Journal of the Minnesota Academy of Science Volume 45 Number Article 1979 In Vitro Experiments on Totipotency of Dacus Carota Karl L Ruser Gustavus Adolphus College Follow this and additional works at: https://digitalcommons.morris.umn.edu/jmas Part of the Plant Biology Commons Recommended Citation Ruser, K L (1979) In Vitro Experiments on Totipotency of Dacus Carota Journal of the Minnesota Academy of Science, Vol 45 No.2, 6-7 Retrieved from https://digitalcommons.morris.umn.edu/jmas/vol45/iss2/3 This Article is brought to you for free and open access by the Journals at University of Minnesota Morris Digital Well It has been accepted for inclusion in Journal of the Minnesota Academy of Science by an authorized editor of University of Minnesota Morris Digital Well For more information, please contact skulann@morris.umn.edu In Vitro Experiments on Totipotency of Dacus Carota KARL L RUSER* ABSTRACT - The intent of this project was to induce the totipotency of carrot cells in tissue culture The information gathered would be applied to work with woody plants Differentiated cells removed from the root were cultured in vitro using a modified White's medium Callus was initiated and successfully subcultured Roots and the production of chlorophyll have been initiated in the cultures Because some specialized equipment and facilities for tissue culture were not available at the beginning of the project, it took considerable time to gather and improvise them A roller tube culture mixer was constructed, using the mechanism from a strip-chart recorder Complete regeneration of the whole plant has not been accomplished, but the tissue and organ formation described indicates that totipotency has been stimulated It shows that successful plant tissue culture studies may be performed in the absence of highly specialized facilities The intent of this project was to induce the totipotency of carrot cells in tissue culture If this could be accomplished at Gustavus, the information gained would be used as groundwork for totipotency studies involving woody plants If the methods used were successfully applicable to woody plants, they might prove to be of value in plant propagation It would also show that experimental tissue culture procedures normally requiring specialized and expensive facilities can be carried on at a college not equipped with such facilities Culture Methods Adapted from Steward The culture method was used by Steward (1958) with carrots The culture medium was White's (1963) modified medium with 200ppm casein hydrolysate and 10 percent coconut milk added No agar was added at first The liquid medium and the cultured cells in it were kept gently agitated to keep the nutrients well mexed and the liquid well aerated The mo tion of the tissues was used for another purpose as well The callus tissue formed from the explants were relatively friable This allowed for the possibility that single cells or small cell groups would become detached from the central mass and proliferate in suspension Steward (1958) found that the regenerative abilities of the cells in suspension were much more pronounced than of those in the callus mass Machinery for keeping the cultures mixed could not be obtained commercially due to budget limitations Two types were developed: a flask mixer and a culture tube mixer The flask mixer rotates the cultures to gently swirl the medium and tissues The flasks are fixed on a tilted rotating platform As the flasks rotate, the tilted surfaces of the media move around with respect to the flasks The motion sets up a gentle swirling action The culture tube mixer consists of an 11-inch removable drum in which culture tubes may be set This drum fits on two parallel rollers, one of which is driven The body of the machine is from a discarded strip-chart recorder The drive roller for the paper chart now functions as the power roller and another roller was made to fit alongside to stabilize the drum Aseptic cultures were initiated from a grocery store variety of carrot, Daucus carota and grown in 50ml of the liquid medium described above 125ml erhlenmeyer flasks were used as the culture vessels The medium was agitated using the tilted rotary flask mixer The cultures were grown in the dark at 25 degrees C They were periodically subcuHured to fresh medium This was done at 24, 68 and 97 days from the start of the cultures There is no significance attached to the exact dates of the transfers; they merely approximate monthly cubculture periods The first and second transfers were to liquid medium The third was to medium supplanted with agar The latter transfer was made after redifferentiation was manifest in the cultures For continued development the cells need an established polarity Roots will form with little or no geotropic polarity present but shoots are much more dependent on this (Steward, 1958) Light was provided for the cu'ltures on agar with a 12 hour photoperiod Two attempts were made to establish woody tissue cultures The first culture was from Populus grandidentata stem tissue and the second was from Populus deltiodes root tissue Root tissue was use d in the latter case to make the parallel to the carrot root control as close as possible Several variations for surface dissinfection and preparation were tried using abrasion, flaming and alcohol Extent of Redifferentiation Noted The carrot cultures initiated callus growth by the ninth day On the 84th day, between the second and third transfers, roots were present on the central callus masses 10 micron sections of the rooted calluses showed that they contained several nodu?es of tracheid regeneration with associated cambium-like organization No free cell suspension was produced although one culture apparently developed two small calluses in the medium in addition to the central one These did not develop roots After 11 days on agar the cultures began to change from the carrot orange they had been to light green The P grandidentata and P deltoid es were both plagued with contamination within two days *KARL L RUSER was a senior biology major at Gustavus Adolphus College, St Peter, Minnesota, at the time of this project He plans on graduate studies ,in horticulture at the Univet"Sity of Minnesota The Minnesota Academy of Science ACKNOWLEDGEMENTS Implications From the Project Cutter ( 1978) reports that external influences such as auxin and high (8%) sugar concentrations are necessary for vascular nodule initiation It has been shown by Steward (1964) that the medium containing coconut milk and casein hydrolysate promotes mainly disorganized growth of cells Since tissue reorganization did occur after 84 days there may also be an internal component related to time in redifferentiation This may be from increased isolation from the medium as considered by Steward ( 1958), or that in time a certain maturation state is reached in the undifferentiated callus cells , or a combination of both It seems most likely that the contamination of the Populus cultures was not due to technique There has been very little secondary contamination of the cultures once they have been established When there was widespread contamination with the carrot it occurred in every flask containing explants from the same parent material Since the Populus contamination was widespread in the same way it is reasonable to suspect the material from which the explants were taken This is the most likely with the P deltoides root cultures due to the possibility of mycorhizal organisms being present No conclusions can be made as to how well the methods used wou 'ld work in propagating woody materials It is evident, though, that this type of work is possible under very low budget conditions With the facilities and equipment set up under this study, further work can now be concentrated on establishing viable woody tissue cultures Journal of, Volume Forty-five, No 2, 1979 The author would like to thank the Gustavus Ad,Jlphus College chapter of Sigma Xi for its financial support, made possible through a W.W Holcomb Research Grant Additionally , the assistance of Dr Charles Mason and Dr Richard Fuller has been greatly appreciated REFERENCES BYCHENCOV A , E A 1963 An investigation of callus formation in certain trees and shrubs by the method of tissue culture in vitro Doklady Bio Sect 151 CUTTER, E.G 1978 Plant Anatomy, Part I: Cells and Tissues Reading, Mass , Addison-Welsey, Co JACQUI OT, C I 966 Plant tissues and excised organ cultures and their significance in forest research J fnst Wood Sci 16 SKOOG, F , and MILLER, C.O 1957 Chemical regulation of growth and organ formation in plant tissues cultured in vitro Soc Exp Bio Symp 11 STEWARD, F.C., and BLAKELY, L.M 1964 Growth and organized development of cultured cells, V The growth of colonies from free cells on nutrient agar Amer J Bot I , MAPES, M.O., and SMffH, J 1958 Growth and organized development of cultured cells, I Growth and division of freely suspended cells Amer J Bot 45 WHITE, P.R 1963 The cultivation of animal and plant cells New York, Ronald Press .. .In Vitro Experiments on Totipotency of Dacus Carota KARL L RUSER* ABSTRACT - The intent of this project was to induce the totipotency of carrot cells in tissue culture The information gathered... very little secondary contamination of the cultures once they have been established When there was widespread contamination with the carrot it occurred in every flask containing explants from the... nodule initiation It has been shown by Steward (1964) that the medium containing coconut milk and casein hydrolysate promotes mainly disorganized growth of cells Since tissue reorganization did