzed directly, without liquid nitrogen exposure. Recovery of seeds from liquid nitrogen ws performed ccording to Stndwood nd Bss (98). To evlute germintion rte, seeds (three replictes of seeds per tretment) were plced on filter pper moistened with ml distilled wter (ph 6.8) for dys (drk, 7± C) in Petri dishes (Ø: mm). For iochemicl determintions, independent smples of seeds ech, recovered from liquid nitrogen ccording to Stndwood nd Bss (98), were ground in liquid nitrogen to produce seed powder. Three independent smples ( mg powder ech) were used to perform every iochemicl mesurement. Levels of chlorophylls (,, totl), phenolics (free, cell wlllinked, totl), mlondildehyde, other ldehydes, proteins (Brdford, 976) nd peroxidse ctivity (Hmmerschmidt et l., 98) were determined. Enzyme ctivity ws lso expressed on the sis of protein s specific ctivity. The electrolyte efflux test ws used for electrolyte lekge determintion (Mrtínez-Montero et l., ) from intct seeds ( replictes of seeds ech per tretment). Fifteen seeds were incuted in ml doule-distilled wter. Conductivity of the wter ws mesured efore (C) nd fter hours of imiitions (C). Smples were then utoclved ( min t C, 7 kp) nd cooled down to room temperture ( C) for hours to determine the totl conductivity (Ctotl). The percentge of electrolyte lekge ws clculted from the rtio: (C C)*/(Ctotl C). Results of this experiment re unpulished. In the third experiment, pinepple trnsgenic plntlets were otined ccording to Espinos et l. (). Agrocterium tumefciens (strin AT6, phca8, r gene controlled y mize Ui promoter, clss-i en chitinse gene controlled y hyrid OCS-S CMV-rice ctin I promoter, tocco p gene controlled y S CMV promoter) ws used to trnsformed emryogenic clli. Phosphinotricin-resistnt plntlets were recovered. Nontrnsformed plntlets (control tretment) were otined following the protocol descried ove ut voiding contct with Agrocterium tumefciens nd phosphinotricin. Sixty non-trnsformed nd 6 trnsformed (one clone) plntlets were trnsferred to greenhouse for hrdening ccording to Ynes et l. (): plstic trys, sustrte: zeolite+filter cke (:), sustrte/plnt: 8 cm, microject utomted irrigtion for s every min nd illumintion: 8 µmol m - s -. Evlutions were mde t, nd dys of hrdening. The experimentl design ws completely rndomized. Plnt height nd weight were recorded. Lef smples were stored in liquid nitrogen until iochemicl nlysis. Ech iochemicl determintion strted from three independent pooled smples ( mg ech). They were finely grounded in liquid nitrogen. Contents of mlondildehyde nd other ldehydes, chlorophyll (, ), nd phenolics (free nd cell wll-linked) were mesured. Results of this experiment were previously pulished y Yor et l. (6). The SPSS (version 8. for Windows, SPSS Inc., New York, NY) ws used to perform t-tests in the first experiment, nd One- nd Two Wy ANOVA nd Tukey tests (p=.) in the second nd the third experiments, respectively. The OCV were clculted s the rte: (Stndrd devition/averge)*. To clculte this coefficient, verge vlues of the tretments, or experimentl fctors, were considered. OCV were clssified in high, medium or low, which is explined in ech tle footnotes. (Mx OCV recorded Min OCV recorded) / produced the clss width. Results nd discussion As fr s Results re concerned, in the first experiment, ccording to the outcome of the t-tests, sttisticlly significnt differences etween tretments were oserved in every single iochemicl indictor evluted (Fig. ). To know which indictors were more ffected y high temperture stress, the OCV were clculted nd clssified s high, medium nd low. The OCV showed tht the most remrkle effects of high temperture were recorded in free phenolics nd chlorophylls (,, totl). We therefore would suggest plnt physiologists to focus their ttention on these four iochemicl indictors. The effect of high temperture stress ws found to e medium on cell wll-linked nd totl phenolics, s well s on other ldehydes. The influence of het stress on mlondildehyde levels ws clssified s low. Tle shows the effects of the second experiment. Electrolyte lekge nd chlorophyll concentrtion were the most modified indictors y the different times of seed exposure

3 Lorenzo CL et l. / Not Bot Horti Agroo,, ():87-9 OCV =.7% OCV = 6.% OCV =.88% stress on common en plntlets): High stress on common en plntlets): Medium stress on common en plntlets): Medium 89 Free phenolics (mg g - fresh Cell wll-linked phenolics (mg g - fresh 8 Totl of phenolics (mg g - fresh 8 OCV =.8% stress on common en plntlets): Low OCV = 7.% stress on common en plntlets): Medium Mlondildehyde (µm g - fresh Other ldehydes (µm g - fresh 8 OCV =.% stress on common en plntlets): High OCV = 9.% stress on common en plntlets): High OCV = 8.6% stress on common en plntlets): High Chlorophyll (µg g? fresh 8 Chlorophyll (µg g? fresh Fig.. Effect of exposure to high temperture on common en plntlets t 9. hours of stress. Indictors were evluted in middle-ged leves. In ech pnel, different letters ove the rs indicte tht differences re sttisticlly significnt (t-test, p.). Overll coefficient of vrition = (Stndrd devition/averge)*. To clculte this coefficient, verge vlues of the two conditions compred were considered. The higher difference etween the two verges compred, the higher the overll coefficient of vrition. Min OCV recorded =.8%; Mx OCV recorded = 9.%; Mx Min =.%; (Mx Min) / =.8%. Clssifiction of the OCVs: low from.8 to.6%; medium from.6 to 87.% nd high from 87. to 9.%. 8 Tle. Biochemicl chnges produced in common en seeds fter different times of exposure to liquid nitrogen Seed exposure to liquid nitrogen (dys) 7 8 OCV (%) ** Totl of chlorophyll (µg g? fresh Clssifiction of the OCV (iologicl effect of the time of exposure to liquid nitrogen) *** Percentge of germintion t dys fter exposure to LN * Low Electrolyte lekge (%) * High Chlorophyll concentrtion (μg g - fresh * Medium Chlorophyll concentrtion (μg g - fresh * High Totl chlorophyll concentrtion (μg g - fresh * Medium Content of free phenolics (µg g fresh c. Medium Content of cell wll-linked phenolics (µg g fresh * Low Totl of phenolics (µg g fresh * Low Mlondildehyde (μmol g fresh * Low Other ldehyde (μmol g fresh * Low Totl protein (mg g fresh * Low Peroxidse ctivity (U mg fresh * Low Peroxidse specific ctivity (U mg of protein) * Low * Results with the sme letter re not sttisticlly different (One-Wy ANOVA, Tukey, p>.). ** Overll coefficient of vrition = (Stndrd devition/averge)*. To clculte this coefficient, verge vlues of the five tretments compred were considered. The higher difference etween the five verges compred, the higher the overll coefficient of vrition. *** Min OCV recorded =.%; Mx OCV recorded = 8.%; Mx Min = 79.%; (Mx Min) / = 6.%. Clssifiction of the OCVs: low from. to 9.78%; medium from 9.78 to 6.% nd high from 6. to 8.%.

4 9 Lorenzo CL et l. / Not Bot Horti Agroo,, ():87-9 Tle. Effects of two experimentl fctors, type of plnt mteril nd time of in vitro-plntlet hrdening, on plnt height nd weight nd levels of mlondildehyde, other ldehydes, chlorophyll pigments (, ) nd phenolics (free, cell wll-linked). NTP: Non-trnsformed plntlets. TP: Trnsformed plntlets. Results of this experiment were previously pulished y Yor et l. (6) Effect of fctors interction) Effect of the fctor type of plnt mteril Effect of the fctor time of in vitro-plntlet hrdening dy of hrdening dys of hrdening dys of hrdening Dys of hrdening Clssifiction of Clssifiction of the Clssifiction of the OCV (effect OCV (effect of the OCV the OCV OCV OCV NTP TP of the fctor type fctor time of in NTP TP NTP TP NTP TP (%) ** (effect of fctors (%) ** (%) of plnt mteril ** vitro-plntlet interction) *** *** hrdening ) *** Plnt height (cm) * 6.c.9c Low Low Low Plnt weight (g) *.c.6c Low.7.. Low Low Mlondildehyde (µmol g - lef fresh.68c.9c.69.6c.6.9. High High High mss) * Other ldehyde (µmol g - lef fresh.9d.6e 6.66.d 9.6.6c.6 High High High mss) * Chlorophyll (µg g - lef fresh Low Low Low mss) * Chlorophyll (µg g - lef fresh.87c.8d.9.d..6d 69.6 Medium High Low mss) * Content of free phenolics (mg g - lef fresh mss) *.d.6 d.d 6. c Medium Low High Content of cell wll-linked phenolics (mg g - lef fresh mss) * 7.9d 8.8c.7 8.8c Low Low Low * Results with the sme letter re not sttisticlly different (Two-Wy ANOVA, Tukey, p>.). ** Overll coefficient of vrition = (Stndrd devition/averge)*. To clculte this coefficient, verge vlues compred were considered. The higher difference etween the verges compred, the higher the overll coefficient of vrition. *** Min OCV recorded =.%; Mx OCV recorded =.%; Mx Min =.7%; (Mx Min) / = 6.79%. Clssifiction of the OCVs: low from. to 6.9%; medium from 6.9 to 7.7% nd high from 7.7 to.% to liquid nitrogen, ccording to their high OCV. The effects of liquid nitrogen exposure on chlorophyll nd totl chlorophyll concentrtions, s well s, on s of free phenolics were clssified s medium. Agin, these indictors with high OCV re the most relevnt effects for plnt physiologists. The comined effects of plnt genetic trnsformtion nd the time of hrdening re shown in Tle. Clcultion of the OCV llowed identifying the most relevnt effects of the interction of the two experimentl fctors: high OCV were found in the levels of mlondildehyde nd other ldehydes. In the fctor type of plnt mteril, the most significnt effects were oserved in the s of mlondildehyde, other ldehydes nd chlorophyll. In the fctor time of in vitroplntlet hrdening, high OCV were recorded in the levels of mlondildehyde, other ldehydes nd free phenolics. The ove mentioned indictors re the most relevnt from the iologicl perspective. We hve previously used the OCV to identify the most relevnt effects of experimentl tretments in plnt reeding (Gomez-Pndo et l., 9; Pérez et l., ), iofertiliztion (González et l., ; González et l., ), cryopreservtion (Cejs et l., ; Cejs et l., ; Zevllos et l., ; Zevllos et l., ) nd production of nturl products (Pérez et l., ). To the est of our knowledge, similr procedure hs not een reported to dte. The proposed method is quite simple nd could e useful in prctice mostly in initil experiments in which lrge numer of vriles re studied providing quntittive nd reltively ojective procedure to focus the nlysis of the results, nd further reserch, on limited numer of prmeters, which re more likely to reflect the most relevnt responses of the plnts to the specific tretment pplied. Acknowledgments This work ws supported y the Bioplnt Centre (University of Ciego de Avil, Cu). We re grteful to Mrs. Myd Arzol, Mrs. Juli Mrtínez, Mrs. Alitz Iglesis, Mrs. Bárr Vlle, Mrs. Lritz Torrelnc, Ms. Meliss Argueds, Mr. Orlens González, Dr. Auror Pérez, Dr. Mrth Hernández, Dr. Crlos Argón, Dr. Ariel Arencii, Dr. Mrcos Edel Mrtínez, Mr. Arnldo Trujillo, Mrs. Leynes Díz-López, Mr. Crlos Leiv nd Mr. Gustvo Llorente. This reserch ws lso prtilly supported y the Austrli Awrd Fellowship through grnt to Dr. José Crlos Lorenzo Feijoo. References Adi H (). Coefficient of vrition, p.-. In: Slkind N (Ed). Encyclopedi of Reserch Design. Thousnd Oks, CA, Sge. Brdford MM (976). A rpid nd sensitive method for the quntifiction of microgrm quntities of protein utilising the principle of protein-dye inding. Anlyt Biochem 7:8-. Cejs I, Vives K, Ludt T, González-Olmedo J, Engelmnn F, Mrtínez-Montero ME, Lorenzo JC (). Effects of cryopreservtion of Phseolus vulgris L. seeds on erly stges of germintion. Plnt Cell Rep :6-7.

5 Lorenzo CL et l. / Not Bot Horti Agroo,, ():87-9 Cejs I, Méndez R, Villloos A, Plu F, Argón C, Engelmnn F, Crputo D, Aversno R, Mrtínez ME, Lorenzo JC (). Phenotypic nd moleculr chrcteriztion of Phseolus vulgris plnts from non-cryopreserved nd cryopreserved seeds. Am J Plnt Sci :8-89. Espinos P, Lorenzo JC, Iglesis A, Yor L, Menéndez E, Borroto J, Hernández L, Arencii A (). Production of pinepple trnsgenic plnts ssisted y temporry immersion iorectors. Plnt Cell Rep :6-. Gomez-Pndo L, Jimenez-Dvlos J, Eguiluz-de l Brr A, Aguilr-Cstellnos E, Flconí-Plomino J, Iñez-Tremold M, Vrel M, Lorenzo JC (9). Field performnce of new in vitro ndrogenesis-derived doule hploids of rley. Euphytic 66: González R, Ludt T, Arzol M, Méndez R, Mrrero P, Pulido LE, Diut B, Lorenzo JC (). Effect of Azotocter chroococcum on in vitro pinepple plnts growth during cclimtiztion. In Vitro Cellulr & Developmentl Biology- Plnt 7():87-9. González R, Serrto R, Molin J, Argón C, Ollde V, Pulido L, Diut B, Lorenzo JC (). Biochemicl nd physiologicl chnges produced y Azotocter chroococcum (INIFAT strin) on pinepple in vitro-plntlets during cclimtiztion. Act Physiol. Plnt. DOI:.7/s78--7-z. Gurr S, McPherson J, Bowles D (99). Lignin nd ssocited phenolic cids in cell wlls, -6. In: Wilkinson DL (Ed). Moleculr Plnt Pthology. Oxford Press, Oxford. Hmmerschmidt R, Nuckleus E, Kuc J (98). Assocition of enhnced peroxidse ctivity with induced systemic resistnce of cucumer to Colletotrichum lgenrium. Physiol Plnt Pthol :6-7. Heth R, Pcker J (968). Photoperoxidtion in isolted chloroplst: I. Kinetics nd stoichiometry of ftty cid peroxidtion. Arch Biochem Biophys : ISTA (). Interntionl Rules for Seed Testing Interntionl Seed Testing Assocition, Bssersdorf, Switzerlnd, p. Ivnov Z (989). The Agriculturl Experimenttion Puelo y Educción, Hvn, p. 9 Mrtínez-Montero ME, Mor N, Quiñones J, González-Arno MT, Engelmnn F, Lorenzo JC (). Effect of cryopreservtion on the structurl nd functionl integrity of cell memrnes of sugrcne (Scchrum sp.) emryogenic clluses. Cryoletters :7-. Pérez A, Ludt T, Mor M, Crvjl C, Argón C, González J, Esclon M, Dquint M, Trujillo R, Hernández M, Lorenzo JC (). Micropropgtion of Hohenergi penduliflor (A. Rich.) Mez. for sustinle production of plnt proteses. Act Physiol Plnt (8):-7. Pérez G, Ynez E, Mogholi A, Vlle B, Sgrr F, Yor L, Argón C, González J, Isidrón M, Lorenzo JC (). New pinepple somclonl vrints: PR nd Dwrf. Am J Plnt Sci :-. Porr R (). The chequered history of the development nd use of simultneous equtions for the ccurte determintion of chlorophylls nd. Photosynth Res 7:9-6. Stndwood P, Bss L (98). Seed germplsm preservtion using liquid nitrogen. Seed Sci Technol 9:. Yor L, Arzol M, Argón C, Hernández M, Arencii A, Lorenzo JC (6). Biochemicl side effects of genetic trnsformtion of pinepple. Plnt Cell Tiss Org Cult 86:6-67. Ynes PE, González J, Sánchez R (). A technology of cclimtiztion of pinepple vitroplnts. Pinep News 7:. Zevllos B, Cejs I, Vlle B, Yor L, Argón C, Engelmnn F, Mrtínez ME, Lorenzo JC (). Short-term liquid nitrogen storge of wild tomto (Solnum lycopersicum Mill.) seeds modifies the levels of phenolics in 7 dy-old seedlings. Sci Hort 6:6-67. Zevllos B, Cejs I, Rodríguez RC, Yor L, Argón C, González J, Engelmnn F, Mrtínez ME, Lorenzo JC (). Biochemicl chrcteriztion of Ecudorin wild Solnum lycopersicum Mill. plnts produced from non-cryopreserved nd cryopreserved seeds. CryoLetters :-.