Abstract
The pioneering attempts of G. Haberlandt (1902) to culture plant cells did not succeed but with the refinement of the in vitro techniques and tissue culture media and the discovery of plant growth regulators it has been possible to establish various types of tissue cultures, starting from embryonic, meristematic and mature tissues. Broadly, there are two types of tissue cultures: (i) unorganized (callus and suspension cultures), and (ii) organized (root cultures, shoot tip cultures, embryo cultures etc.). Organized tissue cultures are dealt with in some of the following chapters. This chapter describes unorganized tissue cultures.
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Suggested Further Reading
Dong J, Bowra S, Vincze E (2010) The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application. BMC Plant Biol 10:239–250
Eibl R, Eibl D (2008) Design of bioreactors suitable for plant cell and tissue cultures. Phytochem Rev 7:593–598
Evans PK, Cocking EC (1975) The techniques of plant cell culture and somatic cell hybridization. In: Pain RH, Smith BJ (eds) New techniques in biophysics and cell biology, vol 2. John Wiley, London
Kohlenbach HW (1966) The potentialities for development of explanted and isolated permanent cells. I. The extension and division growth of isolated mesophyll cells of Macleaya cordata. Z Pflanzenphysiol 55:142–157
Matsubayashi Y, Sakagami Y (1996) Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 93:7623–7627
Reynolds JF, Murashige T (1979) Plant cell lines. In: Jakoby WB, Pastan IH (eds) Methods in enzymology, vol 58. Academic Press, New York
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Vasil V, Hildebrandt AC (1965) Growth and tissue formation from single, isolated tobacco cells in microculture. Science 147:1454–1455
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Appendix
Appendix
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I.
Protocol for mechanical isolation of mesophyll cells from the leaves of Calystegia sepium (after Rossini 1972).
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1.
Surface sterilize the leaves by quick immersion in 95Â % ethanol followed by rinsing for 15Â min in filter-sterilized 7Â % solution of calcium hypochlorite. Wash in sterile distilled water.
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2.
Cut the leaves into small pieces (less than 1Â cm2).
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3.
Homogenize 1.5 g of leaf material with 10 ml of culture medium in a glass homogenizer tube. Filter the homogenate through two sterile metal filters, the upper and lower filters with mesh diameters of 61 and 38 μm, respectively.
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4.
Fine debris can be removed by slow speed centrifugation of the filtrate which would sediment the free cells. Remove the supernatant and resuspend the cells in a volume of the medium sufficient to achieve the required cell density.
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5.
Plate the cells in a thin layer of solid or liquid medium (Table 4.1).
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1.
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II.
Protocol for enzymatic isolation of mesophyll cells from tobacco leaves (after Takebe et al. 1968, as modified by Evans and Cocking 1975).
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1.
Collect fully expanded leaves from 60–80 day-old plants and surface sterilize them by immersion in 70 % ethanol for 30 s followed by rinsing for 30 min in 3 % sodium hypochlorite solution containing 0.05 % Teepol or Cetavlon.
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2.
Wash the leaves with sterile distilled water and peel-off the lower epidermis with the aid of sterile fine tipped forceps.
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3.
Excise 4Â cm2 pieces from the peeled areas with a sterile scalpel blade.
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4.
Transfer 2Â g of the leaf pieces to 100Â ml Erlenmeyer flasks containing 20Â ml of filter-sterilized enzyme solution (0.5Â % macerozyme, 0.8Â % mannitol, and 1Â % potassium dextran sulphate).
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5.
Infiltrate the enzyme into the leaf tissue by briefly evacuating the flasks with a vacuum pump.
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6.
Incubate the flasks at 25 °C for 2 h on a reciprocating shaker with a stroke of 4–5 cm at the rate of 120 rpm min−1.
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7.
Change enzyme solution after the first 30 min. After another 30 min the enzyme solution should contain largely spongy parenchyma cells and after 90–120 min of incubation the suspension should contain predominantly palisade cells.
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8.
Wash the cells twice with culture medium (Table 4.2) and culture.
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1.
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Bhojwani, S.S., Dantu, P.K. (2013). Tissue and Cell Culture . In: Plant Tissue Culture: An Introductory Text. Springer, India. https://doi.org/10.1007/978-81-322-1026-9_4
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DOI: https://doi.org/10.1007/978-81-322-1026-9_4
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