Morphometric analysis of reserve substances and ultrastructural changes during caulogenic determination and loss of competence of Eastern White pine ( Pinus strobus ) cotyledons in vitro

Morphometric analysis of reserve substances and ultrastructural changes during caulogenic determination and loss of competence of Eastern White pine ( Pinus strobus ) cotyledons in vitro
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  IntroductionDuring in vitro organogenesis, ompetent cells within anexplant espond o an organogenic timulus and after an appro-priate nduction period, become determined owards a specificpattern of differentiation (Christianson and Warnick 1983).Adventitious organs arise from groups of small, isodiamet-ric, densely cytoplasmic cells, termed meristemoids. Recently,less complex cell clusters, representing meristemoid precur-sors, were described n Pinus radiata (Villalobos et al. 1985)and Pinus strobus L. (Flinn et al. 1988) explants. n the latter  Author to whom all correspondence hould be addressed.2Present address: Forest Biotechnology Centre, British ColumbiaResearch, 650 Wesbrook Mall, Vancouver, B.C., Canada V65 2L23Present address: Forest Biology Division, The Institute of PaperChemistry, P.O. Box 1039, Appleton, WI 54912, U.S.A. Printed in Canada / lmprim6 au Canada 779study, exposure of cotyledons to [6N]-benzyladenine BA)showed hat minimal levels of caulogenic (shoot) determina-tion were present by the 4th day of culture, and near-maximallevels were attained by day 7 Delaying exposure o BA by pre-culture on basal medium for 2 days caused a significant reduc-tion in competence or caulogenesis. Further delays caused aprogressive decline in competence, and competence was lostafter 14 days.This study also showed hat organized hree- and four-celledclusters were present by day 4 in the presence or absence ofBA. The number of cell clusters, and their complexity, wasenhanced with continued culture on BA. The appearance ffive- and six-celled clusters coincided with increasing evelsof caulogenic determination, using our experimental definitionof determination. This suggested hat these structures epre-sented determined entities. The appearance f three- and four-celled clusters also suggested hat they were target cells for Morphometric analysis of reserve substances and ultrastructural changes duringcaulogenic determination and loss of competence of Eastern White pine(Pinus strobus) cotyledons in vitro Benny S. FrrNN,t DAVrD T. WBne,t AND Wrr-lrau NrwcovrsDepartment of Biology, Queen s University, Kingston, Ont., Canada K7L 3N6Received March 25, 1988FLINN, B. S., WEBn, D. T., and NrwcoMB, W. 1989. Morphometric nalysis f reserve ubstances nd ultrastructural hangesduring caulogenic determination and loss of competence of Eastern White pine (Pinus strobus) cotyledons n vitro.Can. J. Bot. 67: 779-789.Lipid, starch, and protein reserves were monitored cytochemically and, along with certain ultrastructural changes, werequantified morphometrically during adventitious shoot determination or loss of competence n cotyledons rom cultured Pinusstrobus zygotic embryos. nitial explants were rich in lipid, which declined during culture. There was more ipid retention nexplants on cytokinin medium by day 5 than on basal medium. However, by day 7 five- and six-celled clusters showed greaterlipid utilization than most other cells on cytokinin, which may have ndicated a shift towards shoot determination n these cells.Except for an initially greater etention on cytokinin, a similar pattern or storage protein degradation was observed n bothtreatments. Starch evels increased during culture, but did not differ between reatments during the first 7 days. Vacuolationincreased uring culture and was greater on basal medium than on cytokinin. Relative nuclear size also increased, but wasgreater on cytokinin. Relative mitochondrial area ncreased uring culture, except n noncluster ells on basal medium. Whilefew differences were noted between cell types within each reatment, distinct differences n nuclear size, vacuolation, and ipidcontent existed between cells on cytokinin and basal medium. These coincided with the timing of caulogenic determination ncytokinin-treated explants. The loss of lipid reserves, s well as ultrastructural hanges ssociated with the maturation of cellson basal medium, was associated with the loss of competence.FLINN, B. S., WEnn, D. T., et NewcoMn, W. 1989. Morphometric analysis f reserve ubstances nd ultrastructural hangesduring caulogenic determination and loss of competence of Eastern White prne (Pinus strobus) cotyledons n vitro.Can. J. Bot. 67 :779-789.Les r6serves en lipides, amidon et prot6ines ont 6t6 suivies cytochimiquement et, avec certains changements ultra-structuraux, ont fait I objet d une quantification morphom6trique au cours de la d6termination de pousses dventives ou de laperte de comp6tenc chez es cotyl6dons provenant d embryons zygotiques cultiv6 s dt Pinus strobus Au d6part es explantsdtaient iches en ipides, qui ont cependant iminud au cours de a culture. Aprbs 5 ours, il y avait une meilleure 6tention chezles explants cultiv6s sur un milieu enrichi de cytokinine que chez ceux sur milieu basal. Cependant, endu au jour 7, desgroupes de cinq a six cellules ont mont6 une plus grande utilisation de lipides que la plupart des autres cellules disposant decytokinine, ce qui indiquait peut-Otre n changement ers la d6termination caulinaire chez ces cellules. Sauf une meilleurer6tention nitiale en presence de cytokinine, un patron semblable pour la d6gradation des prot6ines de r6serve a6t6 observ6pour les deux traitements. Les niveaux d amidon ont augment6 au cours de la culture mais n ont pas montr6 de diff6rencesentretraitementsaucoursdesseptpremiersjours. avacuolations estaccentu6edurantlaculture tetaitplusprononc6esurlemilieu basal que sur celui enrichi de cytokinine. La taille relative du noyau a aussi augmentd mais 6tait plus prononc6e enpr6sence e cytokinine. La superficie mitonchondriale a augmentd urant a culture, except6 hez es cellules non-group6es urle milieu basal. Quoiqu il y eut peu de diff6rence entre es types cellulaires I int6rieur de chaque raitement, des diff6rencesdans a dimension du noyau, a vacuolation et la teneur en ipides 6taient pr6sentes ntre es cellules sur cytokinine et celles surmilieu basal. Ces diff6rences coincidaient avec a synchronisation e la d6termination caulogbne hez es explants rait6s auxcytokinines. La perte en r6serves ipidiques, aussi bien que es changements ssocids la maturation des cellules sur e milieubasal, 6taient reli6s la perte de comp6tence.[Traduit par la revue]  oo] 302010 b  * + n , Idef *iu 'fif ? cd i o ir +  ln 4 #?.' Toinf r,e ?,E ,+ Y T 'I 780 CAN. J. BOT. VOL. 67,1989 Days n Culture Ftc. l. Percentage f cell cross-sectional rea occupied y lipid droplets n subepidermal *, *), thre€-celled O, a), four-celled tr, I),five-celled ), and six-celled 0) cluster ells after 0, 3, 4, 5, and 7 days on SH (open ymbols) r SHB (closed ymbols). ach data pointrepresents he mean of l0- 1l cells, frcm thrce different explants. Venical lines about each mean eprcsent 1 SE. TrEatments haring he sameletter ale not significantly different at P : 0.05 according o Wilcoxon's sum of rank test. -oO rar o N7 BA, because n the continued presence of the cytokinin theydeveloped into more complex structures. In contrast toexplants on BA, cell cluster number and organization did notchange rom day 4 to 7 on basal medium. Cell clusters wereabsent by day 14. This suggested hat the loss of target cellclusters may have been involved in the loss of competence.The appearance f cell clusters under both caulogenic and non-caulogenic conditions, and their possible roles in both deter-mination and competence, equired urther study o see f therewere any differences between hem under caulogenic and non-caulogenic conditions.The accumulation and utilization of reserve substances reimportant during adventitious shoot formation (Biondi andThorpe 1982; Patel and Berlyn 1983; Patel and Thorpe 1984;Ross and Thorpe 1973; Thorpe and Murashige 1970). How-ever, the mportance of starch eserves n conifer caulogenesisis ambiguous Patel and Berlyn 1983; von Arnold 1987).Furthermore. he lack of reserve substances as been associ-ated with the oss of explant competence Aitken-Christie et al.1985). However, these biochemical and cytochemical studieshave not quantified cellular changes. This is required if thechanges n specific cells, as they become determined Hicks1980) or lose competence, re o be understood. Morphometrictechniques llow the quantification of changes n cellular con-stituents and he comparison of cells exposed o different stim-uli (Rosen t al. 1986).The objectives of this study were to compare changes nlipid, storage rotein and starch eserves, s well as changes nultrastructure within cell clusters and noncluster cells, duringshoot determination or loss of competence. The results pro-vided a quantification of the ultrastructural characteristicsassociated with cell determination or loss of competence.Materials and methodsSeed ources, isinfection rotocols, ulture onditions, nd culturemedia ormulations ere described reviously Flinn et al. 1988). Full-strength growth regulator ree basal medium is hereafter desig-nated as SH. Basal medium containing BA at 1 mg.L-' is hereafterreferred o as SHB.Following overnight seed mbibition, embryos were excised andcultured as described previously (Flinn et al. 1988). After 3 4, 5 7 and 14 days of vertical culture on SH or 3, 4,5, and 7 days of verticalculture on SHB, cotyledons were excised. Cotyledons rom imbibedseeds were used as the 0 day controls. Cotyledons were fixed andembedded as described previously (Flinn et al. 1988).Sections or light microscopy were cut at 0.5 pm using glass knives.Starch and other insoluble polysaccharides were stained using theperiodic acid-Schiff's (PAS) reaction (Jensen 1962). Total proteinswere detected y the aniline blue black method of Jensen nd Fisher(1968), which was used as a counterstain o the PAS reaction. Lipidswere stained with Sudan black B (Bronner 1975). Ultrathin sectionsfor transmission electron microscopy were mounted on formvar-coated copper grids and stained or 30 min in saturated uranyl acetatein 50% methanol, and for 6 min in 0.02% lead citrate (Venable andCoggleshall 1965). To localize polysaccharides, ultrathin sectionswere stained using the pnrcn (periodic acid - thiocarbohydrazide)procedure of Freundlich and Robards 1974). Ultrathin sections wereexamined with either a Zeiss EM9S or l0CR transmission electronmicroscope operating at 60 kV.Median sections of cotyledons rom each treatment were utilizedfor light and electron microscopic analyses. The detection of cellclusters within cotyledon sections was accomplished s described re-viously (Flinn et al. L988) using criteria described y Villalobos et al. (1e8s). Morphometric analyses were performed on transmission electronmicrographs using a Zeiss MOP-3 quantitative mage analyzer. Cellsfrom the first three to four subepidermal layers, which were notmembers of cell clusters, as well as cells from three-, our-, five-, andsix-celled clusters were studied. Each cell analyzed possessed amedian section hrough he nucleus. A total of 10-11 cells, fromthree different explants, were examined or each cell class, when pre-sent. The cross-sectional rea of each cell was measured, and he per-centage ccupied by lipid droplets, storage protein bodies, starch, henucleus, vacuoles, and mitochondria was determined. The data foreach of the above were used to calculate the mean percentage of thecell occupied by each structure. The standard error of the mean (SE)  FLINN ET AL. 781 *t s;*- ffF"-qr ": .e\, ._*. t_ *:" densed mtein; E, epidermis, ER, endoplasmic eticulum; FP, flocculant rctein; G, Golgi body; L, lipid droplet; M, mitochondrion;N, nucleus: CW, outer ell wall ofa cell cluster; PB, prctein ody; PL, plastid; PPL, prcplastid; , ribosome; C, secretory ell; SE, subepi-dermis; SG, starch rain; V, vacuole.Ftcs. 2-7. Light micrographs f cells stained ith Sudan lack B to detect ipids. Fig. 2. Epidermal nd subepidermal ells rom a day 0explant. Note he abundant ipid dmplets within he cells. x770. Fig. 3. Subepidermal ells after 3 days on SH. Note he educed ipid contentcompar€d ith day 0 (Fig. 2). X 790. Fig. 4. Subepidermal ells after 5 days on SH. The ipid content as declined ven urther rom peviouslevels. x660. Fig. 5. Epidemul and subepidermal ells after 14 days on SH. The najority f lipid has been tilized, and cells contain nly afew, small ipid droplets. 290. Fig. 6. Subepidermal ells affer 3 days on SHB. The ipid content as declined rom hat of initial explants(Fig.2). x7'70. Fig. 7. Subepidemal ells after 5 days on SHB. Cells contain more ipid resewes han corresponding ells on SH (Fig. 4).x820. *dE; =ffi}w1 'ffi: r. A&Nlt.'"s It *Y l,TF'rJt.o$n-o'n '.g1| :' k; qi * ; 1 ; S: f"s N-,:t w _Wruxww 3.o #m-- $$ k .q s *- ffi-u"Mfuk-w S| W *S#;ffiW WK ilM qp$#\r$'iss;*,W,, N f--KWN fl$ff;;;-*  Wwv "J u*.u .* ] t ${.*$x"s lis *; "}V,* _* ;'i{" *'''"* foew";dk;&--We ttqffi,i f{.I{,'QXffat\ .}SFq 6 'iff.i E#* Erf str?.:M 6 SX.\** fi-' n BBREVIATIoNS used on all micrographs): AM, amyloplast; CCW, cell wall between cells within a cell cluster; CW, cell wall; DP, con- $l-' . W.ITW  782 CAN. J, BOT. VOL. 67. 1989 Days n Culture FIG. 8. Percentage fcell cross-sectional rea occupied y starch rains n subepidennal *, *), thr€e-celled O, O), four-celled , l),five-celled A), and six-celled O) cluster ells after 0, 3, 4, 5, and 7 days on SH (open ymbols) r SHB closed ymbols). ample umber ndstatistics re as descdbed or Fie. 1. ,q6 \J o o\4was calculated or each mean, and means within an experiment werecompared or statistical ignificance t P : 0.05 using Wilcoxon'ssum of ranks est (Langley 197I). ResultsChanges n storage ipid were followed morphometrically(Fig. 1) and cytochemically Figs. 2-7). Lipids were he prin-cipal reserves n day-0 cotyledons Figs. 1, 2) where hey com-prised more than 40% of cellular area (Fig. 1). The lipidcontent of subepidermal cells decreased markedly during thefirst 3 -4 days of culture on SH and SHB (Figs. I, 3, 6). Therewas a gradual decline n lipids over he next 3 days Figs 1,4,7), and by day 14 on SH, lipids only comprised % of thecellulararea Table l, Fig. 5). During the first 7 days of cul-ture, lipid content decreased more rapidly on SH than on SHB(Fig. l). This was most evident by day 5 when all classes fcells on SHB had more ipids han on SH. By day 7, the major-ity of cells on SHB possessed higher ipid content han onSH. However, the lipid content of three, five, and six-celledclusters was no different from that in cells on SH (Fig. l).During the lst week of culture on SH and SHB, the lipid con-tent of most cell clusters was the same as that of surroundingsubepidermal ells (Figs. l-4).However, on SHB, cells nfive- and six-celled clusters had lower lipid levels than othercell classes fter 7 days Figs. ,20,2l).Starch was a minor component of uncultured cotyledons andaccounted or approximately 1% of cellular area at day 0 (Figs.8, 9). Starch content ncreased uring the first 3 -5 days on SHand SHB, then declined o day 0 levels by day 7 (Figs. 8,11 14). However, after 14 days on SH, starch evels ncreasedto 19% (Table 1, Fig. 12). No significant differences wereobserved etween imilar cell classes n SH and SHB, exceptat day 5, when three-celled clusters on SH contained morestarch han corresponding ells on SHB (Fig. 8). In both treat-ments, by day 7, peripheral cell layers had less starch thancells from deeper mesophyll ayers (Figs. 11, 14).Storage proteins were also a minor component of unculturedcotyledons, but were more abundant han carbohydrates Figs.9, 15) and comprised pproximately % of cellular area at day0. Proteins were stored n prominent protein bodies Figs. 9,16). Protein content decreased apidly during the first 3-4days on SH and SHB (Fig. 15). There was a delay n storageprotein degradation on SHB, and after 3 days, there was nosignificant difference n protein content compared with day 0(Fig. 15). This difference n protein degradation etween ellson SH and SHB was reflected n the ultrastructure of proteinbodies. After 3 days on SH, protein bodies were flocculant andstained ightly (Figs. 10, 17); on SHB, protein bodies wereintact and stained ensely Figs. 13, 18). By day 4, there wasFIGS. - 14. Light micrographs f cells stained ith PAS - Aniline blue black o detect olysaccharides nd proteins. ig, 9. Subepiderrnalcells rom a day 0 explant. Cells contain a few, small starch grains and numerous rotein bodies. x 830. Fig. 10. Subepidermal ells after 3 dayson SH. Starch rains rc morc prcminent. Prctein bodies tain ess ntensely nd are locculant n appeamnce, ndicating egradation. 830.Fig. 11. Epidenml and subepidermal ells after 7 days on SH. Starch rains re smaller n the peripheral ubepidermis, nd arger n the deepermesophyll. Note the absence f protein bodies, and he arge, central vacuoles. X380. Fig. 12. Epidermal and subepidermal ells after 14 dayson SH. Cells contain many prominent tarch rains nd are highly vacuolate. 340. Fig. 13. Subepidermal ells after 3 days on SHB. Starch grains arc arger and more numerous han n day 0 explants Fig. 9). Protein bodies stain ntensely and are morc ntact, n contrast o those on SH(Fig, 10). x740. Fig. 14. Epidermal nd subepidermal ells after 7 days on SHB. Note the few, small starch rains n the peripheralsubepidermis nd the more prominent starch grains n the deeper mesophyll. Cells contain ittle vacuolation and no protein bodies. x360. acd I | ",6 T+ +:+Ti *Tul*+ bcde "?" +","'i" *T +T  FLINN ET AL. 783 spB J**-* wffi ,3.T.-u-€$ 1";-;"q;& *f, q$ w,' 5 e* "' rfu $,, $* N &6x $b t&*G| €*s ryl'ss : &q* )eo. u #.6 k d ..;*$d 8R *" "i;:l',tr$ '' '* s sW N
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