17 Effects of Carbohydrates, Vitamins, and Gelling Agents on Callus — Growth and Plantlet Regeneration stdin ner hgh conpostion oh maul oan nein of he snvrnment og pea oy ‘influences the growth end morphogenesis Neh bam) ene catre. The fects of th rR a ee ner of the evs hares hi Ne ef cutehyites, vimans olin sess eo Sct apna rey ied ad em charted. CARBOHYDRATES: carbohydrate rogulrment for cals intenance were itil inves ee a (end ite (0), The supeintyof cor a8 tae cote wes fst pared by White 28) and Dormer the carbo has been confined by numerous works (6.6.7, wo nua 2) ler coh rcs ao Mh eh 1028 eA lly suppor tstue oth in cule (67,1816 2, a tee imped athe carbbyrse sed to support roth i be mea tough a cxpnon mere, whch ma my be ferent rte depending upon the arbobyast courte rouwced So tte of atk and conversion of the carbohydle (cafe to tn itera could reat owt Se er att for sucrose without Ts ofl goth erase tplopeppes oils) (7) and wide varity of ca eaaeeeerewe been show to suppor embryo formation i carrots Sobre Najecone and salons supperted growth a well 5 8 (20) ce, Bpetcn caltres of wuprane (17) and Pal’ sat rep anal tes gcioa and acto supped pow qual to that obtained with sucrosa during the fist generation; how ‘rer, during the second generation, callus growth was greater with ‘Sucre than wih fructose or glucos, ‘Carbohydrate concestatlon ha lzo been shown to influence callus maintenanee. Sucrose concantrtions between 20 and 60 gitar have boon uted Yo suppor callus growth (7 8,9, 12, 16,17, 23, 24, 28, 20, 31) Callas culture af tobaeca have been shown to be es dependent on Carbohydrate concentation as onthe hormonal rime (11). Tho opti- ‘al sucrove concentrations for in ito growl of carrot Ussues hasbeen reported to be 3% (8). "The method of seizing (autoclaved vs. Alto trlzation) specifi carbohydrates alo affect enl growth in elture. In particular, the use of autoclaved fructose rorults i decreased call rot in several plant Species (1 6, 16,16) Stebsl and Caplin (23) compared the elects of Shtoclaved vs, Altrstrilized facto on caro rot tissue cultures ‘They found that astocled factoe caused both an inhibition of callus ‘owt and a noticeable discoloration of callus, The degree of inhib {ion caused by autoclaved fructose Increased with Ineeaslg fructose {oncentrotions from 1 2% and wae groatst when fructose was auto ‘haved inthe presence of coconut mil. These authors reported siilar tht lose pronounced effects with atoelaved glucose. howove, both fructose and glucose, when fitersterized, supported adequate growth (2a). Autoclaved fructoso hat also Deen roperted to inhibi callus teowth in Paul's scarlet ove (lore sp) the most sigan! inbiion fccurted when frictose was autoclaved inthe presence of micron hts, specifically magnesium slat (26). Moteriols Required arte glass or plastic pete plats (100 mm in diameter) 250-ml beaker 2 acaple with new bla 1 a pe of forceps, and 3 sptulas ‘1 Waterproof pen and stck-on labels 3. Bunsen or etbanal burner 6 Galtre tubes (20> 150 mm) and plastic racks to hold them 7. 180 ml of 95% ethanol {82501 of 20% Clorox soltion supplemented with a few drops of ‘Tween-20 9, 400 ml sterile dail war 10. Stock cllzecultres from one or two species of plans (lily oF ‘Panneroe creping bentazas werk well) 18, 10 medium-stnd healthy Afican volt leavestan CALLUS (MSSUE AND ORGAN CULTURE Procedures Prepare 4 liters ofthe following medion Ms basal alts! Apr 80 giter ‘Thiamin HCL 1.0 mgt Myerinositol 30000 malitsr NAA 04 malter BA 10 miter pa 37 ‘This media wil be usa for the eegenaraton suds. 2. Prepare liters of the following medium, Ms basal sls! Ant 20. gilter ‘Thiamin HCL 10 mpliter Myoriosiol 10000 miter 24D 25 meler Ba a mer it 7 ‘This medium willbe used for the callus maintenance stds 14. Divide each ofthe medin into 250snl aliquots. To each of the liguotsad@ one ofthe following teatments: 0.0 gilitor sucrose 200 gir sucrose 00 gitar sucrose bn. liter sucrose 00 gilt maltose 00 pte glucose ‘9 giiter galactose 200 gyierplaciose 40.0 plier plactose 600 plier galactose ‘Add 10 ml of the above media weatments per culture tube, This wil ive you 25 replletions per Westnet ora total of 400 tubes the media that you prepared. Label all tubes with {Ceatmet date, medium, a issue to be inoculated int the cl ture tube, Prepare African Violet lef disks using the procedures {escrbed Blow forthe egenaraton medium. Prepare calls cl {ure av described below and noua onto the callus matnte- tance medium pea dein in Cpl 2 ng early psa ae pte conditions, remove a portion of callus tissue fom ‘tock ealluscultre of creeping bentgrass Ii. Cut into smal pisces, about 2 mm in diameter 2, Weigh each callie piece and then inoculate into culture tubes {6 pr ie} ong mda propa in he vos 2 Incubate the culture tubes at 25°C inthe dark Periodically moni tor the growth ofthe callus in each treatment Afelean Violet Loaf Cultures Place enough leves to completo the project ina beaker and wash | {ently under renning ap water. Stonizethe leaves for 6-@ min in Pom Clorox solution, then singe thro mes with steno distilled ‘ee Ne corae a ne voy coved th 2, Remove al outer edges of the leaves and section tho remaining portion into I-cm aquare. Inoclate one lec section, adaxial side Aloven, pe clture tube ance the elle ues at 25°C nl ight. Periodically mont tor the regeneration of plantlets from laf poses. Scheduling Event ‘Timing leoltion and inoculation of explants Day 0 cf appearane af planta formation Day 14-21 First notcoeble eects of carbohydrate trentments Day 21-28 ‘Termination of experiment Day 60 Recording Results TRecord all details of sting up the experiment 2, Make visual observations at Heday intervals 5, Determine fresh waht gain af callus cultares and shoot number of laf and cll cultures afar 60 days ‘VITAMINS, ‘Several vitamins including thiamin, pyridoxine, nlotinte acid foie ci ascorbi ald, and myo-nosital~-have been shown tobsessontial {orproper growth metabolism, and division of plant calls in culture 14,19) The Beeomplex vitamins ate particularly important, and thie sin (etc Bi especialy eal In most species. Thiamin func tons ae «coenzyme inthe decarboxylation of keto acids an is impor. tant incelluler metabolism in the whole plant. The omission of thiaminao CALLUS fISSUR AND ORGAN OULTURE from the medium: has boon shown to reduce callus growth in most Species (414). Yeu extoct Wed n air media have been replacod ‘y thin end pyridoxine HCH some species 20, 22,22, 91.32), The Bintoum concervation of thiamin required for satisfactory callus frowth of tobnceo cultivars was 400 yer (14), ‘iootinic aid influences catboydrate metabolism, meristematic tiny, and cell dimension in In vivo cultures. However, in tobacco ‘SIS Cultares, the addition of alcotinie acid showed no beneficial ‘tects an decroaved yelde at the higher concentrations tated (14). Tu hole plant, pyridoxine (vitamin By is ineoved inthe enzymatic reactont dealing with amino acid metabolism and is « coenzyme ia imino sid decarboxylation rections. In tssue culture, lack of py {Toxine has boen shown to decrease mexistomatic activity of roots 2.3, {2 Pyridoxine as been reported to have lit elect on veld o vigr of {bacco callus (14) in whale plants folic ecid functions ag an ntermediate cater of| ‘ontcatvon anit, Linsmaier and Skoog (4) found that fli ald in reas callus yields and improved color im tobacco cultures in the Hight but inhibited callus growth in the dak, *Krcobte acid functions in whole plans asa catalyst in photosyn that phosphorylation and may serve in electron transfer from NADPH tooxiten by undergoing cyclic oxidation reduction. Ascorbic ald (v- {amin €} bad no affect on eat callus yields; however, ts omission {sulted in an incteee in ell umber (27). Ascorbic ac added tothe ‘Sedlum war found to stop melanin formation, which has been shown {o'be detinenta fo callus cultures (4) Callus yields increased and tion of ascorbic aid to tobucco calle ia (4), ‘tn whole plants, myo-inositol involved inthe synthesis of el wall polysaccharides, and in the uptake and ullzation of fone. It also Prevent in the ipl fraction (nostol-phosphosphigolii of cll mam ‘Bianca (13) Deletion of myo-inositol hed no sigficant effect on calls Yields in canots and hpolpappss but resulted in decrased fresh Aight pens in tobacco cultures a all levels of thiamin (14). Myo {noni ka also been reported to promote bud formation in elo (Eimus By calures Presently, ony a Lanted numberof reports are available cee effets of nicotinic wid, ole acid, and ascorbic acid on plant tissue cules Materials Requce rr atrl lass or plastic pot plats (200 mm in diameter) 2. aso bosker 4 acilpls wih blades and 8 spaulas 4 Wuerooa nad tick on ible & Buns or enol bane 8: Gata tes (25 10m) and lant hs for olin thon 7. Stock callus cultures of 4 creeping bentgrass cultivars : Precdare Pepa 2 Iterof th following medium, Ms tsa ae See 200 elie = 20 gtr Pa $0 mgr $0 nefier Motos 1008 lies 2. Divide the moda into 12 qual aliquots of 250ml each To ach of the aliquots dd ono ofthe following Weutments: 00 mgiter 00 nailer thamin Sota acd pyridoxine iter Oni Sr mate Tomaliter Ti maiter eatin ce poridoxine 100 mater 100 mgiter sot acd PYtdsxine 3. Adjustthe pH of ech teatmont then dispense 10m of modi 20% 150 mim culture uber, exp abel each treatment, then {neat 121%, 1 Kgl? for in, There shouldbe 25 culture bes er westinnt “4. est results are obtained inthe studi ifthe callus tobe inoeu- late for the study is “pulsed” by placing the callus on the me «ium Hated below 2~8 weeks prior to the Initiation ofthe study MS basal ele Sweroee 8 300 gle Ape 80. giier 280 5.0 miter 10 merliter "herp fi hal tay in of the vitamin effects. ee = Eos ica 2 ig ay pce ae