MICROPROPAGATlûN OF 'JOHN FRANKLIN' ROSE AND ITS PHOSPHORUS UPTAKE
A Thesis submitted ta the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the Degree of
Master of Science
©
Jihad Abdulnour
Department of Renewable Resources Macdonald Campus of McGill university 21,111 Lakeshore Raad ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9
March 1993 ABSTRACT
Nodal sectlons of the W1.n ter-hardy 1 John Frankll n' rOSQ cul t l V,lr from f ield-grown plants were cul tured on a l1\o,h ( 1 t'cl r-1Ut"<1~,h 1 ~F~ and
Skoog (MS) nu t r1.ent medIum. Very h.HJb levels of c, 'n LIIl1l n,li Ion from the surface of the inl tial sec t 1.011S recl\.llred th,ll pl .,11 t~, lh~ gruwn under greenhouse COnd1.tlons. pLll1t 1ets obt .1111l'd f rom subsequent subcultures were used for the fllS! t l f1ll' ln .l radiotracer experimen t wi th '2p to s tudy the !(lnet les ()f ph()~~pl1()rus
(P) uptake as a funct1.on of tempera ture of the nu tt H'I1t nH'd 1 um. P uptake increased Wl th tin1e for rooted and non-root ('d p Ll!Ü lets in a Ilnear fashlon that d::-..d not rei1ch an equill.brlum v,Llup eVPIl ,I[ler"
96 hours oi. exposure. An analysls of va r iù\1Cc! rpvecd ed th,d ll\p plant lets wi th roots absorbed signiflcantl y greLller éllllOUIIls of P ,ü the 0.01 level compared w1th non-rooted plant lets at 22 n C. P uplLlke was signiflcantly h1gher al the 0.05 level, for rooted versus non
rooted plantlets at 33°C. There was no Slgrllfici1nt (h[ferenc(~ in P
uptake by rooted and non-rooted plantlet!3 al 3 u e. Illt f!!"dctlon
between time of exposures and rootlng was found tn be! s HJJll t LCdn t.
at 22°C and 33°C at the 0.01 level . 'l'he rlc!sult::i ll1 root system, prevIously thought to be InefflcHmt in the nutrü~nt absorptIon, played a key role ln absorblng P from the nutrim1t medium at optimum temperature. Ll. RESUME [J, '; IY)uture~ de nOéuds prélevées sur le rosier rustique 'John f· fdnY l dl' élevé !-!n plc!ln ô.ur Gnt été mlses en culture sur un milieu t'ÎIJJd~,hJ(Jt! I~t Skou(J rnod.lfv:;. Un tùux -levé de contaminatlon a exigé q\H- lI' prÉ:l~vf.:mf~nt dt:~:; boutures so.t effectué sur des plantes cul t Ivép~:; sc)us-serrC!s. La technlqU'.~ de traçùge radioactlf a été clppll ln Vl t rn pdr pluslf:UL~ repiqual)es succe sifs dans le but d'étudier Li C l n~l l que' de l' absorpti on du phosr_ll.ore, en fonction de la tpmp!'rclrure d(: lù solutlon nutrltlve. "n a trouvé que le taux d' ab:,orpt ~on par les pousses enraciné\:,s et non-enracinées a augmenté de manière l.lnéaire, et n'a nas atteint un niveau d'éq\lll.lbn~ rn~/T.E.! après 96 heures de traitemc.:1t. A 22°C, les pousses enrilclll(:C>S onl absorbé slgniflcativement (au niveau 0.01) plus de phosphure, comparées aux pousses non-enraclnées. De même, à 33°C, les pLlIlh.:>~ enraclnées ont absorbé slgnificùtivement (au niveau o . 05) p lu:; de phosphore que les plan tes non-enracinées. Cependant, à 3°e, ilucune dJ fférence sJgluflcatlve dans le taux d'absorptlon du phosphore n'a été décélée entre les plantes ellracinées et les non--enrùcinées. L'anê1lyse de variance révèle une inter.let 1.on slqnlflcatlve (au niveau 0.01) entre la durée du trzlltement et l'enraClnement à 22°C et à 3"~oC. Les résultats lndiquent 'lue le système des racines, Jusqu'à présent. considéré lk1Il-fonct.lonnel d.:ms l'absorption des éléments, joue un rôle lmport~nt dans l'absorpllcr du phosphore du mllieu nutritif, sous condltlons de températ.ure optlmum. iii ACKNOWLEDGEMENTS l would like to express my strong gratltude ta my supervisor, Dr. N.N. Barthakur, for hlS guid<1nce a,~d support tht-owJhout thl') project. l am also especially grateful ta my cO-ddvl~er Dr. N.P. Arno':d for his advlce and cons.i..stent help. The techlHCLll Part of this work was done ln the labor,ltor l(~S of Agricul turp Canada Experlmental Farm at l'Assomption, Québec, under the keell supervision of Dr. Arnold. l wou Id llke ta thank my wife, Linda, for her understanding and support. iv TABLE OF CONTENTS ABS'l'I A(~Kr~()WLEDr:;EMEN'I'S ...... i v rrAI3tJE Of'" CONTENTS ...... V LI s'r ()F" 'l'ABLES ...... " ...... viii LIST UF FIGURES •••••••••••••••••••••••••••••••••••••••••••••••• ix CHAP'I'EH I: INTRODUCTION ..••••••••••••••••••••••••••••••••••••••• 1 1 • 1 Tissue culture ...... 1 1 .2 Ob]ectlves ...... 2 CHAPTEH II: REV l EW OF LITERATURE •••••••••••••••••••••••••••••••• 3 2 . 1 Winter-hardy roses ...... ••.•••••••••••••••••••••••• 3 2.2 Rose ffilcropropagation ...... 5 2.2.1 Plant materials ...•.•...•••••••••••••••••••••.• 7 2.2.1.1 Selectlon ...... 7 2.2.1.2 Surface disinfestation ••••••••..•••••.•• 8 2.2.2 Nutrlent medi\.lm ...... ••.•..••••.••••••••••• 9 2.2.2.1 Inorganic constituents ..••••••••••••••.• 9 2.2.2.2 Organlc constltuents .•..•••••••••.••••. 1 0 2.2.3 Multiplicatlon ...... 11 2.2.4 Rooting ...... 11 2.3 Phosphorus ...... 1 2 2.3. 1 Phosphorus and root system ••...••••••••••••••• 13 v -- 2.3.1 . 1 Root morphology ...... 1 .} 2.3.1 .2 Root physiology ...... 14 2.3.2 Phosphorus .-:lbsorpt lOIl and traI1S1oc .. l t ion ....•.. 15 2.3.3 Temperature and phosphorus uptake ...... 16 2.3.4 Radiotracer studles of phosphorus uptake ...... 18 CHAPT ER III: MATERIALS AND METHODS ...... 20 3.1 Plant material and culture establishment ...... 20 3.2 Preparation of nutrlent media ...... •..... 22 3.3 Plant multiplication ...... •...... 25 3.4 A pilot experlment wlth phosphorus ...... •...... 26 3.4.1 Temperature settlng ...... •.•...... 28 3.4.2 Sample preparation ...... 29 3.4.3 Sample cOuD~ing ...... •...... 29 3.5 Phosphorus ur. :ake experlment •...... •....•...... 31 3.6 Experimental design ...... •...... ••.....•.... 32 3.7 Autoradiography ...... •...... •.....•.••.•...... 33 3.8 Phosphorus determination in different plant parts .... 33 3.9 Dry to fresh weight ratJo ..•...... •...... 34 CHAPTER IV: RESULTS AND DISCUSSION ...•...... •..••...... 35 4. 1 Micropropaga t ion of roses ...... •..•.•..••••.•.. 35 4.1.1 Culture establishment ...... ••.•..••..••..•••.. 35 4.1.2 Multlplication rate .•...... •.••..••...... •.. 39 4.1.3 Rootgrowth ...... •.....•••••...... 40 4 .2 Phosphorus uptake wi th i2p •••••••••••••••••••••••••••• 42 vi 4.2.1 The pllot ex~eriment ...... 42 4.2.2 Rate of P uptake in the main experiment ...... 44 4.2.3 Phosphorus absorption of rooted versus non-rooted plan tlets ...... 47 4.2.4 Phosphorus uptake by plantlets freshly cut at t h (~ i r ba ses...... 52 4.2.5 Autoradiography ...... •••...... • 52 4.2.6 Phosphorus dlstrlbution in plantlets ..•.•....• 52 4.2.7 Ratlo of àry to fresh weights of plantlets ...• S5 CHAPTER V: CONCLUSIONS ...... •...... •..••....•.••••••••••••...• 58 5.1 Selection of plant materiel for rose micropropagation58 5.2 Mlcropropagatlon of 'John Franklin' rose cultivar ...• 58 5.3 Kinellcs of phosphorus '..lptake ...... •.•....••....• 58 5.4 Future research ...... 59 REFERENCES •••.•.••••••••.•••••••••••••••••••••••••••••••••••••• 60 vii LIST OF TABLES Table 1 Murashige and Skoog nutrlent medlllffi versus modified nutrient medium used for ' John 't;'rLlnklln' ...... 23 Table 2 Nutr1ent medium compOS1 t 10n usen ln ' John Frankl in' propaga tion ...... 24 Table 3 Percent contamination of 'John Frank.lin' in vltro cultured nodal pieces colleeted from fleld-grown plants ... " .. " " " " " " " " " " " " . " " " " " " " " " " " " " .. " " " " " . " . " .... " .3t3 Table 4 Percent contamination of 'John Franklln' in vitro cultured nodal pieces colleeted from a greenhouse-grown plant."."""""""""""""""".""""""""."".""."."""."." •• " .38 Table 5 In vitro multiplieatlon rate of 'John Franklin' rose cultivar ...... 41 Table 6 In vitro rooting percent of 'John Franklin' rose eultlvar and survival rate ln the greenhouse ...... 41 Table 7 SClntillation counts of l2p absorbed in in vitro cultured 'John Franklin' rose plantlets eut at thel.r bas es" " " " " " " " " " " " " " " .. " " " " . " " " " " " " " n " " " " " ••• " " " " " " " • " 54 Table 8 Phosphorus conte'1t of 'John Franklln' pL:mtlets grown in vitro."""""""""""."""""""""""""""""""""""""""""""""" .54 Table 9 Percent dry weight of 'John Franklin' plantlets grown in vitro.""""""""""" .. " .. """""""""""""""""""""""""""""""" .56 viii LIST OF FIGURES Flgure 1 Ruoted (A) and non-rooted (B) 'John Franklin' rose plantlets growing l..!l vitro ...... • 37 FHJure 2 P-32 uptake bl' rooted , Johrl Franklin' rose plant lets growlng ln vi tro wi th time a t three different tempera tures ...... •...... •..•..•.. 45 Flgure 3 P-32 uptake by non-rooted 'John Franklin' rose plantlets growing ln Vl tro wi th time at three different tempera tures ...... 46 Flgure 4 P-32 uptake by rooted and non-rooted 'John Franklin' in vltro rose plantlets at 3°C ....•.....••.•.••••••••••• 48 Figure 5 P-32 upt~ke by rooled and non-rooted 'John Franklin' in vitro rose plantlets at 22° ...... •...... 49 Figure 6 P-32 uptake by rooted and non-rooted 'John Franklin' in vitro rose plantlets at 33°C ...... •...•••••.•.•• 50 Flgure 7 Autoradlographs of P-32 uptake, after 24 h of ~xposure at 22°C by A: Rooted ( (1) old leaves, (2) new leaves ) and non-rooted plantlets ( (3) new leaves, (4) old leaves and by B: Control (1) and plant lets exposed to P-32 ( (2) longltudinal stem sections, (3) new leaf, (4) old leaf, (5) stem cross sections ) ...... •••••••••.••• 53 e ix CHAPTER r INTRODUCTION ,., Tissue culture The rose in both wild and cul t i va ted forms is loved and c1d!11 1 n'li around the world. Since ancien t tlffiCS, the genus has bt't'l1 oi interest to sClentists, poets, the rellglous, drisrClL'l',wy and. comrnon people and has been wldl?ly used dS a symbnl by Vdl'lOW.i profeSSIons. Since the beginnlng of the tvJl-'rl r H' t 11 ('t-'Il t ury, contlnuous efforts have been made to Improve and prOp,l (irraclla tion) deslrtlble plant trin ts such in f LUWf-'r color, shape, size and petLll nurr.ber, chc.lmjc ln fu L ld(Jf': slldpr: or color and plant size, and cold or drougl1l reslstanC0. Most of the follage plants sold Hl North America are pUJelucéd in Florida, USA, through in vltro culture and an ever lnCrC~i'1!_ilnq nUIllÛer of cut flowers and woody ornarnr~ntcll, IndU!:> trI cl l ilnd medicInal plants are aiso belng propagated by ttllS m~th0d. The obJectives of the present research proJect were ba~éd an th8 need ta develop knowledge on the uptake of phosphorus. Phosphorus l~ cûnsldered one of the most essent~al elements ln plant growth beCiluse of ~ ts presence ~n RNA and DNA, its involvement in photosynthesls and phosphorylatlon and its llnk to the Krebs cycle ilS weIl as nltrogen metabolism. Phosphorus lS aiso important because of ltS effect on plant maturity, rooting, flowering and its potentlal effects on nitrogen and magnesium content of the leaf and aiso leaf absclssion. The root system of tissue cul tured plantlets is supposed to be inef f lcient because of the absence of root hairs WhlCh are responslble for the uptake of nutrients and other materials from the medlum. A close examination is necessary regarding the truth of thlS statement. Different temperature regimes ar~ used in plant mlcropropagatlon, and cold treatment is sometimes recommended. Therefore, three different temperatures are included in the study. 1.2 Objectives The objectives of the present thesis were: (1) to explore varlOUS expIant source materials ta establish an in vitro culture for the ',John Franklin' rose cultivar (Rosa hybrida) i (2) ta study phosphorus uptake kinetics as a function of nutrient medium temperatures of 3, 22, and 33 oc. (3) ta examlne the raIe of the root system in the absorption of phosphorus by tlssue-cultured plantiets. (4) to determlne the dlstribution of phosphorus concentrations in cultured piantiets. 2 CHAPTER II REVIEW OF LITERATURE. 2.1 Winter hardy roses The majorit~ of modern rose cultivars, mainly hybrid teas, grandifloras, floribundas and large-flowered climbers, are usually grafted to a hardy rootstock. These cultlvùrs cannat be successfully grown in most parts of Canada without exte 1sive Wl.nter protection because extreme cold conditions kill the scion 0r grùft unlon or both. These tender roses rarely survive more than four years and about a thlrd of the plants die each year (Ogllvle and Arnold, 1992). Winter-hardy roses which can resist severe Canùdian winters have been developeà by Agriculture Canada, Central Experlmental Farm, ottawa, Ontario, and recently the Experimental Farm at L'Assomption, Québec, and also at the Morden Research Station, Morden, Manitoba. Most of the 17 roses developed at Ottawa and L'Assomption have been named after famous Canadian explorers and have been aptly called the 'Explorer' serles (Svedja, 1984; Agricul ture Canada, 1986; S4 r 1986; Ogil vie et al., 1993). They are characterized by their ability to surVlve temperatures of -30 to - 40 Oc with only snow cove~ as their wlnter protectlon and also grow on their own roots. They resist powdery miidew (Sphilerot hecil pannosa (Wallr. ex Fr.) Lev.) and blackspot (Dlploci1rpon rOSile Wolf.). The roses developed at Morden called the 'parkland' serles have similar characteristics but have aiso been bred for drought reslstance ta wlthstand the semi-arid western prairies. Sorne of the released varleties ln bath series were highly rated by the 1993 handboak for selectlng roses (Anonymous, 1992). The fIrst 'Parkland' roses released in Morden under the supervisio~ of the plant breeder, Dr. Henry Marshall, were developed using a native praIrie wIld rose species, Rosa arkansana, as a main source of winterhardiness (Ogilvle and Arnold, 1992): .. This wild rose flowers freely on new wood, sa that even when it is killed to the ground after a severe winter, it produces vigorous new growth and flowers abundantly". More varleties of roses, such as 'Morden Blush' and 'Morden Fireglow', with B..:.. arkansana in their lineage have been recently released in the 'Parkland' series under the present breeder Lynn Colicutt. The first roses developed by Dr. FelicItas Svejda in the 'Explorer' series were released as early as 1960. Initially, she concentrated on breedIng ROSél rugosa types, and later used 8. kordesii Wulff. as a source of disease reslstdnce, winterhardiness and climbing growth habIt (Ogllvie et al., 1993; Ogilvie and Arnold, 1993). E.Qg splnosisslmù altalca was also used as a source of hardiness in sorne of the breeding lines used in developing the shrub rose types. The long blooming characteristlc and the fIcHer quality were incorpor~ted from the more tender roses, such as hybrid teas and floribundas. In general, the 'Explorer' roses can be divided into three types, R. rugosa Thun. hybrids (n3.mely 'Martin Forbisher', , ,Jens Munk', , Henr 1. Hudson', 'David Thompson' and 'Charles Albanel' ), climbers (' John Cabot', 'William Baffin' and 'John 4 Davis') and miscellaneous shrubs ('John Franklin' and 'Champlain'). With Svedja's retirement ~n 1986, the 'Explorer' program was transferred from Ottawa ta L'Assomptlon, where two new cllmbers, 'Captain Samuel Holland' and 'Lou~s Jollet', and two shrub types 'Frontenac' and 'Simon Fraser' were recently released (Ogllvie and Arnold, 1992). An example of one of the more outstanding 'Explorer' roses released by Dr. Svedja in 1980 is the 'John Franklin' rose (Svedja, 1980; 1984). It was obtained from a cross between the floribundd 'Lilli Marleene' as a seed parent and a hardy seedllng orlglnating from 'Red Pinocchio' x ('Joanna Hill' and g. splnosissim~ 'Altaica') as a pollen parent (Ogilvie et al., 1993). 'John Franklin' can be described as a hardy and vigorous shrub resembling a floribunda that flowers prolifically throughout the summer (for about 14 weeks). The flowArs are medium red and are barn in compound clusters of up to 30 blooms. The plant has good fleld resistance to powdery mildew (Sphaeretheca pannosa Wallr. ex. Fr., Lev.) and moderate resistance to black spot (Diplocarpon rosae Wolf.), (Osborne, 1991; Fortin, 1991). The plant is hardy to climatic zone three, and can survive the winter (in Ottawa) wlthout protection (Ogilvle et al., 1993). If it lS kliled back to ground levei during a hard winter, it will recover after prunlng of dead wood since it grows on it's own roots (Ogllvie and Arnold, 1992). 2.2 Rose micropropagation Traditionally, roses have been propagated by budding and grafting, 5 which are both expens~ve and time consuming processes. More r8cently, t~SSùe culture, an alternate system of rose propagation (Sklrv~n et al., 1990), has been used for rapid propagation and release of new cult~vars. In this system of micropropagation, plants are proliferated and rooted in autoclavable vessels containing a nutrient med~um that provides aIl of the nutrients, carbohydrates, vitamins and growth regulators. In 1974, Murashige (1974a, b) proposed a three stage protocol of micropropagation (st~ges 1, 2 and 3). Two additional stages (stage 0 and 4) were la ter found essential and included in the protocol. The five micropropagation stages were described by Debergh and Read (1991) as follows: stage 0: preparative stage of the mothar plant to provide least contaminated plant material; stage 1: ~n~tiat~on (establishment) of culture with the adequate expIant; stage 2: proliferation (multiplication) by a number of subculturesj stage 3: elongat~on and root induction to produce cuttings or rooted plantlets; stage 4: acclimation and transfer to greenhouse conditions to ensure a high survival rate of transplants. Several authors have proposed in vitro micropropagation systems for the conunercial production of roses (Bressan et al., 1982; Skirvin and Chu, 1979; Pit tet and Moncousin, 1981 1 1982; Ky te , 1987). However, micropropagation ùf roses in vitro is not without 6 problems, and the systems described in the l~ terature are not sui table for aIl cultivars (Dubois et al. , 1988; and LOllwaars, 1984). For example, w~nter-hardy roses developed by Agricul ture Canada are diff icul t to micropropagLl te Llnd l1\Odl f leLl t ions tù the medium are necessary before multlpllcat~on w~ll occur (Arnold et al., 1992). other problems, which Ky te (1987) descrlbed, lnclude death of explants due to harsh d~sinfectLlnts, strong media salts and contamination or toxic compounds released into the medium. She also suggested that slow plantlet growth mLly be a t tributed to dormancy, stage of growth of the expIant, medium and other physiological factors such as temperature. 2.2.1 Plant materials 2.2.1.1 Selection The condition of the mother plant, from which explants are taken, may affect the establishment of cultures. For example 1 j uvenile and rapidly growing plants are preferred over older Olles since the latter tend to have a decreased regenerative capaclty (Pierlck, 1987). Plants under controlled environment grow contlnuously and are a good source of plant mater~al aIl the yeC:lr round. When fH::ld grown mother plants are selected as source of plant material, expIant collect~on is preferably accornplished dur .1.ng growlng season only (Mamet et al., 1986). Although many different plant parts were reported as expIant source (such as leaves, shoot t~ps, buds and rneristems), axillary buds are preferable because of their easy handl~ng wh~ch results in a high 7 percuntage of success in rose micropropagation (Leffring, 1985). Bulb buc] size an<.l position were investigatedi medium size buds (2-4 mm Hl lf~flgth) develop in onl y 8 days, whereas, large or small buds tend to dIe (Barve et al., 1984). Bressan et al. (1982), found that bud positIon on the stem affected the commencement of bud developmcnt. He also noted that the first and last bud on tbe stem were the last ta grow. Similar f~ndings were reported by Mederos and Rodriguez (1987). 2.2.1.2 Surface disinfestation The first step in the expIant preparation for tissue culture is the removal of surface-borne fungal and bacterial contamination (Sk~rvln et al., 1984). Nodal pleces from woody plants grown under fleld caI1dit~ons tend la be hedvily surface cantaminated and are exlremely dlff~cult to d~sinfect (Hu and Wang, 1983). As green hou~e-grawn plants are usually less contaminated, they are easier ta surf~ce dis~nfect and are, thus, preferred over the field-grown plant s as source for explants. When choice is limi ted, weekly sprL1y"lIlg of f leld-grown plants wi th chemicals, such as fungicides, is recommended. Generally, plant organs can be disinfected with a solut~on contaln~ng NaCla (0.5 to 10%) and then rinsed with sterillzed dlstliled water (Hu and Wang, 1983). A wide variety of microorganisms can be found within plants (specially perennials) and are more difflcult ta remove (Cassels, 1991). These Inlcroorganisms may not express themsel ves for many subcul tures (Sklrvln et al., 1984). 8 2.2.2 Nutrient mediu~ After selecting the proper explants, nutrients and growth regulators are important in the success of micropropagatlon. Many nutrient media have been developed for tissue cul t ure, such as Murashige and S1 Gamborg et al. (1968) B5, Lir.smaeir and Skoog (1965) LS and Whi tp medium (1943). The MS medlum (Table in chapter II), which contains a rela tl vely high total saI t concen trat .10n, rPIll.:llI1S vl'ry popular since it is favourable to the growth of many plant species. Most of the diffe.cent media used currently 111 rose micropropd<)atlon are modlfications of that basic MS medium. Althou<)h many allthors have pI. oposed a gene l'al medium for roses (Sklrvin et al., 19B4; Ky te, 1987; Pi.ttet and Moncousin, 1982), the se were not eftecllve for the propagation of .111 rose cultlvars. 'rhus, the composltion of the medium often had to be modifled to successfully esldblish the culture and to improve p~antlet proliferatlon rate. In sorne cases, rose explants may exudate oXldLzed polyphenol-llke compounds into the medium WhlCh inhlbl t the ].r growth and Iead to their death. Addition of anti-oxidants (e.g. ascorb~c aCld and citrlc acid) or charcoal into the medlum (Plerek, 1987), as weIl as frequent subculturing would prevent thlS problem. 2.2.2.1 Inorganic constituents The composition of the nutrient medlum has been extensi vely discussed in the literature. !t is worth noting that the total salt concentration differs among the various media mentloned earller. 9 The 1-1S médium has the highest salt concentra tJ_on of the media and w~s prlmarll~ developed for herbaceous plants. For micropropagatlon ,)f rnC:Hly woody species, the saI t concentration had to be reduced to 1 /4, 1 /2 or 3/4 st rength to obtain good growth. Lloyd and McCown (1980) developed a low saI t concentration medium (woody plant medium or WPM) sUltab~e for the propagation of many woody pants. The salt concentration reduction usually involves the macro el ement!3 (Tùble 1) only, taklng into consideration the optln1al arrunoIllum/ni tra te ratio (Avramls and Huggard, 1982). Al though the inorganlc constltuents are less crucial to growth than the organic compositlon (Murashlge and Skoog, 1962), concentration of a partlcular salt can be very important for the successful mlcropropùgùtlon of certain species of hardy roses (Arnold et al., 1992). 2.2.2.2 Organic constituents According to Murashige and Skoog (1962), the organic constituents are cruclùl to plantlet growth. Vitamins and amino acids were found to be essentlal (Linsmaler and Skoog, 1965), and are used in very small .:lllloun ts. Growth regula tors, especially auxins and cytokJ_nins, hLlve a drama tic lnfluence on the growth of various plant parts, and were exlensively investlgated. Low conc~ntrations of auxins (naphtaleneacet lC aCld, NAA, and indolebutyric acid, IM) were recomml.mdcd by Hasegawa (1979) and Scott and Sutter (1990), and 0.002 mg/L of NAA was tested by Davies (1980) and Louwaars (1984). Slmllarly, cytoklnins have been reported in varylng concentrations 10 for shoot mu'.tipllcatl.on and the stlmulatl.on of bud development (Rout et al., 1989), but the optlmal concent ra t ion recomm0nded differed among cultlvars (Bressan e';: al., 1982; Khosh-Shul and Sink, 1982). Auxins, in the absence of cytoklnl.nS, were found to induce root formatlon (Hasegawa, 1980). 2.2.3 Multiplication The success of commercial plant mlcropropaga tion depends ta ù large extent on the rate of multiplication (number of shoots per expIant). Shoot proliferation (multiplication) was described by Skirvin et al. (1990) as belng largely the r~ ::.ul t of cytokl.nl.11 appllcation. Arnold et al. (1992) st- ..... Gd tl.~ effeet of growth regulators (cytokinln benzyladenlne, BA, and aux 1[1, NAA ) on the .!..!2. vitro multipllcatlon rate of sorne hardy and hybrld te~ rosas, and reported êI different response from dlfferent cul tlvars, wluch was in agreement with the fl.ndings of Hasegawa (1980). He also suggested that growth regulators, as weIl as other factors (including nutrient medium composl.t.lon), should be eX<.lmlned for each cultivar. With optimum growth regulator concentratlons ln the medium, multiplication rates of up to 5.5 (Khosh-Khul and Slnk, 1982) and six (Hasegawa, 1979) were reported. 2.2.4 Rooting Most reports show that the rootl.ng stage requlres auxin concentrations to exceed cytokl.nin concentrations, to the extent of complete elimination of cytokinl.n in the medium (Khosh-Khul. and 1 1 Sl-nk, 1982; Bressan et al., 1982). Root induction and development in general were enhanced by the absence of cytokinins, reduction of the overall salt strength (Hasegawa, 1980; Hyndman et al., 1982) and the presence of various auxins (Khosh-Khui and Sink, 1982). Effect of auxins (NAA and indolebutyrlc acid, IBA) and media salt concentrations on rooting of selected hardy roses was reported by Arnold (person;:!l cor.ununication) , where optimum rooting requirements vuried wlth rose types. Therefore, he suggested that the effect of plilnt genotype on rooting should not be overlooked. His conclusion is comparable to the one reported earlier ( Arno]1 et al., 1992) for the plantlet multiplication of the same cultivars. Al though rooting roses ln vi tro was considered to be troublesome by Skirvin et al. (1984), and direct rooting of plants in soil was achieved for sorne varleties (Pittet and Moncousin, 1982), rooted plantlets showed a hlgh survival rate after transferring to soil environment (Bressan et al., 1982). Arnolct reported a high rooting percentage (ùp to 100%) in vitro for ha,::dy and hybrld tea roses after 14 days ln a relatively short time, followed by a high survival rate (higher than 95%) after transplanting (personal communication) • 2.3 Phosphorus After nitrogen (N), phosphorus (P) is considered to be the second most important nutrlent for crop production. It is a major essential element and its content in plants ranges from 0.20% to 0.50% of the dry matter (Benton Jones, 1983). This element is 12 indispensable for aIl forms of life because i t is a part of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules which are the bases of the genetic malerlal. It is used in DNA synthesis which occurs prior to chromosome doubllng and ce11 divi~ion during plant growth. In addition, P functions in energy transfer via adenosine triphosphate. Phosphates enter illtO organic combination in plants through esterification of adenoslne to ferm adenosine monophosphate (AMP) r dlphosphate (l\DP) and trlphosphate (ATP). The energy derived from photosynthesis , or releilSl'd throllgh respiration, is trapped mainly ln the ferm of ATP WhlCh is used as energy transfer in aIl cell metabol ism (Ozllnne, 1980). When P enters the plant, it is incorporated into the [lucleic acids, phospholipids, nucleotides, sugar phosphates and protelns. In plants, P is supposed to stimulate root productlon, hasten maturity and flowering and increase resistance to diseases. Inadequate levels of phosphorus, especially wlth the presence of high concentration of N, resul ts in olant stunting, delay in maturity and plant death. Since many soils are poor in readily available P, it is generally desirable to apply commercial fertilizers containing this element on practically aIl soils (Thompson, 1949). 2.3.1 Phosphorus and root system In hlgher plants, roots provide anchorage and support, and play il major role in absorption and translocation of water and nutrlents. They have been the organs most frequently used for studles of 13 general mechanisms of water and ion uptake by plant tissues (Luttge, 1983). Usually, mineraI uptake is affected by both root morphology (l.e. depth, length, branching, and root hairs) and physiology, as explained in the following discussion. 2.3.1.1 Root morphology A larger and weIl branched root system can exploit a greater volume of soils (Nye et al., 1977) and provides a greater contact with nutrients, thus, affecting nutrient absorption (Clarkson and Hanson, 1980). This root branching is especially important for the less mobile nutrients such as phosphate (Barley, 1970). In fact, P uptake increases wi th the root size as measured by length and radi~s, and in the presence of root hairs (Caradus, 1982). Even in solution culture, the size of the root system affects the uptake of phosphate which is usually absorbed at a high rate by the roots of growlng plants and is rapidly depleted in their direct vicinity (Mengel and Kirkby, 1987). Although, according to MarschneJ" (1983), mineraI exhaustion zones are limited in solution cultures, Troughton (1959) reported a 30% increase in P uptake with branched roots compared with nonbranched roots. 2.3.1.2 Root physiology Nutrient uptdke and translocation may be affected by the physiological variations along the length of the root. At root tlpS, the root is covered by an amorphous material (mucigel), containing free carboxyl groups and other material of hydrophillic 14 nature that facilitates the uptake of ions by ion exchange Wl.th solid particles (Lut tge, 1983). In addi t ion, the absence of the suberin layer in the endodermis of the younger p Working with corn roots, Burley et al. (1970), found that al] the regions of the root absorbed phosphorus effectively, and Fer<)uson and Clarkson (1975) reported that suberlzaLlon and endoderllldi thickening had little effect on the radial movemenL o[ phosphorus into the vascular tissue. Bowen and Rovira (1971) showed tllat tlk' root tip and the region where root halrs are developed con t (11 ned il higher concentration of phosphorus, which may be due ta uptake and/or translocatIon. Differences in phosphorus absorptlon between varieties and between lines (intraspeciflc var la t lon) is weIl documented (Lindgren et al., 1977 i Mackay et al., 1990) and suggests that both structural and physiological variations in roots affect nutrients absorption. 2.3.2 Phosphorus absorption and translocation Phosphorus is absorbed by plant roots main ly in the form of monovalent dihydrogen phosphate H~P04 (Ozanne, 1980). Generally, the phosphate content of root cells and the xylem sap JS about 100 to 1000 times higher than that of soil Solutlon. Therefore, the P uptake by plant cells against this steep concentratlon qradlenL suggests that it is a metabollcally driven process (Bleh~skl and Ferguson, 1983). Mengel and Kirkby (1987) suggested that the 15 [Jhospha te anions are moved into the nega ti vely charged cells (by the membrane-bound ATPases), against an electrical gradient. In thlS active uptake, the phosphate ion is exchanged for a hydroxyl lon (OH). Most of the phosphate ions entering the roots are absorbed by the outermost cell layers, and move radially across the root to the endodermis through the 'outer free space' or into the symplasm (by protoplasmic streaming). At the endodermis, the casparian strip restrlcts further inward movement (to the steel) to the symplasm and through the plasmodesmata (Ozanne, 1980). Phosphorus is then loaded into the xylem mainly in the form of inorganic phosphate (Pi) and is then transloca ted to aIl parts of the plant. After Inlgra tion to the leaves, Pi can be retransloca ted through the phloem ta other plant parts, particularly young leaves and growing points (Bieleski and Ferguson, 1983). The occurence of P deficiency symptoms in older leaves reflects the high retranslocation rate of that element (Marschner, 1983). 2.3.3 Temperature and phosphorus uptake In general, temperature affects nutrient uptake and translocation in plants. Root zone temperature (RZT) can alter root and plant growth, nutrient availabllity and uptake, and subsequently the supply of mineraI nutrients to plants. MacKay and Barber (1984) reported a reduction in P uptake at low sail temperatures and attrlbuted it to the limited root growth rather than to uptake at root surface. Shoots, also, were found ta affect the uptake rdte of 1 6 sorne nutrients (such as P) by roots ( Jager, 1979). Tagliavini et al. (1991) explained the increase in P inflow (per uni t root length) to the shoot by a higher shoot demand and absorption capability for P at low RZT. Bouma (1983) suggested that the nutrient uptake is reduced by low temperature, and Zurbuki (1961) showed that exposure ta 12°C decreased P, N and potassium (K) concentration in tomato plants. At three different root temperatures (8, 16 and 24°C), Tùgllavini et al. (1931) reported the highest P concentration in shoots of peach seedlings at 16°C, '"hen the P level in the nutrient solution was adequate to high. Similarly, Turner et al. (1985) showed that uptake rates in banana roats were depressed at both coldest and warmest temperature tested. Both influx (inward movement) and efflux (outward movement) of P can be greatly influenced by temperature. McPharlin (1981), working with water plants, found that low temperature exposure decreased the P influx and increased the efflux, to the extent that a net loss of P was recorded in Spirodela plants. According to MacDuff et al. (1986), the effects of root temperature on growth and uptake of nutrient ions are complex and varies with time, following plant adaptation ta temperature changes. In addition, plants can regulate their rates of ion uptùke over a certain temperature range and within genetically prescribed limits (Macduff, 1989). 17 2.3.4 Radiotracer studies of phosphorus uptake Radloactive tracers of many common elements ( r, P, Ca) have been used for measurlng nutrient movement in plants. As early as 1944, 2 Biddulph and Markle applied radioactive P as phosphate e p04) on a leaf and followed its movement in the phloem. They showed that phosphates move downward in the phloem and laterally from phloem to xylem. L~tnr, the utilization of phosphatic fertilizers by various crops in a series of greenhouse (Dean, et al., 1948) and field experiments (Nelson et al., 1948) was studied with 32p. These ploneering experiments established the use of radioactive tracers in investigating nutrient movement in plants. These authors found 2.5% absorption of the applied 32p-tagged fertilizer by cotton and 10% by potatoes. Rinne and Langston (1960) fed 32p to one part of the root system and studied the inorganic ion transport in the xylem and its redistribution in the leaves of peppermint plants. Of aIl essential macronutrients, P has the lowest concentration in soil solution. This lead to an extensive use of 32p in many soil studies (Larsen and Cooke, 1961; Larsen et al., 1965; Basset et al., 1970; Patel et al., 1975). More recently, radioisotopes labels were involved in most of the work done on nutrient behavior and transport in plants. For example, 32p was used in studies on phosphate uptake (Bahl, 1991; Bahl et al., 1991; Elliot et al., 1984), translocation (Bender et al., 1987) and distribution (Jain et al., 1982). Determination of metabolic fate of phosphate using 32p was also reported (Ashihara et al., 1985). Carleton and Read (1991) fed 14C and 32p to moss 18 shoots, and investigated the nutrient transfer from moss to conifers in the boreal forest ecosystem. It is worth noting that radioisotope tracers are now used in many fields and on a great variety of plants. 19 CHAPTER III MATERIALS AND METHODS 3.1 Plant material and culture establisment Plants of the winter-hardy shrub rose 'John Franklin' developed by Agriculture Canada were used in the present experiments. Lateral branches with 10-12 axillary buds, from field-grown plants, were selected at the end of the growing season (September, 1991). The top and basal buds were discarded, leaves and thorns were removed, and lateral branches cut into nodal pieces (2-3 cm long) each with one axillary bud. The explants were then placed under running tap water for about an hour, and disinfected in 2.5% NaCIO solution for 20-25 minutes. The disinfecting solution was obtained by diluting a commercial bleach with distilled-deionized water (400 mL of 6.1 % NaClO Javex with 600 mL of water). AlI subsequent procedures were carried out aseptically, using a laminar air-flow cabinet ( Model EG-4252 with EdgeGard hood, Baker Co., Maine). The surfaces of test tubes, containers, and the laminar air-flow cabinet were regularly disinfected by spraying 70% ethanol every few mlnutes to prevent contamination. Scalpels and forceps were sterilized by heat, using a bacteriological incinerator (Steri Loop, B9750-1, American Scientific Products). Rubber gloves were worn, and hands were regularly sprayed with ethanol. Great care was taken to avoid contamination inside the cabinet by keeping away the head (or hair) or any other unsterilized objecte Approximately 0.5 cm was excised from bath ends of each expIant to remove damaged 20 tissues, and the 1.5 cm long expIant was transferred ioto 25 x 150 mm culture tubes containing 10 (± 0.1) mL of a modified Murashige and Skoog (1962) medium. The glass tubes were opened ln the direction of air flow in the cabinet to minimize chiv1ces of contamination. The test tubes were lhen transferred ta the culture room where aseptic conditions were maintained with a series of Hepa air filters. The tubes were kept at room temperature (22 ± l)OC, and 16 hours of cool fluorescent llghting per day was provided by Cool White Sylvania fluorescent lamps. The Ilght intenslly at 2 plantlet level was 30-40 !lE m- S-l. In spite of taking aIl necessary precautions to prevent infection, 93% of the tubes were found ta be contaminated after 12 days and were discarded. 'l'hus, the first attempt ta establish the culture was, unfortunately, a failure as the high rate of infection did not allow the experiments ta proceed any further. The main cause of infection was thought to originate from the field-grown plants as the starting malerial. For this reason, a mature 'John Franklin' rose plant was transferred from the field ta a greenhouse in a 37 cm pot (28 L) that contalned a substrate of two parts biosol, two parts turface, and one part perlite. The plant was irrigated twice a day (automatically with a drip irrigation system), and with irrigation water fertilizer was applied ta provide about 200 ppm of N (15-15-18 and 15-30-15 were used on alternate days). An insecticidal soap (Safer) was sprayed on the plant once a week at the rate of 20 mL/L of water. After three months, the new lateral branches were selectnd for expIant 21 preparation by the same procedure as in the first trial. A total number of 145 nodal pieces were cultured, and after 5-7 days buds started to open without showing any sign of infection. In about three weeks tlme, shoots (1-3 per nodal piece) reached a suitable Sl?e for multiplication. 3.2 Preparation of nutrient media Nodal sections, resulting from axillary growth and plant lets were cultured on a Murashige and Skoog (1962) nutrient medium modified to contain three-quarter strength potassium phosphate (KH 2P04 ), potasslum nitrate (KNO j ), magnesium sulfate (MgS0 4 .7H20) and calcium chlor ide (CdC1 2 • 2H"O). Ammonlum ni trate (NH 4 N0 3 ) was adj usted to 1856 mg/L and zinc sulfate (ZnS04 .7H20) and manganese sulfate (MnS04 .H20) were increased to 21.2 and 33.8 mg/L, respectively (Table 1). stock solutions of maeronutrients, calcium chloride, micronutrients, Vl tamins, iron, inosi toI, BA, NAA and IBA were prepared seperately as shown in Table 2. Eaeh stock solution was prepared by adding 500 mL of distilled-deionized water to a one liter volumetrie flask and then adding the eorresponding chemical (s). Weighing accuracy of macro and mieronutrlent was ± 0.01 9 and ± 0.0001 g, respectlvely. The flask was then placed on a magnetic stlrrlng plilte for 15-20 minutes to ensure a homogeneous mixture, broughl ta volume with water, labelled and refrigerated at 10-12 oC. In the case of the growth regulators stock solutions, the BA sùmple was first dissolved in 20-30 mL of 0.1 N HCl, whereas NAA 22 Table 1 Murashige and Skoog nutrient medium versus modified nutrient medium used for 'John Franklin' Compound Murashige Modified and Skoog nutrient medium mg/L mg/L Macronutrients NH 4N0 3 1650 1856.25 KN0 3 1900 1425.00 MgS04 ·7H20 370 277.50 KH 2 P04 170 127.50 CaC12 ·2H20 440 330.00 Micronutrients MnS04 ·H20 16.9 33.8 ZnS04 ·7H2 0 8.6 21.2 H 3B03 6.2 6.2 KI 0.83 0.83 Na2Mo042H20 0.25 0.25 CuS04· 5H 20 0.025 0.025 CoC1 2 ·6H20 0.025 0.025 FeEDTA 36.8 36.8 Vitamins Glycine 2.0 2.0 Thiamine.HCl 0.1 O. 1 Nicotinic acid 0.5 0.5 Pyridoxine.HCL 0.5 0.5 N.B. The original MS medium used FeS04.7H20 and NalEDTA as a source for iron rather than FeEurA. 23 Table 2 Nutrient medium composition used in 'John Franklin' propagation Chemieal Cone. of Cone. of Composition stoek Amount nutrient solution solution g/L mL/L mg/L Mdcronutrients NH 4 NO j 61.875 30 1856.25 KNO j 47.5 30 1425.00 MgS04 ·7H.P 9.25 30 277.50 KH L P04 4.25 30 127.50 11 .0 30 330.0 Micronutrients MnS04 • H,lO 3.38 10 33.8 ZnS04 ·7H"O 2.12 10 21.2 HjBOj 0.62 10 6.2 KI 0.083 10 0.83 Nù;Mo042H 20 0.025 10 0.25 CuS04 ·5H"O 0.0025 10 0.025 CoC1 2 ·6H.P 0.0025 10 0.025 FeEDTA 3.68 10 36.8 Inositol 10 10 100 Vitamins Glycine 0.20 10 2.0 Thiamine.HCl 0.01 10 0.1 Nlcotinic acid 0.05 10 0.5 Pyridoxine.HeL 0.05 10 0.5 6-Benzylaminopurine (BA/BAP) 0.1 10 1.0 NaphtùJene acetic aeid (NAA) 0.01 0.5 0.005 Suc rose 30000 Phytùgar 6000 Indole buteric acid (IBA) 0.2 1 .0 0.2 NB: IBA is omitted in the proliferation medium. In the rooting medium, NAA and BA are omitted. 24 and IBA were first dissolved in 70% ethanol. The nutrient mediun was prepared by adding 500 mL of distilled - deionized water to a one liter Erlenmeyer flask and then pipetting 30 mL of each filacronutrient, 10 mL of each micronut r lCl1t, v llamin, mio-inositol and l.ron stock solutions as given ln Table 2. For the nodal section and multiplication medla, BA WaS added at the rate of 1.0 mg/L and NAA at the rate of 0.005 :ng/L ('1able 2). The rootlng medium was the same as above but only IBA was added (instedd of BA and NAA) at the rate of 0.2 wg/L. Both stock solutions and nutrient solutions were freshly prepared for each subcul ture. Sucrose was added to the medium a t 3% dnd dissolved by stirring. Then the Erlenmeyer flask was brought to volume (leaving sorne space for Phytagar), and the pH of the solution adjusted to 5.7 (± 0.1) using NaOH or HCl as the case may ~e. Phytagar was finally added at 6i . The solution was heated and stirred for about 15-20 minutes, and removed from the heating plate when it became transparent. While still hot, an automatic dispenser was used to fi Il 25 x 150 mm glass test tubes each with 10 (i 0.1 ) mL medium. The tubes were then covered with plastlc caps, and autoclaved for 15 minutes at 12°C and 16 psi. 3.3 Plant multiplication Under aseptic conditions of the laminar air-flow cabinet, the explants were removed, and the shoots that developed from the buds were cut at their bases. Each shoot was cut to 0.8-1 .0 cm long with one or two topmost leaves intact, and transferred to test tubes 25 fl11ed with a fresh modified MS nutrient medium. The plantlets were then incubated in the culture room previously described. After 52 days, the plantlets grew and the proliferated shoots were divided and transferred to a new medium, thus showing an average multiplication rate of 1.43 (per 52 days). In rare cases, shoots were suspected to arise from the callus formation at the base of the plant and were discarded. It took three subcultures, lasting 52 to 55 days each, to obtain the required n'.lmber of plantlets. Fifty days after the third subcul ture, 100 plantlets were randomly selected and used in the preliminary experiments on phosphorus uptake studies. Thus, only non-rooted plantJets were used in the preliminary experiments. Of the remaining plantlets, 288 were subcultered (fourth subculture) onto a fresh multiplication medium and 106 onto a rooting one. The plantlets cultured onto the rooting medium started to root after a few days, and the percentage of rooted plants was recorded. Twelve days after the fourth subculture, plantlets growing on the proliferation and the rooting media were randomly selected for further experiments on phosphorous uptake. The rest of the plants on the proliferation medium were allowed to grow for 40 additional days, and were then subculturpd (fifth subculture) to proliferation and rooting medium. Plantlets thus obtained were used l.n tissue sampling for phosphorus and dry/fresh weight determinations. 3.4 A pilot experiment with radiophosphorus A preliminary experiment was required as no information was 26 available in the literature on radiophosphorus in tissue-cultured rose plants. Phosphorus-32 e2 p) was purchased from ICN Biochemicals, Inc., Costa Mesa, California, as H1P04 in water with 99.9% radionuclide purity and specifie actifv1ty of 10.5 TBq/mg P (total activity = 74 MBq). 32p is a beta-enutter with a maximum energy of 1.71 MeV (1 eV = 1.6 x 10-19 J), and a 11ùlf-life of 14.3 days. Because of the relatively high energy of the betù-pùrticles from 32p, safety measures had to be undertaken to protecl the experimenter and other personnel in the laboratory. Experiments were, therefore, carried out in a medium-Ievel rùdioisotope labora tory designed especially for this type of research. An aseptic transfer chamber with a glass window and a UV germicidal lamp was installed in the radioisotope laboratory, and was used to transfer plantlets aseptically onto a medium containing IIp. For additional protection, a 1.2 cm-thick plexiglass sheet was used to reinforce the glass window of the chamber. Plexiglass lS an ideal shielding material for hard beta-rays that prevents bremmsstrahlung formation. Thus, exposure dose measurements with a monitor showed no beta-particle transmission through the plexiglass. Therefore, it was possible for the plants to be transferred to the radioactive nutrient medium inside the chamber with minimum exposure to radiation. Difficulties, however, were experienced in handling the 150 mm long test tubes to prevent leaves from d1rectly touchHlg the medium. These had to be replaced by shallow and wide-mouthed plastic containers to ensure that 1nside walls were not contaminated with radioactiv1ty while pouring the I 27 Solutlon into these containers and that leaves did not touch the medium. Each plastic container, 10 cm in diameter and 5 cm high, was filled with 50 mL medium containing 32p. The average depth of the medium was 1.5 cm. Plantlets (cultured for 55 days) were cut at their bases and inserted into the medium to a depth of approximately 2 mm. This kept them vertically erect and directed the movement of 12p through the plant tissues. The original radioactive solution was diluted with water so that 0.5 mL contained 37 MBq actlvi ty. This was further diluted in medium solution to provide the concentration of 37 KBq/mL. 3.4.1 Temperature setting The plastic containers were kept at three different tempe rature regimes. Three groups of plants, each with 32 plants, were subjected to these temperatures. The plants were adapted to the corresponding temperatures for 24 hours prior to starting the experiments. The first group of plants was exposed to (0-5)OC in an ice-water bath; the second was kept at (21-23)OC room temperaturei and the third was maintained at (30-35) oC in a temperature controlled water bath. These temperatures settings will be designated as 3, 22 and 33 Oc for the purpose of convenience. For each tempera ture, a control group of plants wi thout 32p was maintained for comparative purposes. Light intensity of 30 ~E m- 2 S-I was provided daily for 16 hours. 28 3.4.2 Sample preparation Four plants were harvested from one container for each temperature setting at time intervals of 0.25, 0.5, 1, 2, 4, 8, 16, and 32 hours. The plants were cut with a sharp razor blade weIl above the level of the medium, weighed with an electronl.c balance (± 0.00001 g), and placed in 22 mL scintillation glass vials. A new blade was used for each cutting. A constant volume (0.45 mL) of 33% hydrogen peroxide and 66% perchloric acid in the ratio of 1:2 by volume was added to a glass vial which was placed in an oven at 60°C for two and a half hours. The plant material was digested during this time, and 15 mL of distilled water was added to the vial that formed a clear solution. The vials were then ready for the scintillation counting. 3.4.3 Sample counting Al though 12p containing samples had to be handled carefully and transported to the counter in a shielded box, the counting itself could be carrl.ed out reproducibly, accura tel y , and relûll. vely inexpensively. Because of its high energy, I~p could be counted by the emission of Cerenkov radiatl.on l.n a liqUld scinLd 1.1 Llon spectrometer. Expensive scintillation cocktail was not necessary, as water could be used as the bulk liquid in the sClntlllaUon vials. The origin and characteristics of Cerenkov radiation have been studied in detail (Horrocks, 1974). Brlefly, Cerenkov radiation i8 produced when charged particles pass through a medium with a velocity greater than the velocity of light ln the medlum. 29 The exchange of energy between the molecules of the medium and the charged particle produces local electronic polarizations. When these polarized molecules return to their ground state, the excess energy lS released as light photons in the violet and near ultra violet region of the spectrum. The light photons interfere constructively when v > cln, where v is the velocity of the particle, c is the velocity of light and n is the refractive index of the medium. Cerenkov rad1ation is highly directional, a~d the threshold energy in MeV required to produce it is given by, 2 2 Em j n = O. 511 [ ( 1 - 1 / n ) -1 / -1 ] ( 1) where 0.511 is the rest mass of an electron in MeV. The minimum energy in water of n = 1.33 to generate Cerenkov rad1a tion is 263 KeV. Thus for 12p wi th 1.71 MeV maximum energy and approximately 570 KeV average energy, 86 % of its beta spectrum is above the threshold energy to produce Cerenkov radiation. A liquid scintillation spectrometer ( Model LKB 1219 Rackbeta, Wallac, Turku 10, Finland) was used to count the samples. The samples were corrected for color quenching, and backround. Chemical quenching was unimportant for Cerenkov counting as water, the heavy quencher in normal liquid scintillation counting with a cocktail, was used as the medium for counting. Optical quenching was avoided by remov1ng condensation and dust particles by wiping the surfaces of the glass vials with tissue papers before counting. Relative counts per nU!1ute (CPM) were obtained by averaging 5 repeat counts for each sample. 30 3.5 Phosphorus uptake experiments The preliminary experiments allowed modifications to be made in the main experiments which followed. 8mall plast1c tubes with caps were used instead of the larger containers for ease of transplantation into the radioactive nutrient medium. Each tube contained only one plant as required from the perspectlve of proper statistical analysis of data on replication. Time intervals of harvest1ng were set at 0, 1, 8, 24, 48, and 96 hours from experiences gained during the preliminary experiments. In these e~{per iments, the or 19 in<11 radioactive solution was diluted ta provide the concentratlon of 18.5 KBq/mL in the nutrient medium, into which the plantle~s were transplanted without cutting their bases. The temperature regimes were not altered, but experiments were conducted on both rooted and non-rooted plants. 8eventy-two rooted and 72 non-rooted plants, cultured for 12 days on corresponding media, were random1zed separately over the three temperalure settings. Within each tempera ture regime, the 24 plants were randomly chosen to fornt four replicates of six plants each. Finally, within each replicate, a plant was assigned for a particular time of harvest by emp10ying a numbering system for identification and ease of randomizalion. The uniformity of Slze of plants was maintained by rejecting plants that were tao small or too big from the populatlon at the very inception of the study. Root number and root length were recorded before transplantation. Two Cool White Sylvanla fluorescent lamps provided 30-35 f-tE m- 2 S-l daily for 16 hours for aIl the plants. The decay correction was made by using the equation, 31 N = NI) exp t-O.0485 t) (2) where N = number of radioactive P at any time t (in days) , and NI) = number of radioactive atorns of P at t = 0 • 3.6 Experimental design A 2 x ~ factorial deslgn was carried out with four replicates to examine the effects of rooting and exposure times (and their interaction) on the rate of phosphorus uptake at any ternperature setting. Two levels of rooting (rooting and non-rooting) and six exposure times were tested. Eight identical trays were available for this experiment, and the two levels of rooting were completely randomized to the trays that resulted in 4 replicates. Each tray was partitioned into S1X sections to which the six exposure times were randomized. An analysis of variance (ANOVA) of the data was computed for every temperature regime experiment (Steel and Torrie, 1960). Furtherrnore, response curves were studied according to the orthogonal polynomial procedure (for unequally spaced treatments) • The random distribution characteristics of radioactivity allows a theoretical relationship to be derived between standard deviation of countHlg error to the count rates and can be represented as, (1 = 1 / ( N" - Nb ) ..j NjT.. + Nb/Tb (3) where N~ = gross count rate of the sarnple, Nt> = background count rate, T~ sample counting time, Th = backround counting time. 32 3.7 Autoradiography Macroautoradiography was performed by using Kodak no-sereen X-ray films (Kodak diagonistie films, X-OMART AR, Eastman Kodak Co., Rochester, New York). A sample (leaf, horizontal and vertical thin sections of stem, whole plant) was plaeed in contact with the film emulsion and held in plaee by pressure withln a wooden cassette. A sheet of Saran wrap was used between the film and the specimen ta prevent ehemical fogqing. After the expasure periad, the s~mple was removed and the film develaped. Sinee the ion densi ty along the track of a partiele is inversely related ta the energy of the partiele, autoradiographs of high resolution eannot be expected from 32p. Unlike 3H and 14C, 32p produees diffuse images. However, autoradiographs were expeeted to provide valuable information on the localization of P in the tissues. 3.8 Phosphorus determination in different plant parts Seventy-two rooted and 72 non-rooted plants growing on fresh nutrient media for 15 days were used to study the P dlstributlon in different plant parts in vi tro. The plants were harvested by cutting the stems above the levels of the media. The two tnpmost leaves, the third, four th and fifth (when dvailable) leaves from the top, the petioles of the two topmost leaves, the petioles of the third, fourth and fifth leaves, and stems were used for analysis. The parts of 72 plants were pooled to<)ether to obtdin a large enough sample for the analysis. Thus, flVS samples each for rooted and non-rooted plant parts were obtained. Tlssues were 33 dlgested using concentrated sulphuric acid and hydrogen peroxide (Thomas et al. , 1962) • The P content was determined colorimetrically with a Technicon auto-analyzer. 3.9 Dry to fresh weight ratio Eighteen rooted and 18 non-rooted plantlets were used lo de termine the ratio of dry to fresh weight of the plants that were exposed to the three temperature regimes. The plants were havested, cut at thelI" bases above the nutrient medium and weighed with an electronic balance. The final dry weights were determined after 72 hours of drying to a constant weight in an oven at 65 oC. The ratio of these two weights was reported. 34 CHAPTER IV RESULTS AND DISCUSSION 4.1 Micropropagation of roses 4.1.1 Culture establishment As discussed in Chapter III, the choice of t.he source p1dnt materia1 to establish an aseptic culture is very important for d successful micropropagation. However, the choice of a sultdb1e source general1y involves the trial and error method winch is time consuming. At first, nodal pieces from 'John Frilnkljn' were selected from field-grovm plants at the end of the grow1.ng seilson (September, 1991) and were found to be heélV1.]y contilmini1ted. Greenhouse-grown roses were not avai1able to us for 11SG at the start of this proj ect. Common surface disinfes ta tion procedure using a solution containing 2.5% sodium hypochlorite (NdC]O) for 20-25 minutes was not effective 1.n eliminatlng entirely tlw surface pathogens of field-grown roses. After one week ln culture, more than 93% of the treated buds (Table 3) were infected dnd hüd la be discarded. Slmilar results of high contamlnation were a1so ohlained in another trial held one year later at the end of the ne .. t 23 were still infected after treatment and showùd COllt.lnlllldtlon only after a few days. Contaminants Wf~re vlsui111y jd(~ntlfjed ilS common saprophytes comprised malnly of Alternarla, P(~nH'l] l1UflI, and Cladosporium spp. In sorne test tubes, yeasts were dlso pre~ent. Complete disinfection of explants taken from the fleld was 35 extremely difficult, and could not be achieved by the commonly used surface sterilization procedures described in the literature (Torres, 1989; Skirvln and Chu, 1984; George and Sherrington 1984). Finally, buds seleeted from new lateral branches from a greenhouse grown plant were easily surfilee disinfested, al though the same explilnt preparation procedures as in the first trial were followed. A very low contamination rate (0.7%) was obtained (Table 4) which allowed il succesful aseptic culture to be established. Figures 1a and lb show typieal non-rooted and rooted plantlets used in our experiments. As in our experiments with roses, high contamination rates were aIse eneountered with peaeh and apricot shoot tips (Skirvin et al. 1979), and w~th pecan buds (Corte-Olivares et al. 1990). Most of our fungi contaminants (penlcillum, Alternaria, and Aspergillus spp.) were also reported by Sanehis et al. (1988). Yeast and bacterlal contaminants were alsu isolated by Leifert et al. (1990, 1989); 78% of the yeast strains were identified as Candidil spp., and 90% of the baeteria as Bacilli, Enterobacter, Mierococcus, Staphylocoeeus, and Pseudomonas spp. Additional measures taken to reduce these contaminants to levels compatible with the use of micropropagation, including the use of different disinfectants and anti-microbial agents, were not always satlsfactory (Enjalrlc et al., 1988). The suggestion for reducing contamlnation in woody speeies by using greenhouse or laboratory grown plilnts (Hu and Wang, 1983) agrees strongly with our findings on roses. Greenhouse-grown plants as source materials are being 36 e e l.ù -...J ------Figure 1 Rooted (A) and (B) non-rooted 'John Franklin' rose plantlets growing in vitro Table 3 Percent contamination of 'John Franklin' in vitro cultured nodal pieces collected from field grown plants (September 1991) Development Contaminated Non-contaminated Total Nodal Nodal Nodal pleces Percent pieces Percent pieces Percent with growth 16 Il.7 6 4.4 22 16.1 No growth 112 81.7 3 2.2 115 83.9 Total 128 93.4 9 6.6 135 100.0 Table 4 Percent contamination of 'John Franklin' in vitro cultured rose nodal pieces harvested from a greenhollse grown plant (January 1992) Developrnent Contaminated Non-contaminated Total Nodal Nodal Nodal pleces Percent pieces Percent pieces Percent with growth o 0.0 138 95.2 138 95.2 No growth 1 0.7 6 4.1 7 4.8 Total 1 0.7 144 99.3 145 100.0 38 increasingly used in micropropagation (Bressan et al., 1982 Mederos and Rodriguez, 1987 and Arnold et al., 1992). In nodal pieces selected from field-grown plants, only 16% of the buds developed (Table 3). Al though conti:lInina t 10n may have been largely respons1ble for the suppressed bud growth (12%), only 67% of the non-contaminated buds developed into shoots. However, 95% of the buds collected from actively grow1ng plants in the greenhouse (Table 4) developed into 1-3 shoots after few days Ùl cul t ure. Mamet et al. (1986), found that the best time for expIant collection is during the growing season, and Hu and Wang (1983) recommended the use of actively growing new shoots in woody species. 4.1.2 Multiplication rate Buds started to develop after 5 to 7 days on the multiplication nutrient medium. An average of 1.43 shoots 1 to 2, and in rare cases 3 shoots) were obtained per expIant (Table 5). Similar findings for different rose cultivars were aiso reported by Khosh Khui and Sink (1982). The shoots were allowed to grow for 21 days till they reached 1.5 to 2.0 cm in length. Shoot t1pS (0.5 lo 1.0 cm) were cut dnd transferred onto fresh medlum and ailowed to grow and proliferate in subsequent subcultures. In the second, third and fourth subcultures, lasting 52 to 55 days each, the mult1pIlcation rates were 1.37, 2.45 and 2.22 respectively (Table 5). A slmilar increase in the mult1pllcat1on rate w1th each subculture was a150 observed by Pellerin (personal communlca tlon) . Arnold et al., 1992, 39 obtained a higher mul tiplication rate (more than 3) for 'John Franklin' by increasing the cytokinin BA 1n the medium and reducing the time of plantlet harvest from 8 to 6 weeks. However, the plantlets in this study needed 50 days to reach the suitable size for subculturing, although four-week subculture intervals have been reported (Khosh-Khui and Sink, 1982) in other varieties. The .iD. vitro multip11cation l"ate varied with plant species (Khosh Khui and Sink, 1982), type of expIant, growth regulators (Hasegawa, 1979) and environmental factors (Bressan et al., 1982) suggesting that optimum requirements should be examlned for each cultivar (Arnold et al., 1992) separately. 4.1.3 Root growth Roots started to develop seven days after the plantlets were lransferred onto the rooting nutrient medium. A high percentage rooting (more than 97%) was obtained in 12 days (Table 6). The average root number/plant was 5.4 (± 1.7), with an average root length of 1.8 (± 0.4) n~. Our results agree fairly weIl with the f indlngs of Arnold et al. (1993) who reported 100% rooting in in v1tro cultured 'John Franklin' plantlets. The percent root 1ng obtau1ed W1 th ln vi tro cul tured rose planl tlets Vilrled in the Ilterature. Skirvin and Chu (1979) obtained 68% rootlng in three weeks with 'Forever yours' roses, and ri t tet et Moncousin (1981) reported 100% rooting wi th other cultivars. Arnold et al. (1993) found that the optimal rooting requlrements for roses varied with rose types and cultivars, and 40 Table 5 In vitro multiplication rate of 'John Franklin' rose cultivar Subculture Number of Proliferated Multiplication explants shools rate 1* 138 198 1. 43 2 138 189 1.37 3 168 411 2.45 4 202 448 2.22 * Nodal pleces Table 6 In vitro rooting percent of 'John Franklin' rose cultivar and survival rate in the greenhouse Rooting Total number Number of Percent of plantlets rooted plantlets rooting 106 103 97.2 Survival Total number* Number of Percent of plantlets living plant lets survival 24 23 95.8 * selected at random from the rooted plantlets 41 concluded that plant genotypes and the interaction between auxins and medium saI ts should not be over looked. Al though the roots produced in vitro were unbranched and lacked root hairs (Davies, 1980), most plants transferred to the greenhouse after a short period of accl~mation have survived (Table 6) and showed vigorous growth. 4.2 Phosphorus uptake wi th 32p 4.2.1 The pilot experiment As mentioned ln Chapter III, since no information on the use of 32p on tissue cultured roses is available in the literature, we had to perform a preliminary experiment for the sole purpose of determining several unknown quantities like the initial and final harvesting times and the amount of 32p to be applied. Furthermore, the small size of the plantlets posed the special problem of handling in so far as the transfer to the radioactive nutrient medium wùs concerned. Using a forcep, plantlets were transferred carefully 50 as to avoid contact between the medium and any other part of the plant lets except the stem length inserted into the medium to a constant depth at the centre cE the medium. We had to make sure that IIp uptake took place only through the stem and not via contamination of any other plant parts. We experimented on test tubes of various sizes and shapes for ease of transfer from the non-radioactive to a radioactive nutrient medium. For example, the 150 mm long test tubes, commomnly used in tissue culture research, were found to be unsuitable for transfer. Plastic test tube caps 42 2.7 cm (i.d) and 3 cm long were found to be suitable and were used in our experiments. Safety considerations restricted us from using bigger containers which would have meant the use of lngher amounts of 32p and increased hazards. The agar provlded enough strength to support the plantlets in a vertical orientation. Elght exposure times were selected at the beginning, ranging from 1~ minutes ta 32 hours. The P uptake appeared to start inunedlately as and within 15 minutes, traces of 12p were detected ln the plantlets growing in aIl three temperature regimes. Harrison and Dighton (1990), Cogllatti and Santa Maria (1990) also reported 32p uptake ln wheat seedllngs from nutrient solutions in 15 and 20 minutes, respectlvely. Other experiments on P uptake used 90 to 180 mlnu tes on ceda r t ree seedlinqs in a 32P-containing solutlons (Bender et al., 1987). Our preliminary experiments shovled an apprecic1blbe amount of P uptake after eight hours of 32p application (data not shawn) for 3 and 22°C medium temperatures. The rate of uptake indlcated thdt an equilibrium value might be arrived at about 96 hours. Bdsed on lhe results of the preliminary experiments, it was declded to start sampling plantlets a t 1, 8, 24, 48, and 96 hours after ILp application. Sampling at zero time was taken as the control. Boaretto et al. (1986) have a1so reported P translocation from leaves to different plant parts Wl thln 8 hours. Autoradlogrilphy was performed a t different intervals (6, 8 24 and 36 hours) ln determine the localization in various plant parts and qualitatlv8ly the amount of P uptake. 43 4.2.2 Rate of P uptake in the main experiment The rate of P uptake increased llnearly and significantly (p < 0.01) wlth tlme at aIl three temperatures and did not reach an equlllbrium value within 96 hours of exposure for rooted plant lets at 22 and 33°C (Fig. 2) as expected from the preliminary experlments. P uptake increased very slowly wlth time at 3°C and no lnteraction between time of exposure and rooting was revealed by using ANOVA. When the plantlets were divided into three groups accard~ng ta time af exposures, i.e. group A (0 ta 8 hl, group B (24 t0 48 h) and group C (96 hl, the intergroup difference was highly sjgnificant (p < 0.01). However, the intragroup difference was found ta be not significant. The non-raoted plantlets at T = 3 behàved slmilar ta the roated ones wi th a linear slope of 6.2. Again, equillbrium values were nat attained within 96 hours. An interpretat.lan for the slaw uptake of P at T = 3 is difficult because many factors may be involved such as wilting, and stress on metabolic activity. Severe wilting was observed and the plantlets looked very sick a t 3 oC. The leaves lost turgidi ty and became sticky to the plastic substrate of the containers. The maximum and the filllUlllum estimated standard deviations in count rates corrected for background were calculted based on equation (3) and are shown (Plg. 2 and Flg. 3). A large biologlcal variability in nutrient uptake was expected in spite of clonally grown experimental plants. Tlus varlab1.lity compounded with the relatively low absorption at on8 hour of exposure resulted in the maximum error when the count Llte was small. The estimated standard deviation was minimum when 44 Figure 3 P-32 uptake by non-rooted 'John Franklin' rose plantlets growing in vitro with time at three different temperatures Specifie activity (CPM/mg) 700 .~.~------, -t-- T • 3 -f- T • 22 -*- T • 33 600 500 400 300 200 Max 3D r 100 Min 80 l o - o 10 20 30 40 50 60 70 80 90 100 Time (H) 46 the count rates were high at 96 hours of exposure. 4.2.3 Phosphorus absorption of rooted versus non-rooted plantlets P uptake by roots was different at diffE'rent temperatures. At T = 3, the rooted and non-rooted plantlets were similar in their uptake behaviour (F . 4). There was no significant difference at 0.01 level of confidence. As mentioned above, plant lets were wilted and seemed to be barely surviving for a large nutrient uptake. The slope of the best fit line is 5.4 CPM/mg fresh weight per h. However, the individual slopes are also shown ln Fig. 4. In contrast, at T = 22 the rooted plantlets were heal thy and absorbed significantly (p < 0.01) more P than the non-rooted ones (Fig. 5). The interaction between rooting and exposure lime waS highly significant (p < 0.01). The effect of rooting became evident at 24 and subsequent hours. The rates of absorption were linear in both cases with different slopes. The slopes were 27.5 and 6.8 (CPM/mg fresh weight per h) for rooted and non-rooted, respectively. At T = 33, the effect of rooting on P absorption was signlficdnt at 0.05 level. There was a highly significant (p < 0.01) lnleraclion between rooting and time of exp0sure as in the case of room temperature (Fig. 6). Again, the rooted and non-rooted plantlets absorbed P Iinearly with time, with slopes of 15.8 and 5.5 (CPM/mg fresh weight per h), respectlvely. As in the case of T = 22, the difference ln uptake started to show after 24 h. This may be due ta the time necessary to recaver fram stress when the plants were 47 Figure 4 P-32 uptake by rooted and non-rooted 'John Franklin' in vitro rose plantlets at 3°C Specifie activity (CPM/mg) 700 ------~------, -4--- N on-rooted -+- Rooted 2 600 Siope = 6.2 T = 3 500 400 300 Siope = 4.6 200 - Max 3D j Min SD 1 100 o o 10 20 30 40 50 60 70 80 90 100 lime (H) 48 Figure 5 P-32 uptake by rooted and non-rooted 1 John Franklin' in vitro rose plant lets at 22°C Specifie Activity (CPM/mg) 3 ------ -- Non-rooted -+- Rooted 2.5 T = 22 Siope = 27,5 2 Max SD 1.5 Min SD l 1 Siope = 6,8-----<1 0.5 ...------ o'"1'F-----'------'-----'----'------'---L---- - _L ______1- --- - L ------o 10 20 30 40 50 60 70 80 90 100 Time (H) 49 Figure 6 P-32 uptake by rooted and non-rooted 'John Franklin' in vitro rose plantlets at 33°C Specifie activity (CPM/mg) (Thousands) 2 -- Non-rooted -+- Rooted 1.5 T = 33 Siope = 15,8 Max SD 1 1 Min SD l 0.5 - ./ ~/ Siope = 5,5 ~~ o 1 ~---~--~----~--~--~--~--~--~ o 10 20 30 40 50 60 70 80 90 100 Time (H) 50 tr~nsferred to the radioactive medium. Thus, in vitro roots increased P uptake with time. l1owever, to grow roots in vitro is not recommended by Skil'vin and Chu (1984) because the process lS expensive and ted1olls. These root sare usually unbranched and lack l'oot hail's, and thel'efol'e the1r l'ole in nutrient absorption was questioned and often underestim.lted (Davies, 1980; George and Sherrlngton, 1984). An alternative approach is to root plantlets directly lnto the soil-llnx which was successful (Pittet and Moncousin, 1982). Marin and Gella (1988) studied the anatomy of i-n vitro roots and they reported of no discantinuity in xylem vessels between these roots and the stem. If this is true, nutrient movement into the plants wl11 not be hindered. Our results support this finding. In additlon, the l'oots provide a greater contact with the medium by exposinq il considerably higher surface area for absorplion. P absorption 15 known ta be an active metabolic process, and honce the uptdke at T = 3 was reduced while at T = 22 and T = 33 the uptdke process was enhanced. Our resul ts are in fair agreement wllh the tacl t hd t T = 22 is narmally considered as the optimum temperdlure for growth of this cultivar of rose (Dr. Arnold, prlvale communication). Thus, rooted plantlets at optlTIlUm temperature absorb more nutrients and show a vigorous growth, WhlCh may result in a high trans~lant survival rate and rapid growth after transfer to sail. 51 4.2.4 Phosphorus uptake by plantlets freshly eut at their bases The non-rooted plant lets were observed to have a thin layer of callus cells at their bases. For this reason, we performed a trial experiment to study the uptake by freshly cut plantlets. At T = 22, the uplake was observed to be high (Table 7) which indicated that this may be the optimum temperature for growth of this rose. However, this needs to br conflrmed by further Experimentations because we did not have a sufficient number of plants for replication. 4.2.5 Autoradiography Autoradiography was performed to compare P uptake of rooted with non-rooted plantlets. The various parts of the rooted plantlets showed hlgher amounts of radioactivity than the non-rooted counterparts (Fig. 7a and Fig 7b). 12p was distributed throughout the plantlet, and although the autoradiograms were diffuse, phosphorus can be seen to be more concentrated in the veins of the leaves. 4.2.6 Phosphorus distribution in plantlets Since the resolulion of autoradiograms is always poor, P distrlbut lon WÙS lnvestigated Wl th atomic absorpt ion, and the results (Tdble 8) showed varlation in P concentrations in (hfferent plant parts. The normal range of P in rose plants is 0.2 to 0.3% of dry matter, and there is a similarity between nutrient concentration of in YlYQ and in vitro plants (William, 1991). 52 ~------ (A) (B) Figure 7 Autoradiographs of P-32 uptake 1 after 24 h of exposure at 22°C by A: Rooted ( (1) old Ieaves, (2) new leaves ) and non-rooted plantlets ( (3) new leaves, (4) old leaves) and by B: control (1) and plantlets expused ta P-32 ( (2) longitudinal stem sectlons, (3) new leaf, (4) old leaf, (5) stem cross sections) 53 Table 7 Scintillation counts of 32p absorbed by non-rooted in vitro cultured 'John Franklin' rose plantlets eut at their bases Average coun ts min- 1 mg-1 fresh weight Exposure time Temperature ( OC) in hours 3 22 33 0 23.2 56.3 36.1 24 433.0 376.1 113.5 96 920.7 1393.6 641 .9 Table 8 Phophorus content of 'John Franklin' plantlets grown in vitro (mg/g dry matter) Plantlet part Non rooted* Rooted* Avg plantlets plantlets Upper two leaves 3.46 5.40 4.43 Upper two petioles 2.97 4.84 3.91 Old leaves 2.40 3.54 2.97 Old petioles 1.28 2.11 1. 70 Stems 1.88 2.84 2.36 Avg 2.40 3.75 * Mean of 72 plantlets. 54 Therefore, the P concentration in our plantlets was adequate for growth. The higher P content recorded in the new plant parts (upper leaves and petioles) agreed with the fact that P is mobile in the plant and is always translocated to new growlng points, which explains the first appearance of P deficiency symptoms in aIder leaves. Another remarkable fact is that P concentration was found to be higher in each individual plant part of rooted compared with non-root~d plantlets. In addition, the higher P concentration in rooted plant lets supports our findings that roots had a positive effect on P uptake. 4.2.7 Ratio of dry to fresh weights of plantlets The growth of plant lets is known ta be greatly affected by medium temperatures. At T = 33, the plantlets were turgid, while ~t T = 3 they were stunted and leaves wil ted. Slnce the resul ts were expressed in terms of fresh welghts, determlning the ratlo of the dry ta fresh weights of plantlets at each temperature was deemed necessary for a better understanding of the results. The percent dry weight decreased with an lncrease of temperature (Table 9) . in bath rooted and non-rooted plantlets. This decrease in the ratio was suspected ta be mainly due ta an increase in fresh weight at high medium tempera tures. Similar resul ts were reported in the literature (Leopold and Kriedmann, 1975). At T ~3, the condensation observed on the foliage could have corltrl~uted partially ta the decreased ratio. The interpreta t ion (JE t h(~ resul ts on P uptake should take this change in fresh weight lnto 55 Table 9 Percent dry weight* of 'John Franklin' plantlets grown in vitro Temperature Oc Nonrooted Rooted 3 27.69 (± 1 .20) 32.31 (±3.25) 22 18.28 (±1.44) 19.47 (±1.63) 33 9.92 (±1.06) 13.65 (±1.80) * (Dry Weight / Fresh Weight) x 100 56 consideration. The slight increase in the percent dry matter observed in rooted plant lets may be attributed to an increased uptake of nutrients. This ous0rvation is in agreement wi th our findings on P uptake. 57 CHAPTER V CONCLUSIONS 5.1 Selection of plant material for ro~e micropropagation Thf;! establlshment of an in vi tro cul ture of roses required careful :-;elect ion of the parent material to avoid excessive fungal and bdcter ial contamlna tians present on plant tissues. Greenhouse-grown plants, wH h relat i "ely less surface microbial infestations, requlred a moderate amount of effort to decontaminate, and should be used, whenever possible, as a source of plant material for mlcropropagatjon. 5.2 Micropropagation of 'John Franklin' rose cultivar The rapid in vi tro mul tiplica tion followed by the high plant let survival rate ln the greenhouse suggested that the micropropagation of 'John Franklin' rose promises to be economically feasible. 5.3 Kinetics of phosphorus uptake Phosphorus uplake J.l1creased WJ.th time but plant!: did not reach an equlllbl-lum value after 96 hours of exposure. Rooted plantlets Jbsorbed more phosphorus lhan non-roo~ed one~ at 22 and 33°C. At 3 Oc , the role of roots J.r1 P uptake was J.nhibi ted, and the non rooted klnet lC curve was above the rooted plant lets . This was .1t trll>uted to the condJ.tJ.on of the plantlets at this unfavourable temperature. An autoradiographic study agreed with the time course ll1L'.1surement s of P uptake. Al thouc.~h the roots developed in vi tro 58 have no root hairs, our results indicated that the root system was active at optimum temperature and enhanced P uptake. 5.4 Future research Whether the increase in nutrient uptake by the in vitro rooted 'John Franklin' is beneficial to plants after transfer to greenhouse conditions, future research should investlgate subsequent growth in the greenhouse and the field. 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