Organogenesis in cultured petiole explants of Begonia × erythrophylla : the timing and specificity of the inductive stimuli

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  Organogenesis in cultured petiole explants of Begonia × erythrophylla : the timing and specificity of the inductive stimuli
  Journal of  xperimental  Botany Vol.  47 No. 297 pp. 557-567 April 1996 Journal ofExperimentalBotany Organogenesis in cultured petiole explants of Begonia  x  erythrophylla:  the timing and specificityof the inductive stimuli David J. Burritt 1  and David W.M. Leung Plant and Microbial Science Department University of Canterbury Private Bag Christchurch New Zealand Received 19 May 1995; Accepted 7 December 1995 AbstractCaulogenesis and rhizogenesis were studied in culturedpetiole explants of  Begonia  x  erythrophylla  in order tolink the developmental stages of primordia initiationwith the physiological requirements of the explant.Petiole sections excised from  B.  x  erythrophylla  plantsgrown  in vitro were highly organogenic, with shootsand roots arising directly from cells of epidermal srcin.Epidermal cells associated with glandular hairsappeared to be most responsive to organogenic stimuli.The point of explant determination for each form oforganogenesis was ascertained by media transferexperiments. Explants became determined for caulo-genesis after 7 d exposure to shoot-inducing medium(SIM), while requiring 3 d on root-inducing medium (RIM)for determination. Explants were strongly canalized forcaulogenesis once determined, but 5 d on RIM wererequired before becoming strongly canalized for rhizo-genesis. No organ specific differentiation was observedat the point of determination for explants exposed toeither shoot- or root-inducing conditions. Preculture ona basal medium containing no growth regulatorsresulted in a gradual loss of competence with time, butpreculture for up to 2 d on SIM or RIM resulted in areduction in the time for determination for both formsof organogenesis.Key words: Organogenesis,  Begonia  x  erythrophylla tissueculture, epidermis, determination.Introduction The ability of organs such as shoots, roots and flowersto arise adventitiously is a widespread natural phenom-enon, which is essentially being exploited in plant tissueculture (Flick  et ah,  1983). Despite commercial applica-tion and significant research, organogenesis is still poorlyunderstood.Organogenesis has been studied histologically in manyplant species (Banks, 1979; Christianson and Warnick, 1983;  Wright  el ai,  1986; Reynolds, 1989); organogenesiscan occur either directly from cells of the srcinal explant,or indirectly via callus formation. Often cells associatedwith the vascular tissues are the srcins of meristemoidswhich give rise to organ primordia, but studies have alsoshown that even specialized cells like those of the epi-dermis can, if exposed to the right conditions, undergoorganogenesis (Tran Thanh Van, 1973).More recently the application of tissue-transfer experi-ments between various culture media has revealed anumber of stages in the organogenic process. Using Convolvulus arvensis  leaf explants, Christiansen andWarnick (1983) divided the process of organogenesis intothree phases. In the first phase the explant gains theability to respond to an organogenic induction, that is itacquires 'competence', which in  C. arvensis  involves theproduction of a small amount of callus along the cutedges of the explant. Christiansen and Warnick (1983)found that competence could be achieved on shoot-, root-or callus-inducing media. The next phase is organogenicinduction; through the influence of the growth regulatorcomposition of the medium competent callus becomesdetermined to follow a particular developmental pathway.Once determined, the tissue can continue on this develop-mental pathway in the absence of growth regulators.Organogenesis in other tissue culture systems can also bedivided into these phases (Flinn  et al.,  1988; Attfield andEvans, 19916). 1  Present address and to whom correspondence should be sent: Department of Botany, Otago University, PO Box 56, Dunedm, New Zealand. Fax:  64  3 479 7583. E  mail:© Oxford University Press 1996   b  y g u e  s  t   onF  e  b r  u a r  y 7  ,2  0 1  6 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   558  Burritt and Leung In this study, the developmental patterns of shoot androot formation in  Begonia  x  erythrophylla  petiole sectionsare outlined and these are correlated to phases in theinitiation of both forms of organogenesis, as identified byvarious media transfer experiments. A. Preculture  on  BM0, 1,2 or 3or RIMdaysfor Materials and methods Plant material All experiments were carried out using explants taken from Begonia  x  ervthrophylla  J. Neuman (Beefsteak begonia) plantsestablished  in vitro  (DJ Burritt, unpublished results).  B.  x ervthrophylla  J. Neuman, also known as  B.  x  feiastii  Hort. exL. H. Bailey, is a hybrid begonia  B. manicatax B. hydrocotyli-folia)  probably of garden srcin.Micro-cuttings consisting of the shoot apex, two or threeexpanded leaves and numerous adventitious roots were subcul-tured every 8 weeks into 250 ml polycarbonate screw-cappedtissue culture vessels, each containing approximately 50 ml ofmaintenance medium. Plants were maintained in a tissue cultureroom at 24 °C under continuous lighting, provided by cool-white fluorescent lamps supplemented with tungsten bulbs. Thefluence rate was approximately 100 /xmol m 2  s~'.  ulture media and procedures Murashige and Skoog (1962) salts plus inositol 0.55 mM,nicotinic acid 40.6  /J.M,  pyridoxine HO 2.43 /xM, thiamine HC11.48 /xM, glycine 26.7 juM, folic acid 1.13 jiM, biotin 0.2 /Jvl, 3%  sucrose, and 0.7% Davis agar was used as a basal medium(BM) for the culture of explants, with the addition of 0.54 /j.Mnaphthalene acetic acid (NAA) and 4.44 (iM benzyladenine(BA) for shoot induction (SIM), and 5.4 ^M NAA and 0.22/j.M BA for root induction (RIM). Axenic plants weremaintained in culture on a maintenance medium (MM)consisting of half-strength Murashige and Skoog salts and 0.7%agar, no organics were included in this medium. All media wereadjusted to pH 5.8 prior to autoclaving.Petioles from the 4th, 5th, and 6th expanded leaves wereexcised from plants grown  in vitro.  The petioles were cut into5 mm sections. The sections were cultured vertically, in 9 cmPetri dishes, with the basipetal surface in contact withthe medium. Media  transfer experiments All basic media transfer experiments were carried out as stated.With the RIM to SIM/SIM to RIM experiments, all explantswere transferred to BM after 22 d in culture to allow organdevelopment. The transfer regime used to ascertain the criticalperiod of exposure to SIM for shoot induction is outlined inFig. 1. To ascertain the critical period for root induction,transfer experiments were conducted as outlined in Fig. I,except that explants were precultured on BM or SIM beforetransfer to RIM. All media transfer experiments were replicatedat least three times.Organ numbers were scored with the aid of a stereomicroscopeafter 6 weeks in culture. Leafy shoots were dissected from theexplant and counted. Roots were easily counted withoutdissection. A treatment was considered to be organogenic if themean value of organs per explant was greater than one. Histology Petiole explants were cut into 1-2 mm sections and fixed atroom temperature for 3-4 h under water vacuum in 2.5%Transfer to SIM   B. 0,1,2Culture on SIM,3,4,5,6,7,10 orfor14days   Transfer to BM 1 C.Culture on BM for organ development Fig. 1. Diagram demonstrating how transfer experiments can be usedto ascertain the critical period during which exposure to SIM is requiredfor shoot determination. (A) Culture of an explant on a medium unableto induce shoot formation for up to 3 d. (B) Transfer of the explant toSIM and culture for 0-14 d. (C) Transfer to basal medium for thedevelopment of determined organs. glutaraldehyde, in a 0.075 M sodium phosphate buffer pH 7.2.Tissues were dehydrated for 2 h in an ethanol series for 20 minin each of 10, 20, 40, 60, and 80%, with 2 x 30 min changes inabsolute ethanol. Specimens were also re-evacuated at a lowersurface tension, in 80% and 100% alcohol changes in order toremove any air adhering to hairs on the surface of thespecimens. Tissues were infiltrated in mixtures of 25, 50, 75,and 100% (x2) LR White acrylic resin (London ResinCompany) dissolved in ethanol, for 2 d each at roomtemperature. Polymerization of the resin was carried out at 4 °Cusing the LR White cold curing procedure. Flat-embeddingcaps were filled with LR White acrylic resin to which the cold-cure additive had been added (1 drop of cold cure additive to12 ml of resin), tissue sections were then quickly positioned inthe resin. The cap was then filled to overflowing and coveredwith a piece of parafilm taking care that no air was trappedunder the parafilm. The cap was then covered with a smallpiece of heavy glass and placed in a refrigerator at 4°Covernight.Transverse sections (2 or 4 ^m) of petiole explants wereobtained with a Reichert rotocut 2000 EX equipped with glassknives. Thin sections were stained with Methylene blue-azureA (Warmke and Sheu-Ling, 1976) and mounted in immersionoil. Bright-field micrographs were recorded on Kodak Ektar 25film using a Zeiss IM35 photomicroscope. Results Light microscopy of organ development Transverse sectioning of the petiole revealed anatomytypical of the Begoniaceae. Petioles consisted of a single-layered epidermis, with underlying corner-thickened col-   b  y g u e  s  t   onF  e  b r  u a r  y 7  ,2  0 1  6 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   lenchyma cells, followed by a cortex, consisting of equal-sized, highly vacuolated cells, with interspersed discretevascular bundles (Plate  1  A).  Epidermal, collenchyma andcortical cells all showed only slight cytoplasmic staining,indicating a low level of metabolic activity. Organogenesis in cultured  Begonia  petiole explants  559 Development on basal medium:  Culture on basal mediumcontaining no growth regulators or on SIM withoutsucrose (SIM-) resulted in no sign of organogenesis.Explants remained green on BM for up to 14 d afterwhich they rapidly turned chlorotic. Sectioning after 14 d \ Plate 1. (A) Transverse section through the petiole at day 0. (B) Transverse section through a petiole explant after 3 d culture on SIM, showingan early epidermal division. (C) Transverse section through a petiole explant after 4 d culture on SIM, showing numerous divisions in the epidermisat the base of a glandular hair and the underlying collenchyma tissue. (D) Transverse section through a petiole explant after 5 d culture on SIM.Meristematic regions composed of small densely stained cells with prominent nuclei form most often beneath glandular hairs. (E) Meristematicdome at the base of a glandular hair (not visible in this section) after 7 d on SIM. Note the lack of differentiation and minimal disruption ofsurrounding tissues. Bars = 50  ym.   b  y g u e  s  t   onF  e  b r  u a r  y 7  ,2  0 1  6 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   560  Burritt and  Leung culture showed no primordia and only rarely were regionsof meristematic activity observed. Explants cultured on(SIM—) remained green for longer than those culturedon BM, with many still green after 28 d in culture. Development on shoot-inducing medium:  The first celldivisions, usually observed after 3 d, were periclinaldivisions in the epidermal and, sometimes, immediatelyunderlying collenchyma cell layers (Plate IB). Thesedivisions seldom resulted in meristem formation.Meristematic regions, composed of clusters of denselystaining, small, mitotically active cells, with prominentnuclei, were first observed after 4 d in culture. Thesemeristematic regions were derived from anticlinal andpericlinal divisions of epidermal cells in close proximityto glandular hairs and were distributed over the entireexplant (Plate 1C). Only rarely were meristematic regionsnot associated with glandular hairs.Continued cell division in these regions resulted in theformation of small discrete zones of highly cytoplasmicrapidly dividing cells (Plate ID), which after at least 7 dculture developed into meristematic domes (Plate IE).The domes appeared continuous with the epidermis withno epidermal rupture. By day 9 the domes had enlargedand were often enclosed by a distinct tunica (Plate 2A).Sometimes a vascular trace was associated with the devel-oping domes.Foliar primordia were observed after 14 d (Plate 2B)and, although cell divisions were observed in the corticalcells surrounding the vascular bundles, the bundles them-selves showed no meristematic activity associated withcaulogenesis. By day 18, numerous apical domes enclosedby developing leaves were present on the explant.By day 24, the most advanced shoots had well organizedapical meristems, with leaf primordia developing fromthe shoot apex. The apex had the oblique orientationtypical of  Begonia.  Most shoots contained some vasculartissue but remained isolated from the vascular bundles ofthe initial explant. Development on root-inducing medium:  Histologically, theinitial stages of rhizogenesis were similar to those ofcaulogenesis. Small meristematic regions mostly, but notalways, associated with glandular hairs were observedafter 4 d in culture (Plate 2C). Unlike culture on SIM,these were confined to the lower half of the explant.Continued cell division in these regions gave rise to rootprimordia. Primordia only developed from the superficiallayers of the explant (Plate 2D). No adventitious rootsof perivascular srcin were observed and no connectionto the vascular tissue had taken place by day 24 of culture(Plate 2D).In undamaged explants, little internal disruption of thesrcinal explant occurred although some cell division inthe cortical and vascular tissues did take place withprolonged culture. Many explants did, however, showregions of random cell division. Most of these regionswere devoid of meristematic structures and consisted oflarge, highly vacuolated, dividing cells, similar to wound callus.  The epidermal and collenchyma layers above theseregions often appeared disrupted, containing collapsed cells.  This localized damage, or wounding of explants,probably occurred during the initial excision and sub-sequent handling of the petiole sections. Such regionswere also observed in explants cultured on SIM. Media transfer experiments Reciprocal transfer experiments were carried out to ascer-tain the duration of exposure to an inductive mediumrequired for determination. Length of exposure to SIM for determination:  Culture for7 d on SIM, before transfer to BM, was the minimumrequirement for shoot induction with most survivingexplants producing at least one shoot (Fig. 2). Prolongingthe period of culture increased the number of shoots perexplant, up to a maximum after 18-21 d on SIM (Fig. 2).Further culture caused little change in the number ofshoots subsequently produced. Transfer prior to day 7resulted in only occasional shoot formation.Experiments involving delayed exposure to SIM werealso conducted by preculturing explants on BM prior toexposure to SIM. Culture for up to 3 d on BM, beforetransfer to SIM, did not affect the number of shootsproduced per explant (Fig. 2). Further culture resulted inreduced shoot numbers with complete loss of competenceafter more than 7 d on BM (Fig. 2). Length of exposure to RIM for determination:  Culture for3 d on RIM, before transfer to BM, was the minimumtime required for determination (Fig. 3). A maximalresponse was observed after 10 d of culture on RIM,further culture did not result in a further increase in rootnumber. Explants could be cultured on BM for up to 3d, with no effect on root number, but prolonged culturecaused a rapid decline with few roots produced fromexplants pre-cultured for 7 or more days on BM (Fig. 3). Effect of BA concentration on the time required for shootdetermination:  To ascertain if BA concentration couldinfluence the time required for determination, explantswere exposed for various lengths of time to differentconcentrations of BA, before transfer to BM. Culture ona medium containing half the concentration of BA inSIM (BA0.5) resulted in few shoots, irrespective of thetime of transfer (Fig. 4). Culture on a medium containingfour times the concentration of BA in SIM (BA4) oreight times that in SIM (BA8) reduced the time requiredfor minimal determination and increased the numberof shoots determined by day 10 of culture (Fig. 4).Increasing the concentration of BA affected neither the   b  y g u e  s  t   onF  e  b r  u a r  y 7  ,2  0 1  6 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   Organogenesis in cultured  Begonia  petiole explants  561 Plate 2.  (A) Dome-shaped bud menstem with a tunica covering a central mass of small, meristematic cells after 9 d on SIM. Note the developingvascular tissue and the division of the underlying collenchyma and cortical cells pushing the bud away from the explant. (B) Foliar primordia areprominent after 14 d on SIM. (C) Divisions of epidermal and underlying collenchyma cells after 4 d on  RIM.  (D) Adventitious root after 24 d onRIM. Note the central cylinder and the minimal disruption of the cortical and collenchyma layers. The epidermis of the srcinal explant is stillcontinuous over most of the explant and the remnants of the glandular hair, beneath which the primordia formed. Bar = 50 ^m. time required for a maximal shoot forming response, northe number of shoots produced (Fig. 4).Despite reducing the time for minimal determination,exposure to increased BA concentrations did not speedup the development of shoot primordia. Both in surfaceview, and histologically, no difference was observedbetween explants cultured on SIM or BA8 for the sameperiod of time. Effects of SIM to RIM transfer regimes:  Although onlyrequiring exposure to SIM for 7 d for determination, thequestion arose whether growth regulator-containingmedia could still influence the formation of shoots beyondthis point. Media transfer experiments were conductedwhere explants were moved from either SIM to RIM,BA8 to RIM or RIM to SIM at various times over a 21d period. After 22 d all explants were transferred to BMto allow organ development. SIM to RIM:  With this transfer regime a similar patternof shoot formation was observed (Fig. 5) to that seenwith transfer from SIM to BM. Transfer prior to day 7   b  y g u e  s  t   onF  e  b r  u a r  y 7  ,2  0 1  6 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om 
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