Parsnip plants (Pastinaca sativa) and carrot plants (Daucus carota) can regenerate from

cultured callus cells in mixed co-cultures.Natalie Marchi, Michael Caron, Peter Bradley

Biology Department, Worcester State University, Worcester, MA

Plant tissue culture techniques are used to culture plant cells in the lab. The presence or absence of plant hormones in the medium can induce regeneration of embryos, shoots and roots from callus cells. In this study, carrot cells were cultured on B5 agar medium containing the auxin “2,4-D”. Transfer to medium lacking 2,4-D induced the formation of carrot shoots and roots. Various different hormone combinations were tried to regenerate the parsnip plants. The formation of shoots was more difficult than with carrot cultures. Parsnip plants were finally regenerated from a mixed culture where plants were also regenerating from carrot cells at the same time. Shoots from parsnip and carrot were recognized by the appearance of the leaves. Did the presence of the carrot tissues stimulate the regeneration of the parsnip? This preliminary study suggests a co-culture method to regenerate shoots from parsnip callus that does not rely on treatments with the usual plant hormones.

Introduction. The techniques needed to produce tissue culture callus masses of cells from carrot roots are well known. It is also easy to regenerate complete plants from carrot callus cells. We first asked if the techniques used with carrot could also be used with another plant in the same family, parsnip. Subsequently, we wanted to know if we could produce mixed cell cultures with the two plants and even regenerate chimera plants from our cells. Chimera plants can occur spontaneously in nature. A chimera is a single individual composed of two different cells in different parts of the plant. Previous research has shown that chimeras can also be produced experimentally by co-culturing calli or graft tissues and then regenerating the plant shoots (i.e. in tobacco species by Carlson & Chaleff 1975, Carlson 1977, Marcotrigiano & Gouin 1984; in Brassica species by Noguchi et al. 1992, Noguchi & Hirata 1993; and in red seaweeds by Cheney & Wang 1994, 1996). The goals of this study were to verify the techniques needed to culture carrot cells and regenerate whole plants from them, to apply the same techniques to parsnip, and then work with mixed cultures containing cells from both plants. When plants were regenerated from mixed co-cultures then we would need to verify the nature of the plants obtained. They could be carrots, parsnips or a mixture (i.e. chimera) of the two. It was possible to regenerate shoots from the co-cultures and attempts were made to verify the cellular make-up of these with isozyme analysis. The details of the experimental work completed so far are in Marchi (2015). Materials and Methods. Plants were grown from seed in the Worcester State University greenhouse in Miracle-Gro Potting mix (i.e. carrot Daucus carota L. cv. Danvers 126 Half Long, and parsnip Pastinaca sativa L. cv. Hollow Crown) and were obtained from Baker Creek (Mansfield MO) (Figures 1-2). Leaf shape of these two plants is different (Figures 2-3). Some work was done with store-bought roots. Tissue cultures for callus growth were initiated by placing root cortical tissue on B5 agar (Gamborg et al. 1968) in Petri dishes or beakers (Figure 4). The medium, containing the usual nutrients, was supplemented with 2 % w/v sucrose and 1 mg/L 2,4-dichlorophenoxyacetic acid (i.e. the auxin “2,4-D”), the pH was adjusted to 5.6 and the medium was autoclaved. Aseptic techniques were used throughout and all transfers were made in a laminar flow hood. The cultures were sealed with Parafilm and kept in an incubator at room temperature under constant light for several weeks. Various other plant hormones were also used in this study including the auxin indole-3-acetic acid, the cytokinins 6-benzyaminopurine, zeatin, kinetin and gibberellic acid (GA3). To regenerate shoots and roots from the calli they were transferred to B5 agar medium lacking 2,4-D but containing all of the usual nutrients (Steward et al. 1964). All the chemicals used in this study were supplied by the Sigma-Aldrich Chemical Company. Attempts were made to verify the nature of regenerated shoots using isozyme analysis. These techniques extract enzymes from the shoots, separate them on polyacrylamide-starch electrophoresis gels and subject them to various staining methods. Isozyme analysis has been shown previously to tell different plants apart (Lee & Fairbrothers 1973; Oelschlegel & Stahmann 1973; Jacobs 1975). In this study the following enzymes were visualized on the gels: PGI, PGM, PGD and EST. The details of these studies are not shown here but are available in Marchi (2015). Results. Carrot callus was grown on B5 agar containing 2,4-D (Figure 5). Parsnip callus was also successfully grown on the same medium (Figure 6). Transferring the carrot callus to medium lacking 2,4-D resulted in the growth of shoots and roots. It proved easy to regenerate shoots and roots of carrot but the parsnip cultures did not respond in the same way. Various hormone combinations were used and some root development or shoot development was obtained from the parsnip calli, but not as good as with the carrot cultures. Then the mixed callus cultures were initiated and cultured and when transferred later to medium lacking 2,4-D the regeneration of the plants occurred (Figure 7). In the mass of tangled shoots obtained from the mixed cultures the characteristic appearance of carrot leaves was seen (Figures 8-9). In addition, a careful examination of these regenerated plants shows some variation in leaf morphology (Figure 9). As well as the leaf clusters characteristic of carrot there are ovate-shaped leaves present. Attempts were made to analyze this material with an isozyme starch gel electrophoresis approach, but those results were inconclusive. If the shapes of the leaves seen in Figure 9 are compared with Figures 2-3 an argument could be made that there are both carrot leaf types and parsnip leaf types present in the shoots from the co-cultures. Conclusions. This study has shown that it is possible to culture callus of parsnip using the same techniques that are used with carrot cultures. However, regeneration of parsnip plants from callus was more difficult than carrot. When callus of the two plants was mixed together and the callus was moved to regeneration medium, many shoots were obtained and the leaf shapes suggested that more than one plant was present. At this time it is not possible to say if the plants are carrot, parsnip, a mixture of the two, or if a chimera was generated in this experiment. A future study might use DNA analysis to answer this question. Does this experimental result suggest that shoots of a “difficult” plant might regenerate from cells by co-culturing them with an “easy” plant? It is interesting to speculate that there could be some unknown hormone activity in the “easy” plant that acts to have an effect on the other. The result in this preliminary study suggests a series of future experiments to investigate this hypothesis.

Acknowledgments. We are grateful to Michael Mayko for some of the chemicals used in this study. Our thanks to Terren Flanders for looking after the Worcester State University greenhouse. The authors. Natalie Marchi graduated with the M.S. (Biotechnology) and Michael Caron graduated with the degree of B.S. (Biology) in December 2015. Peter Bradley was the faculty advisor for this project and can be contacted at pbradley@worcester.edu or pybradley@verizon.net This poster was presented on the campus of Worcester State University on April 13, 2016. Carlson PS & Chaleff S. 1975. Heterogeneous associations of cells formed in vitro. In: Genetic manipulations with plant material (L Ledoux, ed.). 3: 245-260. Plenum Press.Carlson PS. 1977. Novel cellular association formed in vitro. In: Molecular genetic modification of eukaryotes (I Rubenstein, RL Phillips, CE Green, R Desmick, eds.). 43-56. Academic Press.Cheney DP & Wang LZ. 1994, 1996. Method of causing somatic hybridization between two species of algae. US Patents: 5365018 & 5585544.Gamborg OL, Miller RA, Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Research. 50:151-158.Jacobs M. 1975. Isozymes and a strategy for their utilization in plant genetics. Genetic Manipulations with Plant Material. 3:365-378.Lee DW & Fairbrothers DE. 1973. Enzyme differences between adjacent hybrid and parent populations of Typha. Bull. of the Torrey Botanical Club. 100(1):3-11.Marchi N. 2015. A preliminary study of the co-culture of carrot (Daucus carota) and parsnip (Pastinaca sativa) using regenerated shoots from callus cultures. M.S. Thesis. Worcester State University, Massachusetts. 75 pp.Marcotrigiano M & Gouin FR. 1984. Experimental synthesized plant chimeras I. In vitro recovery of Nicotiana tabacum L. chimeras from mixed callus cultures. Annals of Botany. 54:503-311.Noguchi T, Hirata Y, Yagishita N. 1992. Intervarietal and interspecific chimera formation by in-vitro graft -culture method in Brassica. Theor. & Appl. Genetics. 83:727-732.Noguchi T & Hirata Y. 1993. Vegetative and floral characteristics of interspecific Brassica chimeras produced by in vitro grafting. Euphytica. 73:273-280.Oelschlegel FJ & Stahmann MA. 1973. The electrophoretic technique – a practical guide for its application. Bull. of the Torrey Botanical Club. 100(5):260-271.Steward FC, Mapes MO, Kent AE, Holsten RD. 1964. Growth and development of cultured plant cells. Science. 143:20-27.

Figure 1. Working with plants in the Worcester State University greenhouse.

Figure 2. Parsnip plants (left) and carrot plants (right) in the greenhouse. Note the appearance of the leaves. Leaves of carrot (Daucus carota L. cv. Danvers 126 Half Long) have a rosette of 8-12 small leaves in a pinnately compound arrangement where leaves are on both sides of the stalk. Leaves of parsnip (Pastinaca sativa L. cv. Hollow Crown) are also arranged in a pinnate manner and are broad, lobed and ovate in shape.

Figure 3. Leaf arrangement of a store-bought parsnip plant. These shoots are growing towards a light source from a root that was lying on a horizontal surface. Figure 4. Tissue cultures of parsnip and

carrot were growing on agar medium in Petri dishes and beakers.

Figure 5. Carrot callus masses were grown from small pieces of root in a lighted incubator. Standard agar medium was used to induce callus formation. Callus tissues are composed of undifferentiated cells.

Figure 6. Parsnip callus was obtained using the same techniques as used for carrot callus growth. The agar medium contained nutrients as well as a plant hormone known as “2,4-D”.

Figure 7. Callus from parsnip and carrot were mixed in co-cultures and later transferred to medium lacking the hormone 2,4-D or any other plant hormone. After several weeks, shoots grew out of the callus masses. This is the regeneration of adult plants from cells.

Figure 8. A tangle of shoots regenerated from a mixed co-culture of callus cells of parsnip and carrot.

Figure 9. A close look at the shape of the leaves of the regenerated plants show some like carrot shoots and some like parsnips. Using mixed co-cultures might be the best way to obtain shoots from parsnip cells. We were less successful in regenerating parsnip shoots using other methods that were based on treatments with plant hormones. The actual genetic nature of the shoots has not been determined at this time.