Verticordia micropropagation through direct ex vitro rooting

Transcription

1 Edith Cown University Reserch Online Theses: Doctortes nd Msters Theses 2013 Verticordi micropropgtion through direct ex vitro rooting Belind Delney Edith Cown University Recommended Cittion Delney, B. (2013). Verticordi micropropgtion through direct ex vitro rooting. Retrieved from This Thesis is posted t Reserch Online.

2 Edith Cown University Copyright Wrning You my print or downlod ONE copy of this document for the purpose of your own reserch or study. The University does not uthorize you to copy, communicte or otherwise mke vilble electroniclly to ny other person ny copyright mteril contined on this site. You re reminded of the following: Copyright owners re entitled to tke legl ction ginst persons who infringe their copyright. A reproduction of mteril tht is protected by copyright my be copyright infringement. Where the reproduction of such mteril is done without ttribution of uthorship, with flse ttribution of uthorship or the uthorship is treted in derogtory mnner, this my be brech of the uthor s morl rights contined in Prt IX of the Copyright Act 1968 (Cth). Courts hve the power to impose wide rnge of civil nd criminl snctions for infringement of copyright, infringement of morl rights nd other offences under the Copyright Act 1968 (Cth). Higher penlties my pply, nd higher dmges my be wrded, for offences nd infringements involving the conversion of mteril into digitl or electronic form.

3 USE OF THESIS The Use of Thesis sttement is not included in this version of the thesis.

4 Verticordi micropropgtion through direct ex vitro rooting Belind Delney BSc (Biologicl Sciences) School of Nturl Sciences Edith Cown University

5 Abstrct The objective of this study ws to improve the existing shoot multipliction protocol for Verticordi grndis (McComb, Arthur & Newll, 1986; Newell, Growns & McComb, 2005) nd to investigte nd estblish relible root induction nd cclimtistion protocols to enhnce survivl of micropropgted plntlets. It ws envisged tht these protocols would be successful in micropropgtion, growth nd survivl of different V. grndis clones nd possibly pplicble to other Verticordi species. The elongtion of in vitro Verticordi shoots on multipliction medi ws improved by reducing the concentrtion of BAP from 1µM to 0.25 µm, which resulted in more uniform shoot length of cm; necessry for root induction experiments. The root induction protocol ws optimised by determining the pproprite uxin concentrtion (80µM indole butyric cid; IBA) with n exposure time of 6 dys. Acclimtistion nd survivl ws gretly improved by trnsferring the IBA pulsed shoots to ex vitro conditions consisting of free drining nd erted substrte ( mixture of pet nd perlite 1:3) in crck pots. These were initilly plced into greenhouse (with controlled temperture & light conditions) in order to mintin high humidity. Over time humidity ws reduced nd fter 112 dys the plntlets were trnsferred to lrger pots, contining fresh soil (pet/perlite/snd = 1:1:1) nd plced in shde house with regulr wtering regime. Long-term survivl ws monitored nd fter 252 dys survivl ws over 70%. The declining survivl rtes fter this time hs mde it evident tht field performnce nd longterm survivl needs further investigtion. The ppliction of the improved shoot multipliction nd root induction protocols on other V. grndis clones produced survivl rtes of 0 to 62.5% (depending upon clone) over 252 dys. 2

6 Copyright nd ccess declrtion I certify tht this thesis does not, to the best of my knowledge nd belief: (i) incorporte without cknowledgment ny mteril previously submitted for degree or diplom in ny institution of higher eduction; (ii) contin ny mteril previously published or written by nother person except where due reference is mde in the text of the thesis; or (iii) contin ny defmtory mteril 3

7 Acknowledgements I dedicte this reserch to the memory of Edwrd Ted Willims nd Dine Forsythe I hereby grtefully cknowledge the people who supported my reserch for this msters project, especilly my supervisors Dr In Bennett nd Dr Mry Boyce. I thnk them for their support, dvice, time, encourgement nd influence, s these were gretly pprecited. Thnks re lso due to Cly Millr nd his lbortory stff who devoted time nd energy into ll prcticl spects of my working in lbortory, whilst I ws reserching for my project. I consider myself fortunte to hve hd the finncil support for my reserch provided by the School of Nturl Sciences t ECU. I lso wish to thnk my fellow students, volunteers nd friends tht I hve met over the yers t ECU, especilly those who offered encourgement nd were lwys redy to lend helping hnd. This project would hve not been possible without the dediction of Ted Willims who helped me discover Enebb s wildflower tresures, Aln Tinker who shred his vst knowledge of Beekeeper s Reserve nd his mzing botnicl skills with me, nd Dine Forsythe, who lwys encourged my endevours in ny wy possible. I hope the reserch presented here my contribute to the conservtion of the mzing scrlet fether flower Verticordi grndis. 4

8 TABLE OF CONTENTS Use of Thesis Declrtion 1 Abstrct 2 Copyrights nd Access Declrtion 3 Acknowledgements 4 1. Introduction 1.1 Bckground Myrtcee nd Genus Verticordi Verticordi Hbitt Verticordi Morphology Verticordi Seed Germintion nd Reproduction Verticordi Conservtion & Cultivtion Vlue Verticordi Propgtion Micropropgtion of Verticordi grndis Micropropgtion Principles of in vitro Propgtion Composition of in vitro Culture Medi Physicl environment for micropropgtion Selection of Source Plnts for Micropropgtion Juvenility & Rejuvention in Woody Plnts in Tissue Culture Morphologicl Chrcteristics of Micropropgted Plntlets Root Induction Acclimtistion of in vitro Microcuttings to ex vitro Conditions Acclimtistion 30 5

9 1.4 Aims Limittions of conventionl propgtion methods Reserch ims Benefits of this reserch Mterils & Methods 2.1 Plnt mteril Process of initition of new clones into culture Medi preprtion Culture conditions Chnges to shoot multipliction medi composition Testing vrious BAP concentrtions to improve elongtion in clones Originl root induction protocol Optimistion of durtion of IBA tretment for root induction Optimistion of IBA concentrtion for root induction Trils with uxin combintions for root induction Trils with different in vitro substrtes for root induction Comprison of IVS with different nutrients Comprison of in vitro to ex vitro on IVS medi Survivl in greenhouse during cclimtistion phse In vitro shoot multipliction of new GRD clones Root induction of successful new clones Sttisticl nlysis Results 3.1 Survivl of new clones initited into culture Chnges to shoot multipliction protocol 48 6

10 3.3 Chnges to root induction medi composition Optimistion of durtion of IBA pulsing Optimistion of IBA concentrtion Auxin combintions for in vitro root induction Different substrtes Comprison of Agr, blck snd nd IVS Comprison of IVS with different nutrients Ex vitro root formtion nd cclimtiztion Survivl Effectiveness of improved protocols on new clones Shoot growth nd mintennce Roots induction, growth nd cclimtistion Discussion 4.1 Collection nd survivl of new clones initited in to culture Chnges to shoot multipliction protocol Chnges to root induction medi composition Optimistion of durtion of IBA pulsing IBA concentrtion Auxin combintions for in vitro root induction Different substrtes Comprison of Agr, blck snd nd IVS Comprison of IVS with different nutrients Ex vitro root formtion nd cclimtiztion Survivl Effectiveness of improved protocols on new clones 83 7

11 4.8.1 Survivl of new clones Conclusion References 87 TABLES Tble 2.1: Origin of the explnt mteril for new clones 35 Tble 3.1: Survivl rtes of new microcuttings 47 Tble 3.2: Periodicl ssessment of in vitro root induced GRD1 shoots 67 FIGURES Figure 1.1 Mp of the Southwest Botnicl Region in Western Austrli. 10 Figure 3.2 Mens for growth of shoot length 49 Figure 3.3 Optimistion of IBA pulsing time 51 Figure 3.4 Optimistion of IBA concentrtions for root induction 53 Figure 3.5 The effect of different uxin combintions nd concentrtions 55 Figure 3.6 Comprison of different substrtes nd root growth 57 Figure 3.7 Comprison of different substrtes nd root growth 59 Figure 3.8 Comprison of root ppernce from different substrtes 60 Figure 3.9 Comprison of different nutrients nd root growth 62 Figure 3.10 Comprison of sterile soil vs direct soil 64 Figure 3.11 Comprison of in vitro & ex vitro root growth nd survivl 66 Figure 3.12 Survivl of ll clones 69 8

12 ABBREVIATIONS BAP 6 benzylminopurine DDI double de-ionised wter CO 2 crbon dioxide GF Verticordi grndis from lignotuber regrowth fter fire GRD Verticordi grndis clone IAA indole-3-cetic-cid IBA indole-3-butyric-cid IVS in vitro soil MilliQ wter de-ionised wter MS Murshige & Skoog bsl slt mixture NAA nphthlene cetic cid NOH _ sodium hydroxide O 2 - oxygen VOV Verticordi ovlifoli clone VFR Verticordi frgrns clone VDF - Verticordi densiflor clone 9

13 1. Introduction 1.1 Bckground The Southwest Botnicl Province of Western Austrli is situted between ltitudes 27 S nd 35 S nd longitudes 112 E nd 128 E nd extends from Shrk By in the northwest to Espernce in the southest. This region includes the nrrow costl lnd from est of Espernce stretching to the Western nd South Austrlin border nd is confined by ocens in the west nd south nd by rid lndscpes in the north nd est (Cole, 2006). Figure 1.1: Mp of the Southwest Botnicl Region in Western Austrli. The green re shows the eco-region boundry; the drk yellow re shows the trnsitionl zone from Shrk By in the northwest continuing to the SA border in the southest (Cole, 2006). 10

14 It is recognized s one of the world s 34 mjor biodiversity hotspots nd is the only one in Austrli; it consists of more thn km 2 (48.0 million hectres) with over 52% of vsculr plnts nd 12.5% of gener being endemic to the region. This region is lso clssified s centre for plnt diversity by the WWF nd the Interntionl Union for Conservtion of Nture nd Nturl Resources (IUCNN) becuse of its plnt species richness (Hopper & Gioi, 2004). The southwest of Austrli is one of the oldest lndscpes in the world with very diverse vegettion communities tht hve dpted to the leched nd nutrient-poor soils. The species richness nd diversity of flor in this region is probbly due to the combintion of ecologicl nd phylogenetic processes tht hve occurred over long periods of time. This could explin the diverse plnt communities; rnging from kwongn (sclerophyllous shrubs) heth- nd shrub lnds on the costl snd plins to temperte forests in the fr southwest, nd wood- nd shrub lnds further est. All these plnt communities vry gretly over short distnces nd becuse of these vrying fetures the region hs become populr for its wild flower displys in spring, nd eco-tourism hs become n importnt industry (Cole, 2006). Mny of these plnts hve become desirble for the domestic nd export cut flower trde, ornmentl horticulture nd lnd restortion (Seton, 2002). The world s plnt biodiversity is in shrp decline with mny species being listed s criticlly endngered, endngered or vulnerble nd requiring conservtion (Srsn, Cripps, Rmsy, Atherton, McMichen, Prendergst & Rowntree, 2006); this includes mny tx of the Southwest Botnicl Province (Cole, 2006). Over the lst century this region hs been extensively degrded, resulting in lndscpe modifiction, frgmenttion, biodiversity nd hbitt loss (Hobbs & Sunders, 1991). Humn induced processes hve ltered over 90% of the originl vegettion 11

15 (Berd, 1980), which hs been reduced to mostly scttered, frgmented vegettion remnnts in vrious conditions. Lnd clering for griculturl purposes, urbn development, domestic stock grzing, logging nd chnges in fire regimes ccount for most of the ntive vegettion loss nd hbitt destruction (Cole, 2006). Slinistion of groundwter, due to ltered lndscpe hydrology (Hobbs, 1992), susceptibility of remnnt vegettion to weed invsion (Pigott, 2000) nd (ferl) grzing nimls re other threts to the remining bio-diversity of the Southwest Botnicl Province (Denhm & Auld, 2004). Another mjor thret to the flor of this region is disese cused by Phytophthor cinnmomi, soil-borne pthogen (wter mould), which cn cuse irreversible dmge to ffected plnt communities. The pthogen ttcks the roots of susceptible host plnts, thus significntly reducing the host plnt s bility for nutrient nd wter trnsport, nd eventully kills the host (Sherer, Crne, Brrett & Cochrne, 2007). The conservtion significnce of the highly diverse nd endemic flor of the Southwest Botnicl Region hs high priority with government conservtion gencies, s mny criticlly endngered tx hve been identified. Mny of these endngered species require immedite remedil ction to prevent extinction (Cotes & Atkins, 2000), nd in situ conservtion by protecting nturl hbitts nd efficient mngement of wild plnt popultions is very efficient with mny species. But these sometimes need to be complimented with ex situ methods (Srsn, et l., 2006), s numerous species hve reclcitrnt seeds or produce poor qulity seeds; so successful seed propgtion nd plnt estblishment is often very difficult or impossible (Bell, 1999). Mny tx from the Myrtcee, including some Verticordi, re thretened by hbitt loss. 12

16 1.2. Myrtcee nd Genus Verticordi Myrtcee is one of the ten lrgest plnt fmilies in the Southwest Botnicl Province nd of the 785 species 92% re endemic (Berd, Chpmn & Gioi, 2000). The genus Verticordi, belongs to this fmily nd is member of the Austrlin sub-fmily Leptospermoidee, chrcterised by their dry cpsulr fruit (Cochrne, Brown, Cunneen & Kelly, 2001; Clrk & Lee, 2002). It ws nmed by Augustin Pyrmus de Cndolle in 1821, nd is endemic to Austrli (George, 1991). Verticordi belong to the Chmelucium llince with its closest reltives being Chmelucium, Drwini, Homornthus nd Rylstone (George, 1991). These re commonly ssocited with mny other myrtceous gener in Western Austrli s shrub nd heth lnd communities (Cochrne, et l., 2001) Verticordi Hbitt All but three of the species, subspecies nd vrieties of verticordis re endemic to Western Austrli, nd the distribution lrgely depends on the nnul rinfll, with mny dpted to 350 mm to 850 mm rinfll zones (George, 1991; George, 2002; Seton, 2002). They grow in diversity of hbitts nd soils rnging from costl regions to fr inlnd rid regions. They occur s woody perennil understorey shrubs in open forests or s heth shrubs on snd plins nd even s sline tolernt shrubs growing on the edges of slt lkes (George, 1991; Seton, 2002). Although verticordis grow in this diversity of hbitts nd soils, some only occur in very prticulr loctions nd/or specific conditions, mny hve lredy been declred rre nd endngered (George, 1991; George, 2002; Ytes & Ldd, 2004). 13

17 1.2.2 Verticordi Morphology Verticordis generlly grow up to 2 m tll, with few species growing tller, nd hve very diverse forms with considerble vrition depending on species, lthough the bushy shrub is the most common form. Their shrub form my be dwrf, prostrte, erect or widely spreding with compct, strggly or horizontlly lyered ppernce (George, 1991). All verticordis, however, shre one prominent chrcterising feture; the sepls re deeply divided in vrious wys, with most species hving petls tht re often dentted, divided, fimbrited or lobed nd most flowers hving hiry hypnthium (Mrchnt, Wheeler, Rye, Bennett, Lnder, McFrlne, 1987), hence, the common nme - fether flowers Verticordi Seed Germintion nd Reproduction Seed production nd germintion processes re criticl for the survivl of most plnt species (Bell, 1999), but poorly understood for Verticordi species. Bell (1999) emphsised tht Austrlin plnt species rely on severl germintion mechnisms tht interct to interrupt seed dormncy nd initite germintion when environmentl conditions re suitble. Cochrne et l. (2001) reported tht for mny Verticordi species there is no dequte knowledge of the reproductive potentil, lthough some germintion trils with few species hve been successful (Cochrne, et l., 2001). Generlly, nnul reproductive cpcity in the form of seed production nd vibility in mny Verticordi species is exceedingly low (Cochrne, et l., 2001). Mny Verticordi species re cpble of superfluous flower production nd the low quntity of seeds produced is disproportionte to the number of flowers. This low seed to flower rtio is common in the genus (Housten, Lmont, Rdford & Errington, 1993; Cochrne, et l., 2001) nd 14

18 Cochrne et l. (2001) concluded tht the low seed set could be result of the selfincomptibility common in Myrtcee. However, s Tygi, McComb & Considine, (1991) nd others (Bellirs & Bell, 1990; Speer, 1993; McEvoy & True, 1995; Bell, 1999) suggest n rry of environmentl nd physiologicl fctors could contribute to the low seed set nd vibility in the genus (Rye & Jmes, 1992; George, 2002; Ytes & Ldd, 2004). It is not unusul for Austrlin ntive species to hve limited seed production s mny of these plnts reproduce vegettively (Johnson & Burchett, 1996; Kimpton, Jmes & Drinnn, 2002). Cochrne, et l (2001) lso proposed tht nother biologicl constrint tht could ply prt in the reproductive success is tht protein nd lipid cretion required for seed production requires more energy thn flower production. This my be survivl strtegy for tx during dverse environmentl conditions, e.g. chnges in pollintor ctivity, predtion, competition nd other unpredictble events (Cochrne, et l., 2001). Tygi, Considine & McComb (1992) reserched the remrkble longevity of some verticordi pollen t different tempertures for species both with nd without pollen presenter, but did not discover the physiologicl bsis for this. It ws suggested tht this long vibility could fcilitte rtificil hybridistion of species with breeding brriers such s different flowering times, but it ws not determined if in vitro germintion levels could mtch levels of in vivo germintion of the Verticordi species exmined (Tygi, et l., 1992). While verticordis hve limited reproductive success from seed germintion, Bell (1999) sttes, tht the secondry dormncy mechnisms, which cn prevent germintion of mny Austrlin ntive species re not fully understood. Severl Western Austrlin reclcitrnt plnt seeds hve 15

19 been investigted on with seed dormncy-relieving tretments, including smoke tretments, to promote nd increse germintion (Dixon, Roche & Pte, 1995; Roche, Dixon & Pte, 1997; Roche, Koch & Dixon, 1997), nd it ws suggested tht reserch on seed germintion nd reclcitrnce needs to be further investigted. Bell (1999) concluded tht it ws vitl to understnd the eco-physiology of seed germintion processes nd tht further reserch would be needed in order to improve conservtion of Austrlin plnt species for improved lnd mngement, rehbilittion nd for progress in horticulturl prctices Verticordi Conservtion & Cultivtion Vlue The long lsting fether flowers re highly sought fter for the commercil flower mrkets nd mny species hve been bush-picked to the extent tht this hd dversely impcted on wild popultions (e.g. Verticordi eriocephl). Commercil bush-picking, therefore, hs become unsustinble for most ntive popultions (Tygi et l., 1991; Atkins, 1998; Cochrne, et l., 2001) nd the hrvesting from nturl bush popultions is now restricted (George, 2002). Verticordis re not widely cultivted for the cut flower trde (Seton, 2002), but some success hs provided n lterntive to bush-picked plnts. However, this hs been limited to only few species, such s V. plumos (Seton, 2002) Verticordi Propgtion In the pst, sexul propgtion techniques hve been successfully used on some Verticordi species (e.g. V. plumos, V. chrysnthell), with the focus minly on cuttings nd some success 16

20 with tissue culture (George, 2002). In ddition, Egerton-Wrburton, Ghislberti, & Burton, (1998) found tht intergeneric hybridistion fcilitted by synchronous flowering cn be useful (e.g. C. floriferum x V. plumos), nd grfting on to Chmelucium root stock (Yn, 2001) hs lso produced some useful results. Grfting of severl Verticordi species, for commercil pplictions in field conditions, however, hs not been successful (Ben-Jcov, Ackermn & Evenor, 2000). Grfting is lso considered uneconomicl due to its slower production rte nd dditionl costs (Lullfitz, 2001) Micropropgtion of Verticordi grndis The ppliction of ex situ conservtion prctices for plnt species cn compliment in situ methods, or replce them ll together with species tht re too difficult to propgte by conventionl methods (Srsn, et l., 2006). Verticordi grndis, with its lrge scrlet red flowers, is n ttrctive species for the cut flower industry, but hs proven to be difficult to propgte by conventionl methods (Speer, 1993), due to reclcitrnce nd low vibility of its seeds (Bell, 1999). Pollintion of this species is poorly understood (Bell, 1999), but it cn regenerte from lignotuber; it is one of the few verticordis tht possess this cpcity (George & George, 2003). A relible lterntive to conventionl propgtion methods is tissue culture, which cn successfully produce vible clones of difficult to propgte species (Iln & Khyt, 1997). Micropropgtion of V. grndis could produce numerous, disese free plnts for horticulturl purposes, the cut flower trde nd for the restortion of degrded hbitts. 17

21 1.3 Micropropgtion Principles of in vitro Propgtion The development of the elementry methods of plnt tissue culture took plce in the 1950s nd 1960s nd hs since dvnced to commercil plnt biotechnology, which hs become n invluble tool in the plnt propgtion industry; e.g. ornmentl horticulture nd griculture (DeKlerk, 2002). One distinct dvntge of producing plnts by tissue culture is the rising of disese free true-to-type plntlets nd minimising diseses nd pests, which include internl pthogens, phytoplsm, viruses, viroids, prthogenic fungi, moulds, yests, bcteri nd insects. (Bndyopdhyy, Cne, Rsmussen & Hmill, 1999; De Klerk, 2002). In generl it is bsed on the principle of estblishing nd mintining helthy micro-shoots in culture for multipliction so tht these produce rooted micro-cuttings nd plntlets (DeKlerk, 2002; Liu & Bo, 2003). Conventionl propgtion methods re often inefficient for multiplying mny mture nd/or difficult-to-propgte plnt species, but tissue culture cn successfully produce vible clones (Iln & Khyt, 1997). However, it hs lso been well documented tht root induction cn be problemtic in woody plnts (DeKlerk, 2002; Liu & Bo, 2003; Seth, Kendurkr, & Ndgud, 2007). A mjor benefit of micropropgtion pplied to slow growing woody perennils is the propgtion nd multipliction of uniform nd numerous disese free plntlets in short time, s there re no effectul vegettive propgtion techniques for reproducing mture plnts tht cn equl this. Microprogtion lso llows for fster nd greter production of numerous plntlets compred to the slower, conventionl seed or vegettive propgtion methods (Frnclet, 1991; Hpl, Pkknen, Pulkinnen, 2004). 18

22 The bsic principle of plnt micropropgtion of meristemtic shoot tissues is to grow, mnipulte nd multiply identicl plnt cells, tissues nd orgns, which hve been isolted from the mother plnt. This pproch involves techniques tht re conducted in controlled nd septic environment; the cells, tissues nd orgns of selected plnt re isolted, surfce sterilised (Teng, Sin & Teng, 2002) nd cultured in growth-promoting environment (Frnclet, 1991; vn Acker & Scholten, 1995; George, Hll & de Klerk, 2007). There re generlly four distinct phses pplied in micropropgtion; 1. Estblishment, 2. Multipliction, 3. Root Induction nd 4. Acclimtistion. In the Estblishment Phse plnt prt is surfce sterilised nd then introduced to sterile culture medi. This phse determines the suitbility of the explnts; e.g. being free of exo- nd endogenous contmintion. In the Multipliction Phse the explnts re specticlly trnsferred to medi contining growth hormones tht promote shoot prolifertion (usully) nd elongtion. This phse cn generte numerous, stbilised explnts in very short time, which cn be kept in culture (Multipliction Phse) s long s needed by periodic sub-culturing on to new multipliction medi, or the explnts cn be septiclly trnsferred to root induction medi (Root Induction Phse). In the Root Induction Phse elongted explnts re septiclly trnsferred to growth medi tht usully contins uxin/s to initite nd promote root growth in preprtion for utotrophic growth of explnts. In the Acclimtistion Phse rooted explnts re trnsferred to suitble growth substrte nd plced in the controlled environment of glsshouse for grdul cclimtistion to ex vitro conditions. The plnts re initilly kept in high humidity, which is grdully decresed s plntlets cclimtise; they become more cpble of controlling wter loss. 19

23 1.3.2 Composition of in vitro Culture Medi An optimised culture medi is essentil for shoot growth, multipliction nd root induction in tissue culture, s the medi hs to provide ll nutrients nd elements for in vitro growth of plnts. Most culture medi contin the following: Minerls (s mcro- nd micro slts): Most plnts respond fvourbly to the widely pplied Murshige & Skoog (1962) MS slts. Lloyd & McCown s (1980) Woody Plnt Medium is lso widely used. The ppliction quntity depends on the in vitro cultivtion objectives; e.g. high formultions (full strength) re better suited for shoot multipliction, whilst lower concentrtions ( 1 / 2 or 1 / 4 strength) re generlly used to promote root induction depending on plnt species (Kim, Oh, Jee & Chung, 2003). Crbon (energy) source: Sucrose in the form of commercil sugr is the most preferred crbon source in tissue culture becuse it is chep nd redily vilble. Crbon (e.g. sucrose, glucose, mltose or glctose) for in vitro plnt metbolism hs to be dded to mny culture medi s the plntlets re not fully utotrophic nd photosynthesis is not dequte (Rhmn, Islm, Hossin & Islm, 2010). Plnt growth regultors (uxins, cytokinins, gibberellins nd others): Severl growth regultors consist of nturl nd/or synthetic compounds tht mnipulte development nd growth of in vitro plnts. The ppliction nd quntities of growth regultors depends on the in vitro cultivtion objectives; e.g. cllus formtion, shoot multipliction nd elongtion or root induction. Auxins regulte severl physiologicl process; e.g. formtion of shoots nd dventitious nd lterl roots (Ibrhim & DeBergh, 2001). Other orgnics: Vitmins re not necessrily dded to tissue culture medi, but most formultions contin vitmins; e.g. in MS slts the included vitmins re thimine, 20

24 pyridoxine, nicotinic cid, with myo-inositol (sugr lcohol) nd thimine considered to be essentil to plnt growth (Abrhmin & Knthrjh, 2011). ph; the ph is djusted to 5.8 using 1M NOH for shoot multipliction to pproximte wht would be suitble for plnt metbolism nd to ensure gr solidifiction. Gelling gents (purified gr or gelln gum products): In micropropgtion trditionlly solid medi is fvoured over liquid medi with gr often being the preferred gelling gent. The concentrtion of the gelling gent is ssocited with the wter potentil from the medium to the cultured plnts, depending on the objectives of cultivtion; e.g. shoot multipliction or root induction (DeBergh, 1983). Gelln gum (Gelrite) produces cler gel, which llows for ccurte observtion of root induction, nd it hs been observed tht mny Austrlin woody plnts hve greter survivl rte when cultured in Gelrite medium (Willims & Tji, 1987). De-ionised wter; wter is the universl solvent for ll solutes required for micropropgtion culture medi (DeBergh, 1983). The medi needs to then be dispensed into pproprite culture vessels nd sterilised in n utoclve generlly t 121 o C nd 1.2kg/cm -2 for 20 minutes Physicl environment for micropropgtion The physicl environment for micropropgtion is s importnt s the pproprite culture medi formultion. Light nd temperture cycles, necessry for optimum growth of cultured plntlets, re progrmmble nd controllble ccording to the individul plnt species requirements (Johnson, 1996). 21

25 Light: Plnt development nd growth is influenced by wvelength, intensity nd the durtion of light nd this cn be controlled in micropropgtion ccording to plnt species nd the in vitro culture objectives. Light is lso n importnt fctor for phototropism, morphogenesis nd photosyntehsis (Red & Preece, 2003). It hs been observed tht light cn enhnce shoot growth nd root formtion in some species, whilst other species preferred drkness for root induction (Kumr, Plni & Nndi, 2003). Temperture: Plnt development in micropropgtion lso depends on the optimum temperture rnge for the physiologicl process of respirtion, but these optimum tempertures vry ccording to species nd genotypes. Culture tempertures between 20 nd 27 C re most commonly pplied (Red & Preece, 2003). Gs exchnge: In vitro plntlets re grown in culture vessels tht present closed system to prevent microbil contmintion. This cn limit the inflow of CO 2 nd the outflow of O 2 nd ethylene, lthough some gs exchnge my occur depending on the type of culture vessels used (Prk, Jeon, Kim, Prk, Aswth & Joung, 2004). The reltive humidity inside the culture vessel is usully very high, pproximtely % (Gribudo, Restgno & Novello, 2003), which cn be problemtic for initil cclimtistion of plntlets from in vitro to ex vitro conditions. 22

26 1.3.4 Selection of Source Plnts for Micropropgtion Cuttings from different individul explnts re likely to behve differently under culture conditions (Geneve, 1989; Anthony, McLen & Lwrie, 2000). Nevertheless, it is importnt to crefully select plnt source mteril tht is of vigorous, helthy ppernce with the desired phenotypic ttributes (Krtsons & Ppfotiou, 2007). The initil explnt source selection is vitl with regrd to epigenetic nd genetic chrcteristics of the source plnts (Wiltshire, Potts & Reid, 1998), so the creful selection of source plnts is crucil (Idczk, & Brielmier-Liebetnz, 2003). This cn ensure the cloning of the desired true-to-type phenotypic plnt mteril, lthough this cn only be confirmed by close moleculr exmintion (Aror, Shrm, Srivstv, Rnde & Shrm, 2011). Epigenetic nd genetic stbility of micropropgted plnts re n importnt pre-requisite nd this hs been investigted with vrious moleculr techniques. Hop plnts displyed epigenetic differences between the field grown mother plnts nd the micropropgted clones (Peredo, Arroyo-Grci & Revill, 2009). Keppler, Keppler & Rhee (2000) observed tht there ws phenotypic vrition nd temporry ltertions in the physiology of the shoots in micropropgted plnts compred to seed grown plnts. But it ws lso concluded tht over time, with repeted subculture, the phenotypic vrition would initilly ccumulte but then reduce; the micropropgted mteril would become more similr to the source plnt with culture ge (Rodriguez Lopez, Wetten & Wilkinson, 2010). The long-term genetic stbility ws lso confirmed in study for bnn clones (Lkshmnn, Venktrmreddy & Neelwrne, 2007). 23

27 1.3.5 Juvenility & Rejuvention in Woody Plnts in Tissue Culture Severl studies of the different phses in plnt development hve verified tht there re four phses; embryonic phse (estblishment of shoot nd root meristems), juvenile phse (no reproduction cpbilities), dult vegettive phse (estblishment of reproductive cpcity) nd the dult reproductive phse (Hckett & Murry, 1992; Greenwood, 1995; Wiltshire, et l., 1998). These phses re stble nd comprtively inconspicuous s the chrcteristics of one phse re usully replced by trits tht chrcterise the next phse (Conwy & Poethig, 1993; Kerstetter & Poethig, 1998). As with other methods of propgtion, the micropropgtion success of woody plnt species cn depend lrgely on the source plnt s juvenility sttus (Hckett, 1985), s the more juvenile explnt is esier to reproduce thn more mture plnt (Lullfitz, 2001). A cutting from woody plnt in the juvenile phse hs greter bility to root nd this is regrded s evidence tht the plnt hs not yet reched mturity (Hckett, 1985; Jones, 1999). The juvenile stge is defined by grdul chnges, e.g. biochemicl, morphologicl nd physiologicl chrcteristics, tht occur in the young plnt fter germintion (Hpl, et l., 2004). One of the gretest limittions in slow growing woody perennils is their reclcitrnce due to complex sesonl nd life cycles, which cn mke it difficult to estblish stbilised shoot cultures (McCown, 2000). This could be overcome with the genertion of shoot culture by micropropgtion techniques tht utilise the plnt s meristemtic tissues (Irish & Krlen, 1998) tht cn renew nd sustin growth, nd produce new plnts from the existing vegettive structures (Schmidt, 1997; Frnclet, 1991; Hckett & Murry, 1992). Ech step of 24

28 micropropgtion cn be mnipulted nd hs become the preferred method for production of woody plnts tht typiclly hve long juvenility phses nd slow breeding processes (Stokes, 1980; de Jeu & Cdic, 2000), s observed in mny Verticordi species. Tissue culture cn be method to restore the juvenile phse in lrge mounts of explnt mteril, s studies suggest tht sub-culturing over time cn re-estblish juvenility in clones cultured from mture woody plnts (McCown, 2000; Andreu & Mrín 2005). Hckett (1985) stipultes tht juvenility of woody species ends with the onset of flowering. Andreu & Mrín (2005) discussed reinvigortion of mture woody plnts during in vitro culture nd estblished tht the best multipliction results for shoot increse were gined from micropropgted plnts tht originted from cutting derived plnts. These rejuvented plnts differ in physiology nd often hve restored rooting cpbility tht my only be temporry (Hckett & Murry, 1992; Hpl, et l., 2004) Morphologicl Chrcteristics of Micropropgted Plntlets Most reserch suggests tht genotypic nd phenotypic vrition seems to be common occurrence observed in micropropgted plnts. These vritions cn pre-exist in plnt mteril from nturl popultions tht hs been used for tissue culture initition or they cn result from tissue culture conditions. They cn be beneficil, s somclonl vrition cn led to the development of new vrieties nd/or the development nd utilistion of desired trits (Skirvin, 1978; Lrkin & Scowcroft, 1981; Perez, Mbogholi, Sgrr, Argon, Gonzlez, Isidron & Lorenzo, 2011). But repeted sub-culturing of micropropgtion cn lso result in observed 25

29 mlformtion of leves, stems nd roots; e.g. chnges in lef morphology resulting in thinner leves, undifferentited mesophyll, flttened chloroplsts nd other chnges t sub-cellulr level which could ffect some vitl physiologicl processes like photosynthesis (Skirvin, 1978; Sebstini, Minnocci, Vitglino, Gribudo & Novello, 2001; Romno & Louco, 2003). Most micropropgted plntlets lck well-developed nd functionl wxy cuticle, which cts s brrier to prevent wter loss, lthough the cuticle is present. This is problemtic for ex vitro cclimtistion s in vitro plntlets cnnot regulte their wter blnce nd experience wter stress (Skirvin, 1978; Fil, Ghshghie, Horu & Cornic, 1998). Morphologicl chnges observed in stems cn include shorter internodes, thickened stems nd stunted growth (Skirvin, 1978; Mrtin, 2003). Cllus formtion, deformed nd brittle roots hve been observed with in vitro root induction of shoots which cn prove to be problemtic when trnsferring rooted shoots from in vitro to ex vitro conditions, cusing new roots to brek (Kim, Klopfenstein & Cregg, 1998; Metivier, Yeung, Ptel & Thorpe, 2007). After successful cclimtistion most of these described morphologicl deficiencies nd mlformtions improve with plnt ge nd the plnt s functions (lef structure, wter reltions, photosynthetic prmeters) become comprble to the stock plnt in the field (Pospislov, Tich, Kdlecek, Hisel & Plzkov, 1999; Agnihotri, Mishr & Nndi, 2009). DNA nlysis cn be used to confirm tht in vitro cultured plnts re geneticlly comprble nd true-to-type of the wild host plnt (Aror, et l., 2011). 26

30 1.3.7 Root Induction In micropropgtion of woody plnts, dventitious root induction nd formtion re fundmentl for successful production of vible plntlets. Mny woody plnts re reclcitrnt nd do not propgte esily due to the lck of morphologicl plsticity in their cells nd tissues nd require the ppliction of exogenous uxins to induce dventitious root induction. Auxins - The most commonly used uxins for root induction includes indole-3-butyric-cid (IBA), indole-3-cetic-cid (IAA) nd α-nphthlene-cetic-cid (NAA), with IBA being the most effective nd most commonly used. The different effects depend on the plnt species, the concentrtion nd the purpose of ppliction (Ibrhim & Debergh, 2001; De Klerk, Vn Krieken & De Jong, 1999). Different uxins, concentrtions or combintions, my hve to be pplied depending on rooting tretments (in vitro or ex vitro) nd on plnt species (DeKlerk, terbrugge & Mrinov, 1997). The ppliction methods vry gretly, depending on the culture methods; for in vitro root induction the uxin is dded to the culture medium, for ex vitro root induction the stems cn be dipped in highly concentrted uxin formul (Ibrhim & Debergh, 2001). Substrte - For in vitro root induction the microcuttings re mintined in culture medi with low minerl concentrtions (½ MS or less) nd low uxin concentrtions to stimulte dventitious root formtion (Kim, et l., 2003). For ex vitro root induction, microcuttings cn be pulsed on high uxin concentrtions in culture medi with low minerl concentrtions (½ MS or less) for usully 7 dys (Newell, Growns & McComb, 2005) or the cuttings re dipped in high concentrtions of uxins before being trnsferred to n orgnic nd erobic growing substrte (pet/perlite/snd) for root formtion nd simultneous cclimtistion (Mrtin, 2003; Mrtin, 2003/04; Hzrik, 2003; Meiners, Schwb & Sznkowski 2007). 27

31 1.3.8 Acclimtistion of in vitro Microcuttings to ex vitro Conditions The trnsfer of microcuttings from in vitro to ex vitro greenhouse conditions hs been problemtic for number of woody species including those relted to Verticordi, e.g. Euclyptus, Chmelucium nd Scholtzi (Berdsell, 1996; Johnston, 1996; Ben-Jcov, et l., 2000; Lullfitz, 2001). Exmintion of the plntlet condition hs resulted in the recognition tht plnts coming from tissue culture do not necessrily hve norml physiologicl nd morphologicl ntomy (Sntmri & Kertsins, 1994; Dmi & Hughs, 1997). These functionl differences include lck of junction between dventitious roots nd stem vsculr tissue, stomtl mlfunction nd the microcutting s lck of well-developed nd functionl cuticle tht cts s brrier to prevent wter loss; this cn cuse plntlets to die of wter stress (Skirvin, 1978; Fil, et l., 1998; Romno & Louco, 2003). The survivl rte of microcuttings to be cclimtised ex vitro cn be incresed significntly by mintining high reltive humidity (~ 90% - 98%) by using systems tht pply wter vpour, fog or mist periodiclly. In prticulr, the need for high humidity but low wter droplet size hs been emphsised by severl uthors (De Bergh, 1991; Drew, Smith, Moisnder & Jmes, 1991; Red & Preece, 2003; Johnson & Armstrong, 2003). This hs been fcilitted by the vilbility of fog genertors tht cn deliver wter droplets of less thn 5µ, rther thn misting systems. The use of humidity tents cn increse the retention of reltive humidity nd crete microclimte. This wtering regime cn be reduced s the microcuttings cclimtise (Offord & Cmpbell, 1992; Argon, Esclon, Cpote, Pen, Cejs, Rodrigues, Cnl, Sndovl, Roels, DeBergh, & Gonzlez-Olmedo, 2005). 28

32 IBA pulsed in vitro shoots of woody plnts hve successfully been tested for their bility to root under high humidity (Yn & Sedgley, 2006). New res of micropropgtion were being investigted nd in 2003 Newell nd co-workers reported work on shoot microcuttings of Verticordi species in porous gr medium tht contined ir filled porosity from 10% to 29%. The reserchers concluded tht root induction nd length ws significntly incresed compred to stndrd gr solidified medi (Newell, 2003). Newell et l. (2005) hve proposed tht n erobic medium bse my be importnt in fcilitting trnsfer of in vitro plnts to ex vitro conditions. It ws concluded tht for severl woody perennils the rooting performnce in In Vitro Soil (IVS) ws successful nd this method could reduce the stress nd plnt loss during the cclimtistion stge of micro-cuttings from sterile lbortory conditions to field conditions (Newell, et l., 2005). Alterntives to the in vitro shoot multipliction (Phse 2) followed by in vitro root induction (Phse 3) hve lso been exmined. The simultneous ex vitro root induction nd cclimtistion hve been trilled s cost nd time reducing strtegy (McClellnd, Smith & Crthers, 1990; Kim, et l., 1998; Mrtin, 2003; Thoms & Schiefelbein, 2005; Feyiss, Welnder & Negsh, 2007; Xu, Wng & Zhng, 2008). Under these circumstnces root induction nd cclimtistion (Hzrik, 2003) occur simultneously in controlled environment such s greenhouse, under conditions similr to those used for cclimtistion of in vitro rooted plnts. A mjor dvntge of ex vitro root induction is tht root dmge from de-flsking nd trnsfer to soil, tht cn preclude growth nd vigour, is voided (Yn & Sedgley, 2006). 29

33 1.3.9 Acclimtistion The trnsfer of plnts from culture to soil hs, in the pst, cused considerble problems nd hs been the subject of discussion (Rohr, Iliev, Scltsoyinnes & Tsoulph, 2003) nd even conferences on the topic (e.g. Interntionl Society of Horticulturl Science hs orgnised three interntionl conferences on this topic in 2003, 2005 nd gin in 2007). In generl, the requirements re dependent upon two mjor spects: plntlet condition nd the environmentl conditions required for trnsfer. More recently, where these spects hve not been importnt or overcome (Newell, et l., 2005), other spects hve been concentrted upon; such s the medi from which the plnts hve been derived nd/or the number of roots produced per plntlet (McCellnd, et l., 1990; Bonl & Monteuuis, 1997; Kim, et l., 1998; Bennett, McDvid, McComb, 2004). The environmentl conditions hve received considerble ttention nd in recent yers these hve become more stndrdised for mny species. The ultimte requirement of propgted plnts is tht they survive under the field condition for which they hve been produced. To this end, while there hs been some reserch done on the survivl of plnts produced from different modes of propgtion, reltively little hs been produced regrding the long-term growth nd production. Comprisons between micropropgted plnts nd seedlings (Bell, vn der Moezel, Bennett, McComb, Wilkins, Mrshll & Morgn, 1993) nd micrpropgted plnts nd cuttings (Bergmn, 1998; Multy, Wilson, Ong, Dens & Sprent, 2002) hve shown tht ny initil differences between plnts re generlly lost fter long-term growth. This is prticulrly 30

34 importnt for forest species where it hs been suggested tht root development of micropropgted plnts my led to trees being susceptible to wind-throw (Bell, et l., 1993). 1.4 Aims Reserch on verticordis hs been firly limited (McComb, Arthur & Newll, 1986; George, 1991; Tygi, Considine & McComb, 1991; Tygi, Considine & McComb, 1992; Housten, Lmont, Rdford & Errington, 1993; Speer, 1993; McEvoy & True, 1995; Stummer, Smith & Lngridge, 1995; Egerton-Wrburton, Ghislberti & Burton, 1998; George, 2002; George & George, 2003; Ytes & Ldd, 2004), when considering the diversity of this genus nd the common thret of hbitt loss. The min objective of this reserch ws to investigte nd estblish relible nd pproprite micropropgtion, root induction nd cclimtistion protocols for V. grndis in order to increse the qulity nd survivl ex vitro, s results vried in previous studies Limittions of conventionl propgtion methods There re severl limittions in producing verticordis for horticulturl, lnd restortion nd conservtion purposes; e.g. slow growth, limited seed production, seed reclcitrnce nd poor germintion rtes (Bell, 1999; Cochrne, et l., 2001). Conventionl mens of sexul propgtion (cuttings nd grfting) hve been useful for limited number of species, but most re not menble to these processes. This is relted to the vilbility of suitble cutting mteril from wild popultions nd genetic nd environmentl fctors (Johnson, 1996). Often it 31

35 is difficult to obtin n dequte mount of the pproprite mteril to estblish well-replicted nd controlled experiments on the development protocols; e.g. it is very difficult, if not impossible, to obtin lrge number of shoots, t the sme stge of development (sesonl or ontogenetic ge) nd from single genotype, for dequte repliction. Most vilble verticordi clones, such s V. chrysnthell, V. mitchellin nd V. plumos hve limited genetic vrition, nd hve probbly been selected on the bsis of ese of propgtion. In contrst, Verticordi grndis is difficult-to-root species nd micropropgtion with consequent in vitro subcultures cn induce rejuvention or incresed re-invigortion, which cn increse rooting cpcity. Micropropgtion hs become populr technique to overcome mny of these limittions; stock plnt mteril from difficult to propgte plnts cn be used to micropropgte lrge numbers of progeny plnts Reserch ims This reserch investigted the cpcity to produce numerous explnts by improving the existing micropropgtion protocol (McComb, Newell & Arthur, 1986) for Verticordi grndis, s well s investigting the most successful root induction protocol for cclimtistion nd survivl of plntlets. This ws chieved by determining the best method to chieve optiml shoots for micropropgtion from mture woody cuttings. Plntlets from the clones were observed for juvenile growth phse trits in the growth of roots nd shoots nd the different modes of propgtion were compred for the development of prmeters tht cn increse growth nd survivl in field conditions. 32

36 For this study 2 clones of Verticordi grndis, which hve been in culture for severl yers in the tissue culture lbortory t Edith Cown University, were used, nd 21 different Verticordi cuttings were introduced into culture. Cuttings were tken from 4 different Verticordi grndis plnts sprouting fter fire, 10 from new growth on different mture Verticordi grndis plnts from vrious loctions round Enebb in Western Austrli nd cuttings from new growth of 7 different verticordi plnts (5 from V. ovlifoli, 1 from V. frgns nd 1 from V. densiflor). The im ws to initilly experiment with the existing clones of Verticordi grndis to estblish relible shoot multipliction, root induction nd cclimtistion protocols nd then to test the effectiveness of these on the newly introduced clones of verticordis for comprison Benefits of this reserch In prticulr, the findings of this reserch could be beneficil to the reconstruction nd rehbilittion of plnt communities on disused mine sites, drilling explortion sites nd other lnd clered sites in the northern snd plin kwongn vegettion round Enebb, Western Austrli, the nturl hbitt of Verticordi grndis. The results of this reserch could lso benefit growers of West Austrlin wild flowers, s especilly Verticordi grndis is n ttrctive species for the export cut flower mrket (Seton, 2002). 33

37 2. MATERIALS & METHODS 2.1 Plnt mteril All micro-cuttings (shoots) used initilly for the optimistion of the existing protocol for shoot multipliction of V. grndis were obtined from two V. grndis clones (GRD 1; GRD 3) tht hd been in culture (t Edith Cown University s School of Nturl Sciences Tissue Culture Lbortory) for over 5 yers before experiments begn. In July 2007 explnts of V. grndis (GRD) were collected from the Enebb re; loctions were recorded with GPS redings, host plnt nd site descriptions were noted nd ll mteril ws cut from new shoot growth. Explnt mteril from different new shoots of V. grndis (GF) tht hd resprouted fter fire were collected, s well s explnt cuttings from V. ovlifoli (VOV), V. densiflor (VDF) nd V. frgrns (VFR) (Tble 2.1). For ech explnt, minimum of 5-10 cuttings of ~12cm length were collected, depending on the size of the host plnt; e.g. the new shoots of the VGF clones were smll, nd the lignotuber ws exposed ensuring tht cuttings were tken from the sme root. The cuttings were plced in zip lock bg, kept on ice or t 4 0 C nd processed in the lbortory the next dy. 2.2 Process of initition of new clones into culture The cuttings were surfce sterilised in 2% benzlkonium chloride in 10% ethnol solution for 20 minutes, before being rinsed 3x in sterilized milliq wter, fter which they were septiclly cut into ~0.5-1cm pieces t internodes. These were then trnsferred onto the 5 ml of shoot multipliction medi in 50 ml flsks. Initilly, totl of 548 flsks were prepred for ech microcutting. Ech uncontminted, surviving microcutting ws trnsferred dily on to fresh 5ml 34

38 of shoot multipliction medi in 50 ml flsks in septic conditions for 28 dys. After 4 weeks the surviving shoots were trnsferred to 250 ml culture vessels. Tble 2.1: The explnt mteril for clones tht were collected t different loctions nd hbitts re s follows: Clone Loction Hbitt GRD4 S , E Snd over lterite, heth GRD5 S , E Snd over lterite, heth GRD6 S , E Snd over lterite, heth GRD7 S , E Snd over lterite, heth GRD8 S , E Snd, heth, bnksi GRD9 S , E Snd, heth, bnksi GRD10 S , E Snd, heth, bnksi GRD11 S , E Snd, heth, bnksi GRD12 S , E Snd, heth, bnksi GRD13 S , E Snd, heth, bnksi GF1 S , E Burnt heth, woodlnd* GF2 S , E Burnt heth, woodlnd* GF3 S , E Burnt heth, woodlnd* GF4 S , E Burnt heth, woodlnd* VOV1 S , E Snd, heth, verge VOV2 S , E Snd, heth, verge VOV3 S , E Snd, heth, verge VOV4 S , E Snd, heth, verge VOV5 S , E Snd, heth, verge VFR1 S , E Remnnt heth VDF1 S , E Privte Property Clones GRD1, 2 & 3 were in culture t ECU; GRD2 becme contminted nd could not be used in experiments. *New shoot growth from roots fter originl plnt ws destroyed by fire. All others were from new sesonl shoots. 2.3 Medi preprtion All micro-cuttings (shoots) used for the optimistion of the existing protocol for shoot multipliction of V. grndis were obtined from two V. grndis clones (GRD1; GRD3). The initil protocol ws s follows: full strength Murshige & Skoog (MS) (Murshige & Skoog, 1962) medium supplemented with 1µM 6-benzylminopurine (BAP), 1.5 µm Kinetin, 1.25 µm nphthlene cetic cid (NAA), 20 g L -1 sucrose, ph djusted to 5.8, solidified with 2.5 g L -1 35

39 gr nd 2.5 g L -1 gelrite. The medi ws dispensed (50 ml) into 250 ml culture vessels nd utoclved t 121 C for 20 minutes. 2.4 Culture conditions All prepred liquid medi, nutrient solutions nd growth substrtes were sterilised by utoclving t 121 C for 20 minutes prior to ppliction for ll in vitro experiments conducted in the lbortory. All in vitro cultures were mintined in stndrdised conditions (under 16h dy with irrdince of 90µmol m -2 s -1 / 8h night photoperiod, with temperture t 25 C ± 1 C) for 28 dys, before being septiclly sub-cultured for shoot multipliction or utilised for further experiments. All ex vitro cultures (explnts/plntlets) were mintined in stndrdised conditions (10 seconds of wter misting t 7000 lux ccumulted light, with tempertures t 25 C ± 5 C) in enclosed tents for high humidity (~90% - 98%) in the greenhouse for 28 dys. Then the ex vitro cultures were moved out of the tents, ssessed for root growth, nd continued to be mintined in stndrdised conditions without the enclosed tent (10 seconds of wter misting t 7000 lux ccumulted light, with tempertures constnt t 25 C ± 1 C nd reduced humidity). The plnts were ssessed every 28 dys nd t 84 dys the surviving plnts were moved to the shde house for further hrdening. The shde house did not hve temperture or light control, but hd utomted, periodicl wtering (morning nd evening sprinklers for 10 minutes). At 112 dys the surviving plnts were re-potted in soil mixture (pstuerised) consisting of pet, perlite nd white snd (1:1:1) nd survivl ws ssessed every 28 dys. 36

40 2.5 Chnges to shoot multipliction medi composition It ws observed tht using the originl medi formultion resulted in shoot multipliction of GRD1 clones being unvrying in shoot length nd prolific, nd in GRD 3 shoot multipliction ws prolific, but with no consistent elongtion; most of the shoots were less thn 1cm long. Sub-cultured shoots of uniform length (~ 2-5cm) from both clones were needed for further experimenttion nd comprisons Testing vrious BAP concentrtions to improve elongtion in clones The originl shoot sterilised multipliction medi ws used s control (1 µm, BAP) ginst medi supplemented with 0.1 µm, 0.15 µm, 0.2 µm, 0.25 µm, 0.3 µm, 0.35 µm, 0.4 µm, 0.45 µm nd 0. 5µM BAP. Twenty five rndomly selected shoots of clones GRD1 nd GRD3 were kept on the vrious medi for 28 dys nd then hrvested, fter which shoot growth ws mesured; this experiment ws repeted 2 times, with ll the results combined. The men growth for ech concentrtion ws clculted nd the vrince ws nlysed. The shoot multipliction medi ws chnged, ccordingly due to the results tht were chieved, for ll following medi preprtions. 37

41 2.6 Originl root induction protocol The success of micropropgted plntlets depends on vible dventitious root formtion on shoots (with the help of exogenous growth regultors such s uxins), s this determines the survivl of the new plnts (Mrtin, 2003), but root induction for micropropgted V. grndis hs proven to be difficult. The shoot length could lso determine the efficiency of root formtion, s longer shoots re in better physiologicl condition for root induction (Xu, et l., 2008). For root induction experiments shoots of GRD1 (ech cm long) were rndomly chosen from numerous culture vessels, leves removed from the lower prt of the stem (~1cm) s wounding of the stem tissue cn ffect the blnce of growth regultors, which cn enhnce root induction (Mrks & Simpson, 2000). Shoots were grown in sterile rooting medi (50ml) modified from Bennett, et l., (2004); it contined ¼ strength MS mcronutrients nd full strength micronutrients supplemented with 10 µm indol-3-butyric cid (IBA concentrtion depending on experiment), 2% sucrose, ph djusted to 5.5, solidified with 2.5 g L -1 gr nd 2.5 g L -1 gelrite in 250 ml cler culture vessels. Ech vessel contined 50 ml of sterilised medium. The control rooting medium hd no IBA. Ech culture vessel contined 5 shoots nd these were mintined in the growth room t stndrd conditions for 28 dys, fter which the results were nlysed. This protocol ws used for ll experiments uxin pulsing durtion nd concentrtions were chnged in some of the following experiments. The shoots were lwys kept on the rooting mintennce medi with no uxins for 28 dys before being evluted. 38

42 2.6.1 Optimistion of durtion of IBA tretment for root induction IBA hs long been one of the preferred nd ctive uxins to promote root formtion in in vitro cultured plnt shoots (Epstein & Muller, 1993). Previous root initition trils with the existing root induction protocol for V. grndis by McComb, Newell & Arthur (1986) showed inconsistent nd poor root growth of the V. grndis clone for ech tril. In order to determine the optimum IBA pulsing period for root induction, shoot replictes were grown on rooting medi supplemented with 10µM of IBA (s per root induction protocol by Bennett, et l., 2004). For ech dy of tretment 25 shoots of GRD1 were rndomly chosen nd the medi of the control ws not supplemented with IBA. The experiment ws designed for 7 dys; on ech dy 5 shoots, from 5 rndomly selected vessels, were trnsferred to rooting medi tht contined no IBA. These were then kept in this medi for 28 dys, the experiment ws repeted twice; the numbers of roots were counted nd the mens clculted for ech dy of pulsing. For this experiment only the root numbers/shoot were nlysed in order to determine the most effect IBA pulsing time. This ws repeted with GRD3 clones Optimistion of IBA concentrtion for root induction The root formtion performnce is ffected by the concentrtion of uxin, the type of uxin nd the plnt species. IBA is often the preferred uxin for root induction, s plnt tissues cn rpidly oxidise other uxins such s IAA (DeKlerk, et l., 1997). IBA cn either promote the formtion 39

43 of dventitious nd lterl roots, or it cn inhibit this process this vrition depends on the IBA concentrtion nd the plnt species (Muller, 2000). For this experiment different IBA concentrtions were tested to determine the optiml concentrtion to chieve the gretest number of shoots with root induction nd growth. The rndomly selected shoots (GRD1), 25 for ech tretment, were pulsed for 6 dys on medi supplemented with different IBA concentrtions (0, 2.5, 5, 10, 20, 40, 80 nd 160µM). After this, ll shoots were rndomly trnsferred to culture vessels rooting medi without IBA, visible root formtion ws recorded for ech shoot on dily bsis nd then totls scored fter 28 dys for sttisticl nlysis nd evlution Trils with uxin combintions for root induction In contemporry plnt science reserch it hs become evident tht endogenous plnt uxins interct with exogenous uxins resulting in diverse fst nd slow responses (Woodwrd & Brtel, 2005). Different uxin combintions hve been successful for root induction in numerous plnt species due to their vrious properties (Mrtin, 2003; Mrtin, 2003/4; Pti, Rth, Shrm, Sood & Ahuj, 2006; Nndgopl & Kumri, 2007), so severl combintions of uxins were experimented with in order to chieve more overll consistency in root response nd to determine the optiml exogenous uxin tretment for root induction. 40

44 For this experiment it ws ssumed tht 80 µm of n uxin combintion would give comprble result, s ws chieved with 80 µm IBA, in order to determine gretest effectiveness for root induction nd formtion. The rndomly selected shoots (GRD1), 25 for ech tretment, were pulsed for 6 dys on medi supplemented with different uxin tretments: Tretments: no uxin, IAA (80µM), NAA (80µM), IAA (40µM) + NAA (40µM), IAA (40µM) + IBA (40µM), NAA (54µM) + IAA (26µM), NAA (40µM) + IBA (40µM), IAA (54µM) + NAA (26µM), IAA (26µM) + NAA (26µM) + IBA (26µM) or IBA (80µM - control). After this, ll shoots were rndomly trnsferred to culture vessels rooting medi without uxins, visible root formtion ws recorded for ech shoot on dily bsis nd then totls scored fter 28 dys for sttisticl nlysis nd evlution. 2.7 Trils with different in vitro substrtes for root induction In 2003, Newell nd co-workers reported results of experiments on shoot microcuttings of Verticordi species using porous gr medium tht contined ir filled porosity of 10% to 29% to enhnce root formtion. The reserchers concluded tht root induction nd length ws significntly incresed compred to stndrd gr solidified medi (Newell, 2003). Another lterntive in vitro rooting substrte being reserched by Newell et l., (2005) involves in vitro soil medium (IVS), which consists of n erobic rooting medium nd the results were compred with gr-solidified medi. It ws concluded tht for numerous woody perennils (including species from Myrtcee) the rooting performnce in IVS ws successful nd this method could reduce the stress nd plnt loss during the cclimtistion stge of micro cuttings from sterile in vitro conditions to in vivo field conditions (Newell, 2006). Newell, et l., (2005) nd Yn & 41

45 Sedgley (2006) experimented with different substrtes including IVS (in vitro soil medi) insted of gr bsed medi on difficult to root reclcitrnt woody plnts to increse root formtion nd vigour. For this experiment different substrtes were trilled; some were commercilly vilble whilst others were prepred prior to the experiment. The shoots (25 for ech tretment, rndomly chosen) were pulsed on gr/gelrite medi supplemented with 80 µm IBA nd fter 6 dys these were rndomly trnsferred to culture vessels tht contined uniform mount of sterilised substrte (utoclved t 121 o C for 20 minutes for ech different substrte). This ws supplemented with 50 ml sterilised liquid nutrient solution contining ¼ strength MS mcronutrients nd full strength micronutrients supplemented with 2% sucrose, ph djusted to 5.5. The substrtes included: Agr/gelrite, White snd (Ø 1 mm), white snd (Ø 1 mm), sterilised soil (2 pet: 1perlite), Jiffy pots, blck snd (Ø 1 mm) nd rockwool. This ws repeted with fewer tretments tht included: Agr/gelrite, blcks nd sterile soil (2 pet: 1 perlite) Comprison of sterilized soil with different nutrients In order to test if GRD1 clones will initite roots (fter 6 dy IBA pulsing) in sterilized soil without dditionl nutrients, the following experiment ws conducted: 25 shoots for ech tretment (rndomly chosen) were pulsed on gr/gelrite medi supplemented with IBA nd fter 6 dys these were rndomly trnsferred to culture vessels tht contined uniform mount of sterilised sterilised soil (2 pet: 1 perlite). According to tretment some were supplemented with 42

46 50 ml sterilised liquid nutrient solution contining ¼ strength MS mcronutrients nd full strength micronutrients supplemented with 2% sucrose, ph djusted to 5.5 (control), some with just 50 ml sterilised milliq wter supplemented with 2% sucrose, ph djusted to 5.5 nd the others just with 50 ml sterilised milliq wter. These culture vessels were kept in the culture room for 28 dys, fter which the roots were scored. The entire root system of ech shoot ws crefully seprted nd scnned for root surfce re nd length for comprison nd sttisticl nlysis Comprison of in vitro sterilized soil to ex vitro soil In order to test if GRD1 clones will form roots (fter 6 dy IBA pulsing) in soil ex vitro without dditionl nutrients, the following experiment ws conducted: 25 shoots of GRD1 for ech tretment (rndomly chosen) were pulsed on gr/gelrite medi supplemented with IBA nd fter 6 dys these were rndomly trnsferred to crckpots (direct soil) contining the sme soil composition s the control (sterilised soil in culture vessels tht contined uniform mount of soil medi supplemented with 50ml sterilised milliq wter, no nutrients). The culture vessels were kept in the culture room nd the other shoots were trnsferred to the greenhouse in seed trys with vented lids t temperture regime of o C in high humidity (~90% - 98%). After 28 dys the roots were scored; the entire root system of ech shoot ws crefully seprted, scnned for root surfce re nd length nd root numbers were counted for comprison nd sttisticl nlysis. All fresh shoots were seprted from ll roots nd weighed then ll shoots nd roots were dried t 70º C for 24 hours nd weighed gin to determine the shoot nd root biomss for sttisticl nlysis. 43

47 2.8 Survivl in greenhouse during cclimtistion phse For comprison 180 shoots of GRD1 clones (fter in vitro IBA pulsing) were trnsferred to sterilized soil (with just 50ml millq wter) in vitro nd then fter 28 dys were re-potted in to crckpots contining the sme soil mixture, which were plced rndomly in humidity tents tht llowed for retention of high humidity (90-98%) with periodicl misting. In order to fcilitte rooting simultneously with cclimtistion 180 shoots of GRD1 clones (fter in vitro IBA pulsing) were, t the sme time, plced rndomly (rrnged in rndomised complete block design) in crckpots contining uniform soil mixture in humidity tents tht llowed for retention of high humidity (90-98%) with periodicl misting. The survivl rtes for both tretments were scored in intervls of 28 dys for the initil cclimtistion in humidity tents, nd then the shoots were moved out of the humidity tents to reduce humidity (~60%). All shoots remined in the greenhouse until roots were visible t the bottom of the crckpots or until they died. All were scored fter 56 dys. The surviving plntlets were scored every 28 dys, nd then remining shoots were moved to shdy re in the glsshouse (t 84 dys) where the humidity ws less, but periodicl wtering regime ws in plce. After 112 dys the surviving shoots were scored gin nd then moved to the shde house for further in vivo cclimtistion. The shde house hd restricted wter regime of once in the morning (10 mins) nd once in the fternoon (10 mins). These surviving plntlets were then re-potted into lrger pots contining the pet nd perlite mixed with snd (1:1:1) for dditionl dringe. Long-term survivl fter re-potting continued to be ssessed every 28 dys. The pulsed shoots were trnsferred to the greenhouse on Mrch 8 th, 2008, nd this process ws repeted 6 times. Agin fter 112 dys ll surviving plnts were moved to the shde house with 44

48 mbient tempertures nd regulr wtering. Long-term survivl ws monitored for over 252 dys in totl. 2.9 In vitro shoot multipliction of new GRD clones Following the success of the shoot multipliction protocol for GRD1 & 3 the effectiveness ws tested for other clones. Shoot multipliction for new clones introduced into culture (Tble 2.1) ws not chieved for ll clones; VOV, VDF nd VFR did not produce shoots tht could be subcultured over time. Shoot multipliction proved to be difficult for GRD 4, 5, 7, 8 nd 13. The most successful shoots for multipliction were GRD 6, 9, 10, 11, 12 nd GF1, 2, 3 nd 4 (no results shown here), s these clones continuously produced multiple nd uniform shoots. The improved protocol for shoot multipliction ws used on ll new clones to produce comprble shoots with uniform length of ~4.5-5cm for root induction Root induction of successful new clones Root induction trils with the most successful new clones commenced pproximtely fter 1 yer in culture. The optimised root induction protocol ws pplied to shoots tht were pulsed on IBA/gr medi nd then trnsferred to crckpots with uniform soil mixture, plced in the humidity tents in the greenhouse nd monitored. In order to fcilitte rooting simultneously with cclimtistion, 40 shoots of vrious clones (fter in vitro IBA pulsing) were plced rndomly (rrnged in rndomised complete block design) in crckpots contining the uniform soil mixture in humidity tents tht llowed for retention of high humidity (90-98%) with periodic 45

49 misting. The results were scored every 28 dys for nlysis. These trils were repeted 3 times during 2008/09. The mens for survivl were clculted (rounded to complete number) nd results were combined for finl nlysis for ll plntlets tht performed well in culture. The following clones were used becuse of their similr physiology; e.g. firly uniform length, lrge leves nd vigorous ppernce GRD 6, 9, 10, 11, 12, GF1, 2, 3, nd 4 (lthough 1 nd 2 hd longer internodes thn 3 nd 4). All surviving plntlets were re-potted t 140 dys (28 dys lter thn the experiments with GRD1) in pet nd perlite mixed with snd (1:1:1) for extr dringe. Re-potting ws done lter (compred to trils with GRD1) becuse not ll plntlets hd roots visible simultneously by 56 dys Sttisticl nlysis All experiments were rndomised (or complete rndomised block design ws used) nd nlysis of vrince (ANOVA), s liner model, ws pplied to determine differences of mens between clones nd tretments. Levene s test of homogeneity of vrince ws pplied to ll dt sets nd where vrince were found to be unequl log + 1 trnsformtion ws performed. Different post hoc tests were used to determine differences between tretments; Tukey s B multiple-rnge test (P 0.05), or Dunnet s t-test, where ll groups were compred ginst the control group. All root lengths nd surfce res were mesured using n utomtic root nlyser Delt-T-Scn nd Delt-T-Scn softwre. For the nlysis of long-term survivl of rooted explnts in the shdehouse χ 2 test ws used nd resulting vlues were compred to χ 2 criticl tble to determine significnt probbility vlues (P 0.05). SPSS version 17 ws used for ll sttisticl nlysis. 46

50 3. RESULTS 3.1 Survivl of new clones initited into culture A totl of 380 shoots did not survive the first 2 weeks becuse of shoot deth or contmintion. Mny shoots hd vrying xillry bud formtion nd shoot elongtion, some to the extent tht sub-culturing ws not possible; these shoots were just trnsferred to fresh medi every 28 dys. Following subsequent sub-culturing of the clones every 28 dys mny more shoots died; fter 6 months there were no V.ovlifoli (VOV), V. frgrns (VFR) or V. densiflor (VDF) shoots remining (Tble 3.1). Tble 3.1: Percentge Survivl of new microcuttings over 28 dy period with cuttings being trnsferred to new medi in 50ml flsks dily. Survivl (%) Clone Strt Week 1 Week 2 Week 3 Week 4 (n) GRD GRD GRD GRD GRD GRD GRD GRD GRD GRD GRD GRD GRD GRD VOV VOV VOV VOV VOV VFR VDF

51 3.2 Chnges to shoot multipliction protocol It ws observed tht using the originl medi formultion resulted in shoot multipliction of GRD1 clones hving vrible shoot length nd prolifertion, nd in GRD 3 shoot multipliction ws prolific, with but with no consistent elongtion; most of the numerous shoots were less thn 1 cm in length. Sub-cultured shoots of uniform length (~ 2-5 cm) from both clones were needed for further experimenttion nd comprisons. In order to improve shoot elongtion necessry for shoot multipliction nd prolifertion of unvrying shoot lengths it ws decided to modify the BAP concentrtion supplemented to the growth medi. The highest number of shoots with more uniform shoot height ws chieved t 0.25µM BAP, s the shoots of both clones (GRD1 2.3 cm & GRD3 2.1cm) were comprble for shoot elongtion (Fig 3.2). All other BAP concentrtions produced shoots of vrying lengths (the shortest growth 0.7cm for GRD3 t 1µM nd GRD1 ws 1.1cm t 0.45 µm). The new shoots ppered much more vigorous nd helthier in growth ( more visibly uniform lef size nd uniform shoot lengths) compred to most explnt shoots before the experiment. The most unvrying height for both GRD1 & GRD3 shoots were chieved t 0.25 µm of BAP dded to the shoot multipliction medi. These results substntited reserch tht suggests tht xillry bud formtion nd shoot height could be promoted by using low concentrtions of BAP. Consequently the BAP concentrtion ws chnged to 0.25 µm for ll following shoot multipliction medi. 48

52 3.0 Men shoot height (cm) b b b GRD1 GRD BAP concentrtion (mm) Figure 3.2: Comprisons of different BAP concentrtions to improve shoot heights in vitro. The concentrtions of 0.25 nd 0.3µM BAP were significntly different (P 0.042) for GRD 1. For GRD 3 the concentrtion of 0.25µM BAP ws significntly different (P 0.029). Mens were nlysed using Anov; n=25; different letters bove brs indicte significnt differences (ANOVA, Tukey s test P 0.05); Error brs = ± SE. 49

53 3.3 Chnges to root induction medi composition The most commonly used uxins for root induction includes indole-3-butyric-cid (IBA), indole- 3-cetic-cid (IAA) nd α-nphthlene-cetic-cid (NAA), with IBA often being the most common one used for root induction. In previous root initition trils, using the existing root induction protocol (root induction medi supplemented with 10µM of IBA) for V. grndis by McComb, Newell & Arthur (1986), the results showed inconsistent nd poor root growth of the V. grndis clone (GRD1) for ech tril. It ws decided to determine optiml IBA pulsing durtion nd concentrtion Optimistion of durtion of IBA pulsing In order to determine the optimum IBA pulsing period for root induction, shoot replictes were grown on rooting medi supplemented with 10µM of IBA. Root formtion differed for the shoots tht were pulsed with IBA for 5 nd 6 dys; root initils becme visible just 7 dys fter the trnsfer from IBA pulsing medi to non-iba root growth medi, nd most shoots in ll of these culture vessels hd dventitious root growth visible fter 17 dys (Figure 3.3). Shoots tht were pulsed with IBA for 4 dys showed root growth on most shoots fter 23 dys, wheres the shoots tht were pulsed with IBA for 1, 2, 3 nd 7 dys showed poor root induction, with mny of these shoots not forming roots t ll. The highest percentge of root formtion fter 28 dys ws only 45% fter 6 dys pulsing on 10 µm IBA, which ws very low when compred to the originl root induction protocol results (McComb, et l., 1986). 50

54 4 ) b Men number of roots per shoot b b b) 80 Percentge rooting b b 20 0 b IBA pulse durtion (dys) Figure 3.3: Comprison of durtion of IBA pulsing for root formtion nd growth in vitro. ) The mens of roots for dys 1, 2, 3 & 7 were not sttisticlly different (P 0.125), but different to the mens of roots (P 0.031) for dys 4, 5 & 6 which hd no sttisticl difference. b) The gretest percentge of roots fter 28 dys resulted with shoots pulsed for 6 dys. Mens were nlysed using Anov; n=25; different letters bove brs indicte significnt differences (ANOVA, Tukey s test P 0.05); Error brs = ± SE. 51

55 Some shoots of GRD3 clones redily displyed root formtion on shoot multipliction medi so this experiment ws repeted twice for GRD3 clones. For GRD3 only the control tretment hd one shoot with visible roots compred to the other tretments, which hd no results (fter 28 dys ll shoots hd no visible root formtion); consequently no nlysis ws possible. All further experiments for root induction only used GRD1 clones nd 6 dys uxin pulsing for root induction Optimistion of IBA concentrtion Root formtion ws gretest for the concentrtion of 80 µm IBA with men of 7 ± 1.2 roots/shoot, followed by 6.3 ± 1.0 roots/shoot for 40 µm IBA nd 5.7 ± 1.1 roots/shoot t 20 µm IBA (Fig 3.4). Root initils for these tretments becme visible by dy 5 nd 6, fter trnsfer to medi without IBA. Root formtion on shoots pulsed on medi with the other IBA concentrtions becme visible much lter (dy 18-25), if t ll, with mny shoots not producing ny roots. The lower concentrtions (0=control, 2.5µM, 5 µm & 10 µm) of IBA hd little effect on root response nd formtion. All further experiments used 80 µm of uxin for root induction pulsing. 52

56 10 ) Men number of roots per shoot b b b 120 b) Percentge rooting b b b IBA Concentrtion (um) 53 Figure 3.4: Comprison of in vitro root induction nd growth using different IBA concentrtions. ) Men roots per shoot. Tretments with 0, 2.5, 5, 10 nd 160 µm IBA were not sttisticlly different (P 0.237), but different to the mens of roots (P 0.029) for tretments with 20, 40 nd 80 µm IBA were not sttisticlly different. b) Men percentge rooting. Mens were nlysed using Anov; n=25; different letters bove brs indicte significnt differences (ANOVA, Tukey s test P 0.05); Error brs = ± SE.

57 3.3.3 Auxin combintions for in vitro root induction Root formtion in the control (80 µm IBA) ws gin visible 6 dys fter trnsfer to n uxin free medi nd fter 17 dys 24/25 shoots hd roots from this tretment (Fig 3.5). Root formtion for ll other tretments were significntly lower nd most roots ppered visible fter 19 dys. The tretment with no uxin hd 1/25 shoots with 2 smll (>0.3cm) roots. Tretments contining NAA (3, 4, 6, 8 nd 9) produced lrge clluses t the shoot bse nd mens of 0.8 ± 0.3 nd 1.7±0.4 roots/shoot. IAA did ws not very effective for root induction; Tretments 2 nd 4 only produced men of less thn 1 ± 0.2 root/shoot. Tretment 5 nd 7 contined n uxin combintion with IBA nd produced mens of 3.4 ± 0.5 nd 3.8 ± 0.7 roots/shoot, which were more roots thn tretments without IBA, but these results were not sttisticlly different. For ll further experiments 80 µm IBA ws used for root induction. Commercil uxin pplictions (Clonex powder & gel for soft nd semi-hrd wood) nd prepred uxin solutions for dipping the shoots prior to trnsfer to soil for root induction nd simultneous cclimtistion ws lso trilled. However, within few dys ll shoots were ded, so this ws not further investigted nd s no results were obtined, they were not recorded. 54

58 Men number of roots/shoot Tretments: 1. no uxin 2. IAA (80uM) 3. NAA (80 um) 4. IAA (40uM) + NAA (40 um) 5. IAA (40 um) + IBA (40 um) 6. NAA (54 um) + IAA (26 um) 7. NAA (40 um) + IBA (40 um) 8. IAA (54 um) + NAA (26 um) 9. IAA (26 um) + NAA (26 um) + IBA (26 um) 10. IBA (80uM) control b Auxin tretment Figure 3.5: Comprison of in vitro root induction nd growth using different uxin combintions nd concentrtions. There were no differences (P 0.351) between tretments 1 9, compred to the control tretment 10 tht ws significntly different ( P 0.017). Mens were nlysed using Anov; n=25; different letters bove brs indicte significnt differences (ANOVA, Tukey s test P 0.05); Error brs = ± SE. 55

59 3.4 Different substrtes Mny lterntives to gel medi hve been investigted; e.g. fom, vermiculite, vermiculite nd gelrite, rock wool, filter pper bridges, glss beds, coir, luff sponge, jute fibre nd in vitro soil / IVS, s these substrtes cn fcilitte improved gs exchnge conditions. Trils with different substrtes (Figure 3.6) resulted in good rooting response with blck snd (8.1 ±2.7 roots/shoot, mny brnched with lterl roots), rock wool (6.6 ±2.9 roots/shoot) nd gr (4.4 ±2.0 roots/shoot, few brnched with lterl roots). Also shoots grown in blck snd nd rock wool ppered different to those grown in gr medium by hving more, longer nd some lterlly brnched roots. The first roots becme visible in gr fter 7 dys, in rock wool severl roots protruded t the sides fter 13 dys nd in blck snd some roots were visible t the side nd bottom of the culture vessels by dy 10. In the other tretments, the first roots becme visible lter; white snd (0.9 ±2.7 roots/shoot) by dy 17, corse white snd (1.7 ±2.9 roots/shoot) by dy 16, sterile soil (2.7 ±2.8 roots/shoot) by dy 14 nd roots protruded from soil jiffies (3.3 ±2.8 roots/shoot) by dy 14. Overll, the erly visibility of roots in the different substrtes suggests fster rooting response (compred to previous trils with gr). However, the individul roots growing from shoots in gr were the only ones tht could be clerly observed on dily bsis. 56

60 Men number of roots/shoot Tretments: Agr = control Snd 1 = white snd ( 1mm) Snd 2 = white snd ( 1 mm) St soil = sterilised soil (pet:perlite 2:1) Jiffies = commercil brnd Snd 3 = blck snd ( 1 mm) Rockwool = commercil brnd b b b 0 Agr Snd 1 Snd 2 St Soil Jiffies Snd 3 Rockwool Growth substrtes Figure 3.6: Comprison of in vitro root induction nd growth in different substrtes. The mens of roots between tretments white snd ( 1mmØ), corse white snd ( 2mmØ), sterile soil (pet : perlite = 1:3) nd commercil soil jiffies were not sttisticlly different (P 0.276), but different compred to the mens (P 0.022) of the roots between tretments blck snd ( 1mmØ), commercil rock wool cubes (5cm 3 ) nd the control (gr/gelrite medium) which were not significntly different. This experiment ws repeted twice to compre tretments Control, Snd 3 nd Rockwool only, s they hd higher mens of root numbers per shoot, confirming no significnt difference (P 0.153). Mens were nlysed using Anov; n=25; different letters bove brs indicte significnt differences (ANOVA, Tukey s test P 0.05); Error brs = ± SE. The roots from shoots grown in soil jiffies nd rock wool were problemtic to score, s ech contined 5 shoots nd the roots were difficult to completely seprte nd remove intct fter 4 weeks of growth. However, ech shoot hd formed t lest 1 root (often brnched with lterl roots), nd some shoots hd smll white outgrowths from the swelling t the shoot bse; it is possible tht these could hve developed into roots if given more time. 57

61 3.4.1 Comprison of gr, blck snd nd sterile soil The root formtion of GRD1 clones in blck snd ws sttisticlly comprble with gr (Figure 3.6), so the ffect of root induction in these substrtes ws compred to sterile soil rooted shoots. Root numbers lone, where short nd long roots re counted s equl, re not lwys n idel prmeter when compring plnt root systems nd therefore root surfce re nd root length were lso mesured. There were no sttisticl differences for mens of root numbers (P=0.012) between tretments (Figure 3.7). The root surfce re mens in Figure 3.7b (13.9cm 2 ± 1.1) for shoots mintined in sterile soil were sttisticlly different compred to root surfce re mens of roots grown in gr (8.4cm 2 ± 1.6) nd in blck snd (11.9cm 2 ± 1.8). The sterile soil nd blck snd roots (less in blck snd) were visibly different s well; these hd more brnched lterl root formtion compred to roots from gr tht consisted of single roots of vrying lengths. It ws lso observed, whilst seprting the roots from the substrte, tht not ll roots were submersed in the nutrient liquid. The men lengths (Figure 3.7c) were for roots grown in sterile soil (6.2cm ± 2.0) were sttisticlly different compred to roots grown in blck snd (5.7cm ± 1.8) nd gr (4.3 ± 1.7). Roots grown in gr were mostly single roots with no brnching, compred to the brnched roots of vrious thicknesses from the other tretments of in vitro blck snd nd in vitro sterile soil (Fig 3.8). 58

62 Men number of roots/shoot ) Men root surfce re (cm 2 ) b) b Men root length (cm) c) Agr Blck snd Sterile Soil b 2 0 Substrte Figure 3.7: Comprison of in vitro root induction nd growth in different substrtes ) Men roots per shoot. nsd (P=0.012). b) Men root surfce res grown in gr/gelrite = control were sttisticlly not different to blck snd (P=0.113) compred to roots grown in sterile soil (P=0.014). c) Mens of root length grown in gr/gelrite = control were sttisticlly not different to blck snd (P=0.245) compred 59 to roots grown in sterile soil (P=0.037). Mens were nlysed using Anov; n=25; different letters bove brs indicte significnt differences (ANOVA, Tukey s test P 0.05); Error brs = ± SE.

63 ) b) c) Figure 3.8: Comprison of hrvested nd wshed roots grown in different in vitro substrtes. ) Shoot with root formtion grown in gr. b) Shoot with root formtion grown in blck snd. c) Shoot with root formtion grown in sterile soil. For further in vitro experiments only sterile soil ws used. 3.5 Comprison of sterile soil with different nutrients The previous experiment chieved good root induction on GRD1 clones using the erted, porous substrte of sterile soil insted of n gr medium. This led to experimenting root induction with sterile soil supplemented with just sucrose, with complete nutrients nd with just DDI wter s control. The men root numbers for sterile soil +DDI (4.1 ± 1.2), sterile soil + sucrose (4.6 ± 1.8) nd for the control, sterile soil + nutrients (5.6 ±1.7) were not sttisticlly different (Fig 3.9). The mens for root surfce re (Fig 3.9b) for shoots grown in sterile soil + DDI (3.9 cm 2 ± 1.6) nd for shoots grown in sterile soil+ sucrose (5 cm 2 ± 2.0) were sttisticlly different from shoots grown in the control of sterile soil + nutrients (11.8 cm 2 ± 1.9). The root surfce re for the control tretment (sterile soil + nutrients) ws lso less thn in the previous experiment (Fig 3.7b, 13.9 cm 2 ± 2.0). The men root lengths for sterile soil +DDI (4.4 cm ±0.4), sterile soil + sucrose (4.6 cm ±0.1) nd for 60