This research work is dedicated to my beloved parents.

Transcription

1 An exmintion of the reltionship etween NO, ABA nd uxin in lterl root initition nd root elongtion in tomto A thesis sumitted in prtil fulfillment of the requirements for the degree of Mster of Science in Plnt Biotechnology By Mlini Sivnnthn t the 2006

2 This reserch work is dedicted to my eloved prents. ii

3 iii ABSTRACT The length of the primry root nd the density of lterl roots determine the rchitecture of the root. In this thesis the effect of NAA, ABA nd the NO donor SNP lone s well s the comintion of ABA or NAA with SNP on lterl root development ws investigted. The interction etween CPTIO, NO scvenger, nd NAA or SNP is lso reported. Following preliminry experiments in which it ws oserved tht the eril prt of the seedling influenced LR growth nd tht there ws possile inhiitory effect of light on cultured root tips, experiments were conducted with excised roots tips in the drk. NAA ws shown to hve the potentil to initite LRs cross wide concentrtion grdient with the totl numer of LRs nd initited lterl root primordi (LRP) remining constnt cross the rnge of concentrtions tested. Over the lst decde, nitric oxide (NO), ioctive molecule, hs een reported to e involved in the regultion of mny iologicl pthwys. The presence of NO in the system provided vi sodium nitroprusside (SNP), promoted LRP initition sed on the NAA concentrtion grdient; ut without chnging the totl LR initition, tht is LRs plus primordi density remined constnt long the concentrtion grdient of NAA. The sence of LR nd LRP in the tretments of CPTIO ( NO scvenger) with SNP or NAA suggests tht NO regultes LRP initition triggered y NAA, which is in greement with the recent pper pulished fter the commencement of this study (Corre-Argunde et l., 2006). In greement with previous studies, ABA inhiited lterl root development y reducing LR density nd the numer of LRs. The experiments with fluridone, n ABA iosynthesis inhiitor, my indicte tht endogenous ABA ws t sufficient concentrtions in the excised root tips to inhiit primordi initition. In this study, evidence is presented for the first time to show tht SNP cn relieve the inhiitory effect of ABA on LR density nd numer of LRs suggesting the NO, relesed from SNP, cts downstrem of ABA. Overll these dt confirm criticl role for NO in LR initition.

4 iv CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF PLATES ABBREVIATIONS i ii iii iv CHAPTER 1.0 INTRODUCTION Overview Structure nd formtion of lterl roots (LRs) Fctors ffecting lterl root initition nd elongtion Environmentl fctors Hormones Auxin nd LR initition/elongtion Ascisic cid (ABA) nd LR initition/elongtion Cytokinin nd LR initition/elongtiongierellins Gierellins nd root initition/elongtion Ethylene nd root initition/elongtion Nitric Oxide (NO) NO in niml kingdom Involvement of NO in plnts Mesurements nd nlysis of results Mesurements from the experiments Density of LRs Aims nd ojectives 17

5 v CHAPTER 2.0 MATERIALS AND METHODS Mterils The plnt mteril Sources of plnt growth regultors nd other chemicls Medi Methods Preprtion of sl medium Steriliztion Seed germintion The effect of ABA on seedling roots nd excised root tips The effect of SNP on seedling roots nd excised root tips The effect of SNP on seedling roots in growth room with 14 h of light nd 10 h of drkness The effect of SNP on excised root tips in complete drkness Interction etween ABA nd SNP on excised root tips of tomto The effect of NAA on excised root tips of tomto The effect of fluridone on excised root tips of tomto Interction etween NAA nd SNP on excised root tips of tomto The effect of CPTIO on NAA nd SNP on excised root tips of tomto Imge nd dt nlysis Imge scnning Sttisticl nlysis 29

6 vi CHAPTER 3.0 RESULTS Morphology of seedlings nd n excised root tip grown in White s medium Development of lterl root primordi The effect of ABA on seedling roots nd excised root tips The effect of SNP on seedling roots nd excised root tips The effect of SNP on seedling roots in growth room with 14h of light nd 10h of drkness The effect of SNP on excised roots in growth room with 14h of light nd 10h of drkness The effect of SNP on excised root tips in complete drkness Interction etween ABA nd SNP on excised root tips of tomto The effect of NAA on excised root tips of tomto The effect of fluridone on excised root tips of tomto Interction etween NAA nd SNP on excised root tips of tomto The effect of CPTIO on SNP nd NAA on excised root tips of tomto 78 CHAPTER 4.0 DISCUSSION Preliminry experimentl studies Seedling roots versus excised root tips The effect of initil length of the root segments The effect of light on LR elongtion Confirmtion of pulished dt ABA inhiits LR emergence SNP promotes LR density NAA promotes LR initition nd elongtion Interction etween NAA nd NO 93

7 vii 4.4 NO opertes downstrem of ABA The effect of CPTIO on SNP nd NAA Conclusions Directions for future study 98 CHAPTER 5.0 REFERENCES 100 CHAPTER 6.0 APPENDICES 109 Appendix 1 Preprtion of White s medium 109 Appendix 2 Prepring the ABA solution 111 Appendix 3 Prepring the SNP solution 112 ACKNOWLEDGEMENTS 113

8 i LIST OF FIGURES Figure 1.1 Digrmmtic illustrtion of LRP formtion in Aridopsis 5 Figure 2.1 Figure 3.1 Figure 3.2 Figure 3.3 Typicl digrm to illustrte the mesurements of different prmeters on seedling nd n excised root tip 23 The effects of ABA on 4-dy-old, 2 cm long seedling roots nd 4-dy-old, 2 cm long root tips of tomto, cultured t 25±1 C in complete drkness. 33 The effect of SNP on 3-dy-old, 1 cm long tomto seedling roots in growth room with dily photoperiod of 14 h of light nd 10 h of drkness. 37 The effects of SNP on 4-dy-old, 2 cm long excised root tips of tomto cultured in complete drkness. 43 Figure 3.4 Interction etween ABA nd SNP on LR density. 49 Figure 3.5 Interction etween ABA nd SNP on the numer of LRs. 50 Figure 3.6 Interction etween ABA nd SNP on the PR length. 51 Figure 3.7 Interction etween ABA nd SNP on the length of the longest LR. 52 Figure 3.8 Interction etween ABA nd SNP on picl distnce. 53 Figure 3.9 Figure 3.10 The effect of NAA on 4-dy-old, 2 cm long excised root tips of tomto. 55 The effect of fluridone on 4-dy-old, 2 cm long tomto excised root tips. 60 Figure 3.11 (i) Interction etween NAA nd SNP on LR density. 67 Figure 3.11 (ii) Interction etween NAA nd SNP on primordi density. 68 Figure 3.11 (iii) Interction etween NAA nd SNP on LR plus primordi density. 69 Figure 3.12 (i) Interction etween NAA nd SNP on the numer of LRs. 70 Figure 3.12 (ii) Interction etween NAA nd SNP on the numer of primordi. 71 Figure 3.12 (iii) Interction etween NAA nd SNP on the numer of LR plus primordi. 72

9 ii Figure 3.13 Interction etween NAA nd SNP on the PR length. 73 Figure 3.14 Interction etween NAA nd SNP on the length of the longest LR. 74 Figure 3.15 (i) Interction etween NAA nd SNP on LR picl distnce. 75 Figure 3.15 (ii) Interction etween NAA nd SNP on primordi picl distnce. 76 Figure 3.15 (iii) Interction etween NAA nd SNP on LR plus primordi picl 77 distnce. Figure 3.16 Figure 3.17 The effect of CPTIO on SNP nd NAA on the LR, primordi nd LR plus primordi density. 80 The effect of CPTIO on SNP nd NAA on the numer of LRs, primordi nd LR plus primordi 82 Figure 3.18 The effect of CPTIO on SNP nd NAA on the PR length. 84 Figure 3.19 Figure 3.20 The effect of CPTIO on SNP nd NAA on the length of the longest LR 85 The effect of CPTIO on SNP nd NAA on the LR, primordi nd LR or primordi picl distnce 86 LIST OF TABLES Tle 3.1 The effects of SNP on 3-dy-old, 1 cm long excised root tips of tomto incuted in growth room with 14 h of light nd 10 h of drkness. The vlues re mens ± SE from 3 independent experiments (n=16). 41

10 iii LIST OF PLATES Plte 2.1: Tomto seedlings germinted for four dys in drkness t 25±1 C 21 Plte 3.1: Morphology of seedlings nd n excised root tip grown in White s medium 30 Plte 3.2: Lterl root primordi 31 Plte 3.3 Plte 3.4 Plte 3.5 Photogrphs of 2 cm long excised tomto root tips cultured for 7 dys in complete drkness t 25±1 C. 42 Photogrphs of 2 cm long tomto root tips incuted for 7 dys in different concentrtions of NAA in drkness t 25±1 C. 54 Photogrphs of 2 cm long excised root tips of tomto treted with NAA or SNP nd together with CPTIO 78

11 iv ABBREVIATIONS ABA - Ascisic cid C - Celsius CPTIO - 2-(croxyphenyl)-4,4,5,5-tetrmethylimidzoline-1-oxyl-3-oxide DAF-2 DA - 4,5-diminofluorescein dicette dh 2 O - Distilled wter g - grm H - Hour L - Litre LR - Lterl root LRP - Lterl root primordi M - Molr mg - milligrm Min - Minutes ml - millilitre mm - millimeter M.W - Moleculr weight NAA - 1-nphthylcetic cid NO - Nitric oxide NR - Nitrte reductse NOS - Nitric oxide synthse PR - Primry root SNP - Sodium nitroprusside Soln - Solution µm - Micro molr

12 1 CHAPTER 1 INTRODUCTION 1.1 Overview Roots re of equl importnce to shoots s the life support system of plnts. In ddition to providing nchorge, roots help to sor wter, fcilitte the extrction of micro- nd mcro-nutrients nd trnsport nutrients to the oveground prts. Also roots re one of the sites tht synthesise mny metolites tht re essentil for plnt (Durovsky et l., 2006). The length of the primry root (PR) nd the density of lterl roots (LRs) determine the rchitecture of the root system (Mlmy nd Benfey, 1997). Three mjor processes tht ffect the overll rchitecture of the root system re: (1) cell division t the primry root meristem tht enles continuous growth y dding new cells to the root, (2) LR formtion, which increses the explortory cpcity of the root system, nd (3) root hir formtion, which increses the totl surfce of the PR nd LRs (Lopez-Bucio et l., 2003). The ntomy of the root is very consistent throughout the higher plnts, ut the numer, plcement nd the direction of growth of ech root in the system re highly vrile, even mong geneticlly identicl plnts (Mlmy, 2005). The focus in this thesis is on lterl roots. Lterl root initition is influenced y complex interctions mong different hormonl nd environmentl fctors. The numer nd loction of LRs re not predetermined: ech plnt integrtes informtion from its own environment nd this influences root initition nd elongtion (Mlmy nd Ryn, 2001). For exmple, vilility of nutrients plys n importnt role in oth the numer nd the plcement of LRs (Leyser nd Fitter, 1998). In soil or medium with ptchy nutrient distriution, LRs preferentilly proliferte in the nutrient-rich zone (Drew et l., 1973; Drew nd Sker, 1975; Roinson, 1994).

13 2 Lterl root initition nd elongtion is lso under hormonl control. Genetic nd physiologicl evidence suggests tht uxin plys n importnt role t severl specific developmentl stges in LR formtion (Csimiro et l., 2003). Overproduction of uxin or ppliction of exogenous uxin leds to incresed numers of LRs (Blkely et l., 1988; Celenz et l., 1995; Boerjn et l., 1995; Himnen et l., 2004). Further studies showed tht endogenous nitric oxide (NO) is lso involved in LR initition, suggesting possile interction of NO with uxin ( Corre-Argunde et l., 2004; Corre-Argunde et l., 2006). Another plnt hormone, scisic cid (ABA), hs een shown to ffect LR development y inhiiting LR initition (De Smet et l., 2003). The ltest studies show tht the inhiitory effect of ABA is likely to e medited y n uxin-independent pthwy (De Smet et l, 2003). Root growth is n importnt component of plnt growth ut hs received little ttention from plnt reeders ecuse of difficulties ssocited with oserving roots in situ. Understnding the fctors ffecting LR formtion nd their interctions is crucil for modulting the rchitecture of the root system. The ility to modulte root rchitecture helps to overcome the inility of plnts to move towrds wter or nutrients nd thus mximize crop production. However, the mechnisms y which plnts incorporte these fctors into LR initition nd elongtion re poorly understood. In this thesis the focus is on the interction etween uxin, NO nd ABA.

14 3 1.2 Structure nd formtion of lterl roots (LRs) Most of the higher plnts hve three types of roots. They re the min root (tp root), lterl root nd dventitious root (Fitter, 1991). The first root to emerge from germinted seed is the rdicle which, in most dicotyledons, enlrges to form prominent min root. But in monocots, the rdicle is short-lived nd is replced y mss of dventitious roots ( Dicotyledonous plnts produce one or more orders of lterl root rnches. The min root produces the first order lterls nd these produce the second order lterls nd so on. The different orders of roots vry in mny wys, for exmple, in their thickness, rnching ptterns, growth rtes, cpcity for secondry growth, life spns nd structurl fetures. These vritions will influence their cpcity to otin wter nd nutrients, support mycorrhizl ssocitions nd survive dverse conditions. Higher order lterl LRs re generlly thinner, shorter nd do not live s long s those of lower orders ( The meristem of the min root is formed during emryogenesis, wheres in mny plnts, the meristems of lterls nd dventitious roots re formed post-emryogeniclly (Mlmy nd Benfey, 1997). However, in some groups such s the Cucuritcee, lterl root primordi (LRP) re initited during emryogenesis (Durovsky, 1986). The process of LR development tkes plce in three steps. These steps re initition, orgnistion nd emergence. In this thesis, mesurements of elongtion incorported emergence hs een evolved. Mny studies hve een done on LR development, ut the sic mechnisms tht control LR initition re poorly understood (Durovsky et l., 2006). The exct mechnism vries etween ech plnt species, ut the sic fetures re common to ll species. In most plnts LRs re initited from single lyered pericycle. Generlly the pericycle is the exterior cell lyer of the provsculr cylinder which is surrounded y the ground meristem (cortex + endodermis) nd epidermis, ut in cucumer, the pericycle ecomes multilyered (Durovsky, 1986). In some Pteridophytes, second tissue, the endodermis prticiptes in lterl root primordil

15 4 (LRP) formtion (Lin nd Rghvn, 1991). Rrely, the cortex cn lso prticipte in LRP formtion. In Aridopsis thlin, which hs simple root ntomy composed of single lyers of epiderml, corticl nd endoderml cells surrounding the vsculr tissues, LRP re derived from the pericycle. LR initition in tomto is similr to tht in Aridopsis (Lskowski et l., 1995). Lterl root primordi cn e initited in two different wys. In Aridopsis nd some other ngiosperms, longitudinl icellulr type of LRP initition ws first discovered. In this type, two djoining cells long cell file opposite to the xylem poles ecome founder cells. Another type of initition, the longitudinl unicellulr type, occurs when only one cell long the file ecomes founder cell nd prticiptes in primordium formtion (Durovsky et l., 2001). However, longitudinl unicellulr type of LRP initition is rrely found (Blkely et l., 1982). Pericycle founder cells re descried s cells tht cquire developmentl fte different from tht of their mother nd, s consequence, ply principl role during the first stges of LR initition (Durovsky et l., 2000). Initilly, the two pericycle founder cells within the sme cell file undergo lmost simultneous polrized symmetric trnsverse divisions nd crete two short cells flnked y two longer cells in Aridopsis (Lskowski et l., 1995). These cells undergo symmetricl nd symmetricl division nd crete group of short cells tht re similr in length s descried in Figure 1.1 (Csimiro et l., 2001; Durovsky et l., 2001). A similr series of mitotic division lso occurs in oth pericycle cell files followed y rdil expnsion (Csimiro et l., 2003). The centrl short dughter cells divide severl times periclinlly in the meristemtic zone nd form dome-shped LRP. These cells rpidly elongte nd differentite towrds the se within the prent root, giving rise to n orgnised primordium (Mlmy nd Benfey, 1997; Csimiro et l., 2003). This LRP forms functionl meristem nd elongtes, cusing the LR to emerge through the epidermis. LR emergence ppers to e due to expnsion of existing cells rther thn cell division (Mlmy nd Benfey, 1997). Orgnistion into LRP requires sufficient cell

16 5 divisions so tht there re t lest three pericycle founder cell lyers (Durovsky et l., 2001). Figure 1.1 Digrmmtic illustrtion of LRP formtion in Aridopsis. Two types of lterl root primordium initition in Aridopsis thlin. A, unicellulr longitudinl; LRP cells re enclosed in cell wll of the founder cell 6-8 mm from the root tip. B, Longitudinl icellulr. Erliest stge of LRP initition on histologicl section of the root portion 2-4 mm from the root tip. Note, the first symmetricl division in two pericycle cells leding to LRP formtion. Anticlinl cell wlls of the founder cells re mrked with sterisks. Loction of the cell wll resulting from the first division of founder cells is mrked y rrowhed. Blck r represent 20 µm. (Durovsky et l., 2001, reproduced with permission of Springer-Verlg, cited in As descried ove, in most plnts LRs re initited t the protoxylem poles. In tomto, primordi initited either opposite to the protoxylem pole or slightly to one or other side of it, which gives dirch pttern (Brlow nd Adm, 1987). But the position of LRs long the min root is poorly understood. Since the spcing etween the LRs is regulr, it hs een suggested tht there must e positionl mechnism tht cuses regulr spcing

17 6 (Chrlton, 1991). Further study hs shown tht the presence of LRP gurntees the initition of the other primordium in different pole (Michel et l., 2006). Generlly, LRs re initited cropetlly long the root, where the primordi re initited distl reltive to the lredy initited primordi. However, erlier stges of LRP initition cn e found sipetlly in the roots, where the primordi re initited proximl reltive to the lredy initited primordi (Durovsky et l., 2000). Primordi re initited t certin distnce ehind the pex nd this distnce remins lmost constnt s the PR elongtes. Therefore primordium initition dvnces cropetlly in tomto (Brlow nd Adm, 1987). 1.3 Fctors ffecting lterl root initition nd elongtion There re two different mechnisms or pthwys tht determine the rchitecture of the root system: n intrinsic pthwy nd response pthwy. The former is essentil for orgnogenesis nd growth tht determines the chrcteristic rchitecture of the plnt. The ltter determines how plnts respond to externl signls nd modultes the intrinsic pthwy (Mlmy, 2005). The intrinsic determinnts determine LR initition, the developmentl pttern of the primordium, LR formtion nd growth. However, the morphology of the root system is lso drmticlly influenced y environmentl signls (Forde nd Lorenzo, 2001; Lopez-Bucio et l., 2003) Environmentl fctors Environmentl fctors such s nutrients, wter, light, temperture, soil texture, ph nd grvitropic signls hve significnt influence on root rchitecture (Durovsky nd Rost, 2003). Soil nutrients re criticl elements for plnt growth nd productivity. The ility of plnt to respond ppropritely to nutrient vilility is essentil for it to dpt to the environment (Lopez-Bucio et l., 2003). Nutrients such s nitrte, phosphte, sulphte nd iron ct s signls nd ctivte moleculr mechnisms tht modify cell division nd cell differentition processes within the roots which, eventully, chnge the root

18 7 rchitecture. Nutrients such s nitrte nd phosphte hve n importnt role in LR elongtion. Zhng nd Forde (1998) hve shown tht n incresing nitrte concentrtion reduces PR elongtion ut does not cuse ny chnge in the LR density ecuse the - numer of LRs re lso reduced. The NO 3 signl ppers to hve two distinct effects. Exposure of Aridopsis roots to high concentrtion of nitrte (10 mm) retrded the elongtion of LRs. However, when split root system ws used nd when the plnt ws grown on low nitrte concentrtion (10 µm), ut with prt of the root system exposed to high nitrte concentrtion (10 mm), LR elongtion ws promoted only on the low - NO 3 side. This suggests tht the inhiitory effect is response to nitrte sufficiency (Zhng nd Forde, 1998). Studies on Aridopsis hve shown tht high concentrtions of phosphte (P) increse PR length nd no chnge in the numer of LRs results in reduction in LR density (Linkohr et l, 2002). A moderte concentrtion of P (100 µm) results in more LR growth thn PR growth. Low sulphte concentrtions incresed the numer of LRs (Kutz et l., 2002). The response of root rchitecture to nutrients cn e modified y plnt growth regultors, suggesting tht the nutritionl control of root elongtion my e medited y hormones (Lopez-Bucio et l., 2003). The grvity vector ppers to e n importnt fctor in controlling the position of the PR nd LRs. The initil growth of LRs is nerly horizontl in orienttion. But when the LRs elongte, they chnge to ner-verticl position (Mullen nd Hngrter, 2003). This chnge in orienttion coincides with the increse in growth rte (Mullen et l., 2005). Light is n importnt fctor which controls growth nd differentition of plnt cell, tissue nd orgn cultures. Mny studies show tht light controls in vitro root formtion. Light stimultes root formtion in rhizome frgments of Helinthus tuerosus (Gutheret, 1969). Light hs different effects on root initition nd root elongtion (Seko nd Nishimur, 1996). For instnce, light stimultes root initition nd inhiits root elongtion in whet (Vinterhlter et l., 1990). However, trnsfer of plnts from drkness to light induces elongtion fter short lg period. The reverse trnsfer from light to drkness

19 8 efficiently rrests root elongtion ut promotes the ppernce of LRs (Vinterhlter et l., 1990) Hormones LR development is lso under hormonl control. Since most hormones exist t some sl level in plnts under growth sitution, hormonl effects on root system rchitecture cn e considered to e intrinsic. However, hormones re lso importnt components of response pthwys, since their levels cn e djusted in response to environmentl signls. Mlmy (2005) suggests tht overlpping hormone signlling pthwys provide the complexity tht is constntly integrting informtion from mny sources into decision to initite LR. There re mny different hormones involved in LR initition nd elongtion Auxin nd LR initition/elongtion Among the plnt hormones, uxin is the only sustnce tht is ctively distriuted throughout the entire plnt. Auxin is iosynthesised typiclly in young, proliferting prts of the shoot nd is more undnt in shoots thn in roots (Ljung et l., 2001; Ljung et l., 2005). It is trnsported sipetlly down the plnt, tht is, sipetlly down the shoot nd cropetlly to the root tip (Bhlero et l., 2002). Accumulted uxin in the root tip is trnsported sipetlly towrds the elongtion zone of the root (Rshotte et l., 2000; Csimiro et l., 2001; Ottenschlger et l., 2003). Indole-3-cetic cid (IAA), phenyl cetic cid (PAA), chlorinted form of IAA (4-Cl-IAA) nd indole-3-utyric cid (IBA) re some of the nturlly occurring compounds tht show uxin-like effects (Ludwig- Muller nd Cohen, 2002). Auxin plys n importnt role in LR initition nd elongtion. Synthetic uxin such s 1- nphthylcetic cid (NAA) nd 2,4-D-relted compounds hve een used commercilly for mny yers. In severl experiments on different plnts, similr uxin effect on LR

20 9 elongtion ws found. Incresing the uxin level resulted in significnt increse in the numer of LRs (Blkely et l., 1998). A recent study showed tht exogenous uxin incresed the proility of inititing LRP within the sme vsculr pole from two to six times (Michel et l., 2006). Internl uxin overproduction y trnsgenic plnts lso resulted in n incresed numer of LRs (Boerjn et l., 1995; Lskowski et l., 1995). Csimiro et l. (2001) confirmed the effect of uxin on LRP initition y growing Aridopsis seedlings with n uxin trnsport inhiitor N-1-nphthylphthlmic cid (NPA), which reduced the numer of primordi initited y inhiiting the uxin trnsporttion from shoot to root tip (Csimiro et l., 2001). In ddition, LR initition ws decresed in n uxin-insensitive mutnt, ux 1 (Csimiro et l., 2003). Auxin regultes ll three stges of LR development. In the presence of exogenous uxin, t ny developmentl stge, LRP cn develop into LR. However, in the sence of exogenous uxin, only LRP tht hve reched the stge of hving t lest 3-5 cell lyers cn develop into LRs. There ws no development oserved in the LRP which hd fewer thn three cell lyers (Lskowski et l., 1995). These oservtions suggest tht the development of LRP from 3-5 cell lyers is uxin-dependent nd eyond this stge, they re either uxin-independent or they produce the required mount of uxin themselves. Experiments with n errnt lterl root (lf3-1) mutnt show the importnce of uxin for LR initition eyond the 3-5 cell lyer stge (Celenz et l., 1995). Elongtion of the first LR coincides with the emergence of the first true leves. Experiments with Aridopsis seedlings showed tht young seedlings required shootderived uxin to develop LRs. As the root system develops, mture seedlings re less dependent on lef-derived uxin nd strt to produce their own uxin (Bhlero et l., 2002). This ws further supported y recent study, of oth the roots of seedlings nd excised roots of Aridopsis, showing tht uxin synthesis rtes nd levels in excised roots re very much less thn those of seedlings. In ddition, it strongly suggests tht developing LRs cn supply uxin t lter developmentl stges (Ljung et l., 2005).

21 10 The removl of the picl segment of the min root prior to the formtion of the first true leves hd little effect on LR initition ut inhiited LR emergence, suggesting tht uxin lso regultes LR emergence (Bhlero et l., 2002). Auxin intercts with other hormones nd nutrients in most kinds of developmentl processes. Reduction of LR initition with high C:N rtio ws correlted with the ccumultion of uxin in the hypocotyls (Mlmy nd Ryn, 2001). However, the ddition of exogenous uxin overcme this nutritionl effect, suggesting tht nutritionl cues were not locking the ility of the root to respond to uxin (Mlmy nd Ryn, 2001). Therefore, it is possile tht the reduction of LR initition could e due to lockge in the movement of uxin from the shoot to root. Studies in the pst mostly concentrted on the effect of uxin in its regultion of root initition nd elongtion. However, there is little informtion out the influence of uxin on the other spects of root rchitecture, such s the length of LRs nd picl distnce Ascisic cid (ABA) nd LR initition/elongtion ABA regultes norml root initition nd elongtion. A study showed tht endogenous ABA my inhiit LR initition ut it is possile tht ABA ffects different processes of root development differently, vi differing sensitivities to ABA (Hooker nd Thorpe, 1998). An exogenous supply of ABA inhiits LR initition y locking the ctivtion of LR meristems t the 3-5 cell lyer of LRP development. However, this inhiition is reversile in the sence of ABA (De Smet et l., 2003). Exogenous ppliction of uxin filed to rescue this ABA-induced rrest, suggesting tht the inhiitory effect of ABA is medited y n uxin-independent pthwy (De Smet et l, 2003). A recent study on legumes hs shown tht ABA exerts n opposite effect on LR elongtion in legumes. It reduced LR density y reducing the numer of LRs elongted in non-legumes ut promoted LR density y incresing the numer of LRs elongted in legumes regrdless of whether they were nodulting or non-nodulting legumes (Ling nd Hrris. 2005).

22 11 Hooker nd Thorpe, (1998) found tht the ppliction of fluridone, n ABA iosynthesis inhiitor, promoted oth the numer of emerged LRs nd LRP in the presence of ABA on tomto excised root tips. Furthermore, they showed tht ABA is involved in the regultion of picl dominnce in roots. Considerle evidence suggests tht ABA intercts with other signlling pthwys. It intercts with ethylene to control shoot nd root growth under wter stress (Shrp nd LeNole, 2002). ABA intercts with NO nd ethylene in gurd cells (Neill et l., 2002; Grci-Mt nd Lmttin, 2002; Desikn et l., 2004; Tnk et l., 2005). Recently it hs een shown tht ABA intercts with uxin signlling pthwys in roots (Rock nd Sun, 2005). In ddition, ABA is required to medite the regultory effects of nitrte on - root rnching. The inhiitory effect of high NO 3 ws reduced in ABA-insensitive - mutnts, suggesting tht the inhiitory effect of NO 3 my operte through n ABA signl trnsduction pthwy in mediting the nitrte effect on LR elongtion (Signor et l., 2001). However, the effect of ABA on LR initition nd elongtion is poorly understood. The interction of ABA with NO in gurd cells suggests tht there is possiility tht internl NO could regulte the effect of ABA on LR elongtion Cytokinin nd LR initition/elongtion Cytokinins re synthesised oth in shoot nd root, ut minly in the root. It is trnsported to shoots vi the xylem (Aloni et l., 2005), lthough it could lso e trnsported from shoots to roots vi the phloem (Gessler et l., 2004). Reduction in the cytokinin level in erly LRP development suggests potentil role for cytokinin in LR initition (Lohr et l., 2004). Mny reserches hve found tht, unlike uxin, cytokinin inhiits LR initition (Hinchee nd Rost, 1986). Further study with cytokinin deficient trnsgenic Aridopsis, which over expressed cytokinin oxidse/dehydrogense (AtCKX) gene nd incresed cytokinin rekdown confirmed the opposite function in the regultion of shoot nd root elongtion, tht is, reduced shoot picl meristems nd lef primordi growth nd incresed root initition (Werner et l., 2003). A recent study on rice y Dei et l. (2005)

23 12 shows tht the effect of cytokinin vries depending on developmentl stges of LR formtion. This effect is restricted to initition of LRP. They found tht no inhiitory effect ws evident on LR elongtion from the primordi tht hd lredy formed. Glis et l. (2005) further supported the inhiitory effect of cytokinin in LR initition in trnsgenic tocco plnt. Activity of AtCKX promoter ws oserved in the region where primordi developing nd this is supporting the previous finding tht cytokinin exerts n inhiitory role in the LR initition (Glis et l., (2005). Cytokinin not only inhiits LR initition ut lso promotes LR elongtion y incresing cell length (Dei et l., 2005). Cytokinin, together with uxin, plys n essentil role in the formtion of roots, shoots nd their reltive growth (Skoog nd Miller, 1957 in Werner et l., 2001). The inhiitory effect of cytokinin on LR initition nd promotion in elongtion cn e overcome y the ddition of exogenous uxin, suggesting tht cytokinin cts on n uxin-dependent pthwy (Dei et l., 2005). This finding grees with the previous studies tht the uxin:cytokinin rtio plys n import 98(18): nt role in LR elongtion (Hinchee nd Rost, 1986) Gierellins nd root initition/elongtion Gierellins (GA) re importnt regultory fctors in the control of shoot nd root growth (Richrds et l., 2001). GA is iosynthesised minly in hypocotyls nd root tips. The requirement of GA for norml root growth ws reveled y the use of chemicl inhiitors nd mutnts of GA iosynthesis. Experiments with muttions such s d5 in mize nd gi-1 in tomtoes showed tht root elongtion is slower in these mutnts thn in the wild type (Blusk et l., 1993). However, GA is ineffective on its own, lthough it regultes ethylene-medited growth responses in shoots nd roots (Steffens et l., 2006). GA, together with ethylene, promotes the numer of roots nd the growth rte of emerged roots.

24 Ethylene nd root initition/elongtion Ethylene is lso involved in the growth nd differentition of shoots nd roots. During flooding, more ethylene is produced nd it promotes LR formtion (Ymmoto et l., 1995). A decrese in the ethylene level with the ppliction of n ethylene iosynthesis inhiitor reduced the numer of dventitious roots produced y Rumex plusris ffected y flood (Visser et l., 1996). Aloni et l. (2006) hve found tht ethylene lso medites cluster root formtion under iron deficiency. Ethylene intercts with uxin signlling pthwys. Moreover, elevted ethylene concentrtion might interrupt polr uxin trnsport (Visser et l., 1996). Ethylene is lso involved in the effect of ABA on root growth. The inhiitory effect of ABA requires functionl ethylene-signlling cscde (De Smet et l., 2003). However, ethylene production is not the purpose of ABA ction; rther, ABA inhiits ethylene synthesis in order to mintin root growth t low wter potentil (Shrp, 2002). 1.4 Nitric Oxide (NO) NO is gseous free rdicl which serves s signl in plnts nd nimls. The iologicl significnce of NO ws recognised y Science in 1992 which nmed NO the Molecule of the yer (Koshlnd nd Koshlnd, 1992). Initilly it ws thought to e toxic y-product of oxidtive metolism. Lter it ws reveled tht it regultes mny cellulr functions. NO hs een more comprehensively studied in nimls thn in plnts, even though it ws first discovered in plnt (Klepper, 1979) NO in niml kingdom In mmmls, NO is n intercellulr nd intrcellulr signlling molecule with rod spectrum of regultory functions in the centrl nervous system, in pltelet inhiition, in progrmmed cell deth, in host response to infection nd in crdiovsculr nd immune

25 14 systems (Moncd et l., 1991; Lloyd-Jones nd Bloch, 1996). It is lso involved in oxidtive stress in geing nd ge-relted diseses (Bln et l., 2005). Nitric oxide synthse (NOS) is responsile for the primry source of NO, which hs three different isomers: enos (endothelil NOS), nnos (neuronl NOS) nd inos (inducile NOS) (Alderton et l., 2001). NOS oxidizes L-rginine to NO nd citruline Involvement of NO in plnts Although NO reserch in plnts is not s dvnced s in nimls, in the lst decde NO hs een shown to prticipte in mny physiologicl processes nd it hs ecome n incresingly populr trget of investigtion in plnts. NO synthesis in plnts ppers more complex thn in mmmls. It is synthesised vi oth nitrite- nd rginine- dependent mechnisms. Initilly high NO emission ws correlted with high nitrite levels nd nitrte reductse (NR) ctivtion (Rockel et l., 2002). However, more recently severl sources tht contriute to NO-medited responses hve een suggested (Corps et l, 2004). Nitrite-medited sources re NR, mitochondri, the poplst, nitrte-no reductse nd clss-2 hemogloin. The rginine-medited source is NOS (AtNOS1), which is different from known niml NOS protein (Guo, 2003). In ddition, NO is lso relesed from soils nd the mount vries depending on temperture, oxygen vilility, ph nd N- fertilistion rtes (Stohr nd Stremlu, 2006). The NO donor, sodium nitroprusside (SNP) ws shown to induce LR formtion in mize (Gouve et l., 1997), tomto seedlings (Corre-Argunde et l., 2004) nd dventitious root formtion in cucumer plnts (Pgnusst et l., 2002). The effect ws dosedependent nd the optimum concentrtion of SNP vried depending on plnt species nd the experimentl conditions. For exmple, the mximum iologicl response ws t 10 µm SNP for dventitious root formtion in cucumer hypocotyls nd 200 µm SNP for LR formtion in tomto seedlings.

26 15 A study pulished fter this project commenced showed tht NO modultes the expression of cell cycle regultory genes in tomto pericycle cells resulting in induced LR initition (Corre-Argunde et l, 2006). A further study with 2-(croxyphenyl)- 4,4,5,5-tetrmethylimidzoline-1-oxyl-3-oxide (CPTIO) demonstrted tht NO ws required for LR initition, ut not for LR emergence (Corre-Argunde et l., 2006). Gouve et l. (1997) reported tht NO hd no effects on IAA-induced cell expnsion, ut, s mentioned ove NO intercts with IAA in dventitious root elongtion in cucumer plnts (Pgnusst et l., 2002). Furthermore, trnsient increse in NO concentrtion occurred during dventitious root initition following tretment with IAA, suggesting role for NO in uxin-signlling pthwys. Antomicl studies exmined the formtion of dventitious root primordi in the IAA or SNP tretments, wheres none were found in the control (Pgnusst et l., 2004). The results of recent experiments crried out y dding CPTIO on different dys to 0.1 µm NAA-treted tomto seedlings suggest tht NO ws required for cell cycle progression nd estlishment of LRP in the pericycle ut not for the elongtion nd emergence of LRs (Corre-Arngunde et l., 2006). However, this study did not consider oth LRP nd LRs together in reltion to the effect of NAA or SNP. In ddition, similr effect on primordi development ws reported in either 0.1 µm NAA or 200 µm SNP. It would e interesting to find the reltionship etween SNP nd different concentrtions of NAA on oth LR initition nd elongtion. Different mechnisms hve een suggested for the involvement in stomtl closure vi ion chnnels nd chemicl messengers (Schroeder et l., 2001). More recently, it hs een suggested tht NO ccumultion in gurd cells ws necessry for the ABA-induced stomtl closure, plcing new component in the ABA signlling trnsduction pthwy. This ws further confirmed y the externl ppliction of ABA to gurd cells, which led to incresed ccumultion of NO. Tretments of gurd cells with NO scvenger, CPTIO, inhiited stomtl closure wheres tretment with NO donor, SNP, incresed stomtl closure (Grci-Mt nd Lmttin, 2002; Neill et l., 2002). The interction of ABA with NO in gurd cells suggests tht there is possiility tht internl NO could regulte the effect of ABA on LR elongtion.

27 16 NO cn e monitored y vrious techniques such s electron prmgnetic resonnce nd NO electrodes (Crwford nd Guo, 2005). NO-sensitive fluorophore 4,5- diminofluorescein dicette (DAF-2 DA) llows the detection of the presence of NO in oth nimls nd plnt cells (Kojim et l., 1998; Foissner et l., 2000). DAF-2 DA is hydrolysed in living cells y cytosolic esterses to relese 4,5-diminofluorescein (DAF- 2) which rects with NO to produce the fluorescent trizole derivtive trizofluorescein (DAF-2T), giving rise to incresed green fluorescence. Further confirmtion cn e done with 4-minofluoresceindicette (4-AF DA) which lcks one of the mino groups tht constitutes the NO specific domin of the DAF-2 DA molecule. Cesstion of incresed levels of green fluorescence with 4-AF DA confirms tht the green fluorescence corresponds to n ccumultion of endogenous NO, nd not to unspecific rection of the proe (Grci-Mt nd Lmttin, 2002). Although the dvntges of this technique re ovious, the vritions of DAF-2T nd DAF-2 re hrd to control in living ojects (Stohr nd Stremlu, 2006). They re highly dependent on ph, the vilility of oxygen, the presence of ntioxidtive sustnce nd of C 2+ (Zhng et l., 2002). However, such mechnisms were eyond this project. Severl lines of experiments will e needed to otin etter understnding of NO interction with the phytohormones. The identifiction of genes tht re responsile for the production of NO could led to the discovery of new signlling functions for mny metolic enzymes. In this thesis, source of NO (SNP) s well s limited supply of NO scvenger (CPTIO) ws used. 1.5 Mesurements nd nlysis of results Mesurements from the experiments In recent decdes, either dt otined from vrious experiments on seedlings or excised roots hve een presented in different wys. Some reserchers considered only the percentge of seedlings or excised roots with LRs s function of different concentrtions of hormones (Creus et l., 2005). Some reserchers considered the numer

28 17 of LRs, which were greter thn 0.5 mm while others considered LRs greter thn 1 mm (Hooker nd Thorpe, 1998). Some other experiments considered oth the numer of primordi nd the numer of LRs in order to find the effect of hormones on LR initition nd elongtion. Most of the experiments considered the PR length ut only few rticles considered the length of the longest LR Density of LRs LR density is customrily clculted s rtio of the numer of LRs to the PR length. Most studies in the pst did not include LRP in their nlysis, where LRP hve the potentil to develop s LR (Lopez-Bucio et l., 2003). This method of estimtion is pplicle only when in ll ecotypes nd t ll ges, the rtio etween the root portion covered y LRs nd the PR length remins constnt. This hppens vry rrely nd therefore, it is necessry to evlute the LR density in terms of the portion of the PR where LRs re present (Durovsky et l., 2006). 1.6 Aim nd ojectives NO ppers to e involved in most spects of plnt growth, development nd in response to oth iotic nd iotic environmentl system. In this reserch the im ws to exmine whether NO, ABA nd NAA interct to control LR initition nd elongtion. In the present study, severl ojectives were pursued using seedling roots nd excised root tips of tomto. They were: 1. To determine if tomto seedling roots or excised root tips were suitle model for studying LR initition nd elongtion y incuting seedlings nd root tips in White s medi (control tretment).

29 18 2. To determine if ABA inhiits LR initition nd/or elongtion y monitoring the response to n ABA iosynthesis inhiitor 3. To determine if NO opertes downstrem of NAA in LR initition nd elongtion y supplying source of NO, SNP. 4. To determine if NO opertes down strem of ABA in LR elongtion y supplying source of NO, SNP. 5. Confirm tht NO is key component of the signl trnsduction of NAA nd ABA y incuting root tips in medi contining the NO scvenger, CPTIO. Hypothesis NO is criticl downstrem component of the signl trnsduction pthwy leding to LR initition nd the effects of NAA nd ABA re medited y NO.

30 19 CHAPTER 2 MATERIALS AND METHODS 2.1 Mterils The plnt mteril A 25 g pcket of untreted tomto seeds (Lycopersicon esculentum Mill. cv. Money mker) were purchsed from Eco Seeds, Christchurch, New Zelnd nd stored t 4 C. Some experiments (2.2.6, 2.2.8, nd ) were crried out with seeds purchsed from Asin Seeds, Trdle, Npier, New Zelnd Sources of plnt growth regultors nd other chemicls Plnt growth regultors nd chemicls used in this work nd their sources re listed elow. Plnt growth regultors Source (±) cis, trns- scisic cid (99%) (ABA) Sigm Tissue culture regent, St.Louis, USA 1-Nphthylcetic cid (98.5%) (NAA) BDH Chemicls Ltd, Poole Englnd. chemicls Fluridone Duchef Biochemie, Netherlnds Sodium nitroprusside (99.0%) (SNP) BDH Chemicls Ltd, Poole Englnd. Chromium trioxide BDH Chemicls Ltd, Poole Englnd. CPTIO Sigm, St.Louis, USA

31 Medi White s modified medium supplemented with White s orgnics (Butcher nd Street, 1964) ws used for ll experiments. 2.2 Methods Preprtion of sl medium Stock solutions of mjor slts, White s orgnic supplement nd iron solutions were mde nd stored t 4 C (Appendix 1). The required volumes of the stock solutions were mixed nd sucrose (8 g/l) ws dded to the medium. Then the ph ws djusted to 5.7±0.1. Required volume of medi for ech tretment ws dispensed into ech Schott ottle (500 ml) nd sterilised Sterilistion Medi used for plnt tissue culture work were sterilised y wet utoclving, which is wet het sterilistion of 20 min t 121 C nd 120 kp. Petri dishes, filter pper nd pipette tips were sterilised y dry utoclving where dry ir is used for 20 min t 121 C nd 120 kp. Solutions such s NAA nd ABA were filter-sterilised using n ultr-filtrtion unit Millex GP, Millipore Corportion (22 µm) under septic conditions. SNP, CPTIO nd fluridone were lso filter-sterilised. Sclpels nd forceps were sterilised in 100% ethnol nd left to dry efore nd fter trnsferring root tips to ech of eight Petri dishes. Tomto seeds were surfce sterilised in lminr flow work sttion for 10 min with 5% (v/v) household lech contining 4.8% (w/v) sodium hypochlorite s n ctive gent. Then they were thoroughly rinsed three times with sterile distilled wter.

32 Seed germintion Surfce sterilised seeds were trnsferred to sterile Petri dishes (30 seeds per dish) contining one lyer of filter pper soked with 8 ml of sterile distilled wter (Plte 2.1). After sowing, seeds were incuted t 25±1 C in the drk. Plte 2.1: Tomto seedlings germinted for four dys in drkness t 25±1 C The effect of ABA on seedling roots nd excised root tips Fresh ABA stock solution (0.264 M) ws prepred y dissolving mg of ABA with severl drops of 1 N NOH, dded to dh 2 O, nd then the ph ws djusted to 5.7±0.1 efore it ws mde up to 50 ml with dh 2 O. The required mount of the ABA stock solution to e trnsferred to ech ottle of medium ws clculted. See Appendix 2 for the clcultions. In lminr flow cinet under septic conditions, ABA stock solution ws filter-sterilised efore it ws dded to ech ottle of the medium. For control, no ABA solution ws dded. Ech ottle of utoclved sl medium ws dispensed

33 22 (pproximtely 25 ml ech) into 16 sterile Petri dishes. ABA concentrtions tested in this experiment were 0.1 nd 1 µm. Apicl root segments ech of two cm long were excised from 4-dy-old tomto seedlings nd trnsferred to medi in eight Petri dishes (90 mm dimeter). Two root tips were plced in ech dish. Sixteen replictes per tretment were prepred. Similrly, 2 cm long seedlings were chosen nd trnsferred to medi in eight Petri dishes. Trnsferring of picl segments or seedlings ws crried out rndomly. All the Petri dishes were wrpped long the edge using polyethylene film. Both excised root tips nd seedlings were incuted for seven dys in drkness t 25±1 C. After seven dys, the numer of LRs which emerged (>1 mm long) from ech root tip ws counted nd ll the Petri dishes were scnned for further nlysis. The PR length, the picl distnce (the length from the root tip to the most picl lterl root) nd the length of the longest LR were mesured s descried in Figure 2.1 nd, using Imge Pro. LR density (the numer of lterl roots per cm of the portion of primry root xis where LRs re present) ws clculted s follows: LR density = No of LRs 10 (Length of PR LR picl distnce) This experiment ws repeted t lest three times nd the men vlue ws tken s the finl result. Dt were nlysed s descried in Section

34 23

35 The effect of SNP on seedling roots nd excised root tips The effect of SNP on seedling roots in growth room with 14 h of light nd 10 h of drkness The concentrtions of 50, 100 nd 300 µm SNP nd control were chosen to perform this experiment. Three-dy-old seedlings were used in this experiment. Stock solution (59.58 M) ws prepred y dissolving g of SNP in 10 ml dh 2 0. The required mount of this solution to e trnsferred to ech ottle of utoclved sl medium ws clculted. See the Appendix 3 for the clcultions. The rest of the experiment ws crried out exctly the sme s descried in Section ut 1 cm long picl segments nd 1 cm long seedling roots were used. All the Petri dishes were kept in growth room under 14 h of light:10 h of drk photoperiod t 25±1 C. Light period (60 µmol/m 2 /s) ws given y white fluorescent tues (Philips, the Netherlnds). After five dys of tretment, mesurements were tken s descried in Section The effect of SNP on excised root tips in complete drkness A rnge of concentrtions of SNP (50, 100, 300 nd 500 µm) ws chosen in ddition to the control (no SNP). The experiment ws crried out s descried in Section 2.2.4, ut with excised root tips only. After seven dys, mesurements were tken s descried in Section Interction etween ABA nd SNP on excised root tips of tomto The following grid ws designed to test the effect of comintions of different ABA nd SNP concentrtions. ABA nd SNP stock solutions were prepred nd filter-sterilised under septic conditions. Eight ottles of medi (contining 200 ml ech) were prepred nd utoclved. The required mount of stock solution to e trnsferred to ech ottle

36 25 ws clculted nd trnsferred under septic conditions. Ech ottle of medi ws dispensed into eight sterile Petri dishes. Apicl root segments (ech 2 cm long) were excised from 4-dy-old seedlings nd trnsferred to Petri dishes. All the Petri dishes were incuted t 25±1 C in complete drkness. After seven dys, mesurements were tken s descried in Section ABA 0 1 µm SNP µm µm µm The effect of NAA on excised root tips of tomto This experiment ws crried out with 50, 100, 500 nm NAA nd control (without NAA). NAA stock solution (0.186 M) ws prepred y dissolving 18.6 mg of NAA with severl drops of 1 N NOH nd the ph ws djusted to 5.7±0.1. This solution ws mde up to 100 ml with dh 2 O. The required mount of this solution to e trnsferred to ech ottle of utoclved medium ws clculted. The rest of the experiment ws crried out exctly the sme s descried in Section After seven dys of tretment, mesurements were tken s descried in Section

37 The effect of fluridone on excised root tips of tomto Fluridone stock solution (6 M) ws prepred y dissolving 9 mg in 1.5 ml of 100% ethnol. Therefore sterilistion ws not necessry for this solution. Two different controls in ddition to 1 nd 0.1 µm fluridone were prepred for this experiment. Neither fluridone nor ethnol ws dded to one of the controls. The equl volume of 100% ethnol s fluridone stock solution ws dded to nother control in order to check whether the ethnol used to dissolve fluridone might hve ny influence on the result. The experiment ws crried out s descried in Section After seven dys, mesurements were tken s descried in Section After the root mesurements were completed the roots were soked in 5% (W/V) chromium trioxide for 5 min nd the numer of lterl root primordi (LRP) ws counted using light microscope. Emerged LRs tht were less thn 1 mm long were lso counted s primordi. Primordi picl distnce (the length from the root tip to the most picl primordium) ws mesured s descried in Figure 2.1 (). LR plus primordi picl distnce ws lso mesured (the length from the root tip to the most picl LR or primordium). Primordi density nd lterl plus primordi density ws clculted s descried in Hooker nd Thorpe (1998) nd the formule used re shown elow: Primordi density = (Numer of primordi) 10 (Length of primry xis Primordi picl distnce)

38 27 (Numer of lterls + Numer of primordi) 10 Lterl plus primordi density = (Length of primry xis The shortest length of either picl distnce or primordi picl distnce) LR density ws clculted s descried in Section Interction etween NAA nd SNP on excised root tips of tomto The following grid ws designed to test the effect of the comintions of different concentrtions of NAA nd SNP: SNP µm NAA nm nm nm nm 9 10

39 28 SNP nd NAA stock solutions were prepred (Sections nd 2.2.7) nd filtersterilised under septic conditions. Ten ottles of sl medi (contining 200 ml ech) were prepred nd utoclved. The required mount of stock solution to e trnsferred to ech ottle ws clculted nd trnsferred under septic conditions. The medium in ech ottle ws dispensed into eight sterile Petri dishes. Apicl segments (ech 2 cm long) were excised from 4-dy-old seedlings nd trnsferred to Petri dishes. All the Petri dishes were incuted t 25±1 C in complete drkness. After seven dys, mesurements were tken s descried in Section The numer of LRP ws counted s descried in Section The effect of CPTIO on NAA nd SNP on excised root tips of tomto The following experiment ws designed in order to test the effect of CPTIO on SNP nd NAA. The tretments of SNP (500 µm), NAA (50 nm), CPTIO (1 mm) nd control, in ddition to the mixture SNP with CPTIO nd NAA with CPTIO were crried out in this experiment. SNP stock solution ws prepred y dissolving g of SNP in 10 ml dh 2 O nd NAA stock solution ws prepred y dissolving mg in 2 l dh 2 O. CPTIO stock solution ws prepred using 26.2 mg in 10 ml dh 2 O. All the stock solutions were filter-sterilised under septic conditions. Six ottles of sl medi (contining 25 ml ech) were prepred nd utoclved. The required mount of stock solution to e trnsferred to ech ottle ws clculted nd trnsferred under septic conditions. The medium in ech ottle ws dispensed into five glss vils (5 ml ech). Apicl segments (ech 2 cm long) were excised from 4-dy-old seedlings nd trnsferred to glss vils (ech with totl cpcity of 25 ml). One root tip ws trnsferred to ech glss vil nd five replictes were prepred for ech tretment. All the glss vils were incuted t 25±1 C in complete drkness. After seven dys, mesurements were tken s descried in section Then LRP ws counted s descried in section

40 Imge nd Dt nlysis Imge Scnning All smples were scnned using ScnMkerX12 USL (MICROTEK, Chin) nd ScnWizrd 5.85 (Microtek Interntionl, Inc) s the softwre Sttisticl nlysis Dt were nlysed ccording to stndrd error of the men, where dt were grouped from replicte experiments. One-wy ANOVA ws performed to test whether the tretment groups differed significntly. Tukey s lest significnt difference test ws conducted using Sttistix 8 (Microsoft Corportion, Settle, USA) to perform multiple comprisons for determining which men scores were significntly different.

41 30 CHAPTER 3 RESULTS 3.1 Morphology of seedlings nd n excised root tip grown in White s medium Photogrphs of seedlings nd n excised root tip grown in culture medi (control) with 14 h of light nd 10 h of drkness (Plte 3.1 ) or complete drkness (Pltes 3.1 nd c) re shown elow. Seedlings exposed to light developed thick rigid stem, roots nd green leves, wheres seedlings cultured in complete drkness were etiolted nd hd soft stem nd roots. () () (c) Plte 3.1: Morphology of seedlings nd n excised root tip grown in White s medium. () Photogrph of 1 cm long seedling root grown in culture medi in growth room with 14 h of light nd 10 h of drkness t 25±1 C. () Photogrph of 2 cm long seedling root grown in culture medi in complete drkness t 25±1 C. (c) Photogrph of 2 cm long excised root tip grown in culture medi in complete drkness t 25±1 C.

42 Development of lterl root primordi LRP developed in 2 cm long excised root tips treted with 1000 nm NAA re shown elow. Presence of LRP on the root tip is shown in Plte 3.2. Initition nd elongtion stges of primordi oserved under light microscope re shown in Plte 3.2 (i) nd (ii) respectively. () () (i) (ii) Plte 3.2: Lterl root primordi () A photogrph to descrie the position of LRP on excised root tip treted with 1000 nm NAA ws tken with Olympus Cmedi 5.1 Meg pixel. () A typicl imge of lterl root primordium treted with 5% (W/V) chromium trioxide. Photogrphs were tken with Olympus Cmedi 5.1 Meg pixel under compound light microscope (Olympus BH2). Primordi development t (i) n erly stge (ii) lter stge. Br = 50 µm

43 The effect of ABA on seedling roots nd excised root tips in complete drkness Both seedling roots nd excised root tips, which were cultured in complete drkness showed similr responses to the ABA concentrtions used in this experiment. The presence of 1 µm ABA in the culture medium significntly reduced the LR density compred with the control in oth seedlings roots nd the excised root tips (Figure 3.1 ). However, LR density for the excised root tips ws less thn the seedling roots for the concentrtions tested ove. Similr responses were oserved in the numer of LRs (Figure 3.1 ). The PR length of seedling roots ws reduced t 1 µm ABA lthough no sttisticl difference ws oserved within the rnge tested in excised root tips (Figure 3.1 c). The PR length of excised root tip is less thn the seedling roots t the sme concentrtions except t 1 µm ABA, where, it ws slightly longer in excised root tips thn in seedling roots. The length of the longest LR of excised root tips ws reduced t 1 µm ABA. However, no sttisticl difference ws oserved within the rnge tested in seedling roots (Figure 3.1 d). No sttisticl difference ws oserved in picl distnce of oth seedling roots nd excised root tips (Figure 3.1 e).

44 33 Figure 3.1 The effects of ABA on 4-dy-old, 2 cm long seedling roots nd 4-dy-old, 2 cm long excised root tips of tomto, cultured t 25±1 C in complete drkness. () lterl root density, () numer of lterl roots, (c) primry root length, (d) length of the longest lterl root nd (e) picl distnce. The vlues re mens ± SE from three independent experiments (n=16). Dimonds re used for seedlings nd squres re used for excised root tips. Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

45 34 () LR density (No. of LRs/cm) Concentrtions of ABA (µm) () 30 No. of LRs/root Concentrtions of ABA (µm)

46 35 (c) 180 Length of PR (mm) Concentrtions of ABA (µm) (d) 30 Length of longest LR (mm) Concentrtions of ABA (µm)

47 36 (e) 70 Apicl distnce (mm) Concentrtions of ABA (µm) 3.4 The effect of SNP on seedling roots nd excised root tips The effect of SNP on seedling roots in growth room with 14 h of light nd 10 h of drkness A NO donor, SNP, incresed LR density in dose-dependent mnner. The presence of 300 µm SNP in the culture medium significntly promoted the LR density compred with the control (Figure 3.2 ). However, no sttisticl difference ws oserved in the numer of LRs (Figure 3.2 ). In contrst, 300 µm SNP significntly decresed the PR length (Figure 3.2 c). Consequently, opposite ptterns were oserved with LR density nd the PR length (Figures 3.2 nd c). No significnt difference ws oserved in the length of the longest LR nd picl distnces. However, n incresing trend ws oserved in the length of the longest LR nd decresing trend ws oserved in picl distnces (Figures 3.2 d nd e).

48 37 Figure 3.2 The effect of SNP on 3-dy-old, 1 cm long tomto seedling roots in growth room with 14 h of light nd 10 h of drkness. () lterl root density, () numer of lterl roots, (c) primry root length, (d) length of the longest lterl root nd (e) picl distnce. The vlues re mens ± SE from 3 independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

49 38 () 8 LR density (No. of LRs/cm) Concentrtions of SNP (µm) () No. of LRs/Seedling Concentrtions of SNP (µm)

50 39 (c) Length of PR (mm) Concentrtions of SNP (µm) (d) Length of longest LR (mm) Concentrtions of SNP (µm)

51 40 (e) 60 Apicl distnce (mm) Concentrtions of SNP (µm)

52 The effect of SNP on excised root tips in growth room with 14 h of light nd 10 h of drkness A single 1 cm long root tip ws excised from 3-dy-old seedlings nd cultured in different concentrtions of SNP. Compred with the seedling roots, excised root tips responded to the SNP tretments differently. There were no LRs oserved in the control. Only few root tips developed LRs nd the numer of LRs formed ws smll in the concentrtions of SNP tested. LR development ws not oserved t the highest concentrtion tested (500 µm). A decresing trend in PR length ws lso oserved with the incresing concentrtions of SNP. A summry of the dt is given in Tle 3.1. LR density (Numer of LRs/cm) Numer of LRs/ root primry root length (mm) Length of the longest LR (mm) Apicl distnce (mm) Control ± µm SNP 0.18 ± ± ± ± ± µm SNP 0.13 ± ± ± ± ± µm SNP 300 µm SNP 0.09 ± ± ± ± ± ± ± ± ± ± µm SNP ± Tle 3.1 The effects of SNP on 3-dy-old, 1 cm long excised root tips of tomto incuted in growth room with 14 h of light nd 10 h of drkness. The vlues re mens ± SE from 3 independent experiments (n=16).

53 The effect of SNP on excised root tips in complete drkness The sme experiment (s descried in Section 3.4.2) ws crried out except tht 2 cm long excised root tips were incuted in complete drkness t 25±1 C. Excised root tips treted with different concentrtions of SNP re shown in Plte 3.3. The PR of SNP treted root tips were thicker nd shorter thn the control. () () (c) (d) Plte 3.3 Photogrphs of 2 cm long excised root tips of tomto cultured for 7 dys in complete drkness t 25±1 C. Root tips were treted with () 0 µm SNP (the control), () 50 µm SNP, (c) 100 µm SNP nd (d) 300 µm SNP. Br = 2 mm

54 43 Figure 3.3 The effects of SNP on 4-dy-old, 2 cm long excised root tips of tomto cultured in complete drkness. () lterl root density, () numer of lterl roots, (c) primry root length, (d) length of the longest lterl root nd (e) picl distnce. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

55 44 It ws found tht 500 µm SNP in the culture medium significntly promoted the LR density compred with the control (Figure 3.3 ). No sttisticl difference ws oserved in the numer of LRs within the concentrtions tested ove (Figure 3.3 ), ut decresing trend ws oserved. 100 µm or higher concentrtions of SNP significntly reduced the PR length nd the length of the longest LR (Figures 3.3 c nd d) while 50 µm or higher concentrtions of SNP significntly reduced the picl distnces (Figure 3.3 e) () LR density (No. of LRs/cm) Concentrtions of SNP (µm)

56 45 () No. of LRs/Root Concentrtions of SNP (µm) (c) 120 Length of PR (mm) Concentrtions of SNP (µm)

57 46 Length of longest LR (mm) (d) Concentrtions of SNP (µm) (e) Apicl distnce (mm) Concentrtions of SNP (µm)

58 Interction etween ABA nd SNP on excised root tips of tomto In order to evlute the interction etween ABA nd SNP, different concentrtions of SNP were mixed with 1 µm ABA. Tretments with 500 or 1000 µm SNP led to significnt increse in the LR density with respect to the control ( sl medium without ny plnt growth regultors), lthough decresing trend of the stimultory effect of these three SNP tretments ws oserved (Figure 3.4). Compred with the control, 1 µm ABA reduced the LR density. The comintion of 1 µm ABA with 500, 1000, 1400 or 2000 µm SNP showed significntly incresed LR density compred to ABA lone. However, 1 µm ABA with 2000 µm SNP showed significntly reduced LR density thn the other concentrtions. One µm ABA dded to 500 or 1000 µm SNP significntly decresed LR density compred with the respective SNP concentrtions lone, ut 1 µm ABA dded to 1400 or 2000 µm SNP did not significntly decresed LR density compred with the respective SNP concentrtions lone. A similr numer of LRs ws oserved in 500 µm SNP compred with the control. However, sttisticlly significnt decrese ws oserved in the numer of LRs (Figure 3.5) s function of the incresing concentrtions of SNP (500, 1000, 1400 µm nd 2000 µm SNP). The comintion of 1 µm ABA with 500 or 1000 µm SNP resulted in n incresing trend in the numer of LRs compred with1 µm ABA lone. There ws no significnt difference etween the control nd 1 µm ABA s fr s PR length ws concerned (Figure 3.6). All other tretments resulted in significntly shorter PR compred with the control. There ws no significnt difference mong the control, 1 µm ABA nd 500 µm SNP in the length of the longest LR (Figure 3.7). Also there ws no sttisticlly significnt difference mong the different concentrtions of SNP tested (500, 1000, 1400 nd 2000 µm SNP). In ddition, the comintion of 1 µm ABA with 500 µm or higher concentrtions of SNP did not show ny significnt difference in the length of the longest LR compred with the respective SNP concentrtions lone.

59 48 As in the PR length, no significnt difference ws oserved etween the control nd 1 µm ABA on picl distnces (Figure 3.8). All the other tretments resulted in reduced picl distnces nd there ws no significnt difference mong them. Except from the preliminry experiments with either ABA or SNP, the experiment to find the reltionship etween ABA nd SNP ws crried out with the sme vriety of tomto seeds, ut purchsed from different compny, Asin Seeds. Some of the effective concentrtions (0, 300, 500 µm SNP) were repeted nd confirmed tht the new seeds re ehving the sme s the previous seeds.

60 49 LR density (No. of LRs/cm) cde g e cde c cd ef f 0 0 ABA 500 S ABA S 1000 S ABA S 1400 S ABA S 2000 S ABA S Concentrtion Figure 3.4 Interction etween ABA nd SNP on LR density. The effect of 1 µm ABA (ABA) nd different concentrtions of SNP (µm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

61 No. of LRs/ root de cd c cde cde cde e ABA 500 S ABA S 1000 S ABA S 1400 S ABA S 2000 S ABA S Concentrtion Figure 3.5: Interction etween ABA nd SNP on the numer of LRs. The effect of 1 µm ABA (ABA) nd different concentrtions of SNP (µm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

62 Length of PR (mm) c cd c d d cd cd 0 0 ABA 500 S ABA S 1000 S ABA S 1400 S ABA S 2000 S ABA S Concentrtion Figure 3.6: Interction etween ABA nd SNP on the PR length. The effect of 1 µm ABA (ABA) nd different concentrtions of SNP (µm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

63 52 Length of longest LR (mm) cd c cd cd d cd cd cd 0 0 ABA 500 S ABA S 1000 S ABA S 1400 S ABA S 2000 S ABA S Concentrtion Figure 3.7: Interction etween ABA nd SNP on the length of the longest LR. The effect of 1 µm ABA (ABA) nd different concentrtions of SNP (µm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

64 Apicl distnce (mm) ABA 500 S ABA S 1000 S ABA S 1400 S ABA S 2000 S ABA S Concentrtion Figure 3.8: Interction etween ABA nd SNP on picl distnce. The effect of 1 µm ABA (ABA) nd different concentrtions of SNP (µm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

65 The effect of NAA on excised root tips of tomto Root tips treted with different concentrtions of NAA re shown in plte 3.4. The PR of NAA treted root tips were thicker nd shorter thn the control. () () (c) Plte 3.4 Photogrphs of 2 cm long excised root tips of tomto incuted for 7 dys in different concentrtions of NAA in drkness t 25±1 C. Tretment of root tips with () 0 nm NAA, () 50 nm NAA, (c) 100 nm NAA nd. Br = 2 mm

66 55 Figure 3.9 The effect of NAA on 4-dy-old, 2 cm long excised root tips of tomto. () lterl root density, () numer of lterl roots, (c) primry root length, (d) length of the longest lterl root nd (e) picl distnce. The vlues re mens ± SE from four independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

67 56 There ws dose-dependent increse in LR density nd the numer of LRs in response to the NAA tretments. It ws found tht 100 nm NAA significntly promoted the LR density (Figure 3.9 ). A decresing trend ws oserved t 500 nm compred with 100 nm, even though there ws no sttisticl difference oserved (Figure 3.9 ). A similr effect of NAA on the numer of LRs ws lso oserved (Figure 3.9 ). The PR length nd picl distnces were reduced in response to the NAA tretments in dose-dependent mnner (Figures 3.9 c nd e). It ws found tht 100 or 500 nm NAA resulted in significnt decrese in the PR length compred with the control (Figure 3.9 c). However, no sttisticl difference ws oserved in the length of the longest LR (Figure 3.9 d) mong the concentrtions of NAA tested.

68 57 LR density (No. of LRs/cm) 7 () Concentrtions of NAA (nm) () No. of LRs/root Concentrtions of NAA (nm)

69 58 (c) Length of PR (mm) Concentrtions of NAA (nm) (d) Longest length of LR (mm) Concenretions of NAA (nm)

70 59 (e) 60 Apicl distnce (mm) Concentrtions of NAA (nm) 3.7 The effect of fluridone on excised root tips of tomto The effect of fluridone, n ABA iosynthesis inhiitor, on excised root tips of tomto is shown in Figure There ws no sttisticl difference oserved in the LR density mong the concentrtions of the fluridone tested (Figure 3.10 (i)). However, 1 µm fluridone significntly incresed primordi density nd therefore, LR plus primordi density (Figure 3.10 (ii) nd (iii)). The sme pttern of response ws oserved in the numer of LRs or primordi (Figure 3.10 ). However, there ws no significnt difference oserved in the PR length or the length of the longest LRs or picl distnces in response to the concentrtions of fluridone tested (Figures 3.10 c to e).

71 60 Figure 3.10 The effect of fluridone on 4-dy-old, 2 cm long tomto excised root tips. () (i) lterl root density () (ii) Primordi density () (iii) Lterl plus primordi density () (i) numer of lterl roots () (ii) numer of primordi () (iii) numer of lterl plus primordi (c) primry root length (d) length of the longest lterl root nd (e)(i) lterl root picl distnce. (e)(ii) primordi picl distnce (e)(iii) lterl root or primordi picl distnce (distnce to either first LR or primordi from the root rip) Et 100 % ethnol 0.1 Flu 0.1 µm fluridone 1 Flu 1 µm fluridone The vlues re mens ± SE from 3 independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

72 61 () (i) LR density (No. of LRs/cm) E t 0.1 F lu 1 F lu Concentrtion Primordi density (No. of primordi/cm) (ii) E t 0.1 F lu 1 F lu Concentrtion LR plus primordi density (iii) 0 Et 0.1 Flu 1 Flu Concentrtion

73 62 () (i) No. of LRs / root Et 0.1 Flu 1 Flu Concentrtion (ii) 3 No. of primordi/ root Et 0.1 Flu 1 Flu Concentrtion No. of LRs plus primordi/ root (iii) Et 0.1 Flu 1 Flu Concentrtion

74 63 (c) Length of PR (mm) Et 0.1 Flu 1 Flu Concentrtion (d) Length of longest LR (mm) Et 0.1 Flu 1 Flu Concentrtion

75 64 (e) (i) LR picl distnce (mm) E t 0.1 F lu 1 F lu Concentrtion primordi picl distnce (mm) (ii) 0 Et 0.1 Flu 1 Flu Concentrtion LR or primordi picl distnce (mm) (iii) c 0 0 Et 0.1 Flu 1 Flu Concentrtion

76 Interction etween NAA nd SNP on excised root tips of tomto In order to evlute the interction etween NAA nd SNP, the effect of vrious concentrtions of NAA (50, 100, 500 or 1000 nm) comined with 500 µm SNP ws evluted. It ws found tht 50, 100, nd 500 nm NAA resulted in n incresing trend in LR density in dose-dependent mnner, with the response to 500 nm NAA eing significntly different compred with the control (Figure 3.11 (i)). However, 1000 nm NAA reduced LR density. No sttisticlly significnt difference ws oserved in the primordi density mong the control nd the NAA tretments, except the concentrtion of 1000 nm NAA where significnt increse ws noted. However, there ws no significnt difference oserved in the lterl plus primordi density mong ll the concentrtions of NAA tested. The comintions of 500 µm SNP with 50 or 100 nm NAA reduced the LR density compred with the respective NAA concentrtions lone. The tretments of SNP with 500 or 1000 nm NAA completely inhiited LR formtion. In contrst, the tretments of NAA with SNP showed dose-dependent increment in the primordi density. In ddition, ll the tretments resulted in sttisticlly significnt different in primordi density compred with the respective NAA concentrtions lone (3.11 (ii)). No sttisticl difference ws oserved in LR plus primordi density compred with vrious NAA concentrtions lone or the control. Similrly, no sttisticl difference ws oserved in LR plus primordi density within the tretments of NAA with SNP. However, the tretments of NAA with SNP resulted in sttisticlly significnt difference compred with the respective NAA concentrtions lone nd the control (Figure 3.11 (iii)). Among the different NAA tretments, only 1000 nm NAA significntly reduced the numer of LRs compred with the control (Figure 3.12 (i)). The opposite response ws oserved in the numer of primordi which ws promoted in response to 1000 nm NAA (Figure 3.12 (ii)). No sttisticl difference ws oserved in the numer of LR plus primordi density in ll NAA concentrtions tested (Figure 3.12 (iii)). A mixture of 500 µm SNP with 50, 100, 500 or 1000

77 66 nm NAA decresed the numer of LRs compred with the SNP lone nd with the respective NAA concentrtions. However, in response to SNP nd NAA, the numer of primordi drmticlly incresed compred with the control, SNP nd the respective NAA concentrtions lone. The tretments of NAA comined with SNP resulted in incresed numer of LRs plus primordi, ut no sttisticl difference ws oserved mong them (Figure 3.12). NAA decresed the PR length in dose-dependent mnner. The tretments of SNP with different concentrtions of NAA resulted in no sttisticl difference compred with 500 µm SNP. However, the tretments with SNP nd 50 or 100 nm NAA reduced the PR length compred with the respective NAA concentrtions lone. However, there ws no sttisticl difference oserved in the tretments with SNP nd 500 or 1000 nm NAA (Figure 3.13). The length of the longest LR ws reduced t the concentrtion of 500 nm nd 1000 nm NAA concentrtions. The tretments of SNP with the different concentrtions of NAA reduced the length of the longest LR compred with the respective NAA concentrtions lone (Figure 3.14). NAA lone decresed the picl distnces of LRs nd picl distnces of LRs plus primordi in dose-dependent mnner compred with the control. Although they were not significntly different, decresing trend ws oserved in picl distnces of LRs nd picl distnces of LRs plus primordi in the tretments of different concentrtions of NAA with SNP, compred with the respective NAA lone (Figure 3.15). However, there ws no difference oserved in picl distnce of LRs nd picl distnce of LRs plus primordi in the tretments of NAA nd SNP, compred with the SNP lone. No sttisticl difference ws oserved in the picl distnce of primordi in either NAA lone or in comintion with SNP.

78 67 LR density (No. of LRs/cm) c cd cd cd cd d d 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.11 (i): Interction etween NAA nd SNP on LR density. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

79 68 Primordi density (No. of primordi/cm) c c d d d d d 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.11 (ii): Interction etween NAA nd SNP on primordi density. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

80 67 LR plus primordi density (No. of LRs + primordi/cm) c c c c c c 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.11 (iii): Interction etween NAA nd SNP on LR plus primordi density. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

81 No. of LRs/root c c c 2 0 c c 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.12 (i): Interction etween NAA nd SNP on the numer of LRs. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

82 69 No. of primordi/root c d d d d d 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.12 (ii): Interction etween NAA nd SNP on the numer of primordi. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

83 70 No. of LRs plus primordi/root cd c c cd cd cd 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.12 (iii): Interction etween NAA nd SNP on the numer of LRs plus primordi. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

84 Length of PR (mm) c cd cd cd d d d d 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.13: Interction etween NAA nd SNP on the PR length. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

85 74 Length of longest LR (mm) c c c c c c c 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.14: Interction etween NAA nd SNP on the length of the longest LR. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p<0.05 (t-test).

86 75 LR picl distnce (mm) c c c 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion c c c c c Figure 3.15 (i): Interction etween NAA nd SNP on LR picl distnce. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

87 76 Primordi picl distnce (mm) S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion Figure 3.15 (ii): Interction etween NAA nd SNP on primordi picl distnce. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test).

88 77 LR or primordi picl distnce* (mm) c c c 0 S 50 NAA 50 NAA + S 100 NAA 100 NAA + S 500 NAA 500 NAA + S 1000 NAA 1000 NAA + S Concentrtion c c c c c Figure 3.15 (iii): Interction etween NAA nd SNP on LR or primordi picl distnce. The effect of 500 µm SNP nd different concentrtions of NAA (nm) on 4-dy-old, 2 cm long excised root tips of tomto cultured in drkness t 25±1 C. S represents 500 µm SNP. The vlues re mens ± SE from three independent experiments (n=16). Different letters indicte the significnt difference with respect to ech other t p 0.05 (t-test). * Distnce to either first LR or primordi from the root rip.

89 The effect of CPTIO on SNP nd NAA on excised root tips of tomto Photogrphs of excised root tips treted with control, 500 µm SNP nd 50 nm NAA lone nd together with CPTIO re shown in plte 3.6. Root tips pper rown in colour due to the stining process. () () (c) (d) (e) (f) Plte 3.5 Photogrphs of 2 cm long excised root tips of tomto treted with () 0 µm SNP or NAA (Control), () 500 µm SNP, (c) 50 nm NAA, (d) 1 mm CPTIO, (e) CPTIO+500 µm SNP, (f) CPTIO+50 nm NAA nd incuted for 7 dys in complete drkness t 25±1 C. Then roots were stined with 5% (W/V) chromium trioxide. Br = 2 mm