Tissue and Cell Culture

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.

Appendix

• I. Protocol for mechanical isolation of mesophyll cells from the leaves of Calystegia sepium (after Rossini 1972).

  • 1. Surface sterilize the leaves by quick immersion in 95 % ethanol followed by rinsing for 15 min in filter-sterilized 7 % solution of calcium hypochlorite. Wash in sterile distilled water.

  • 2. Cut the leaves into small pieces (less than 1 cm²).

  • 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.

  • 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.

  • 5. Plate the cells in a thin layer of solid or liquid medium (Table 4.1).

• II. Protocol for enzymatic isolation of mesophyll cells from tobacco leaves (after Takebe et al. 1968, as modified by Evans and Cocking 1975).

  • 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.

  • 2. Wash the leaves with sterile distilled water and peel-off the lower epidermis with the aid of sterile fine tipped forceps.

  • 3. Excise 4 cm² pieces from the peeled areas with a sterile scalpel blade.

  • 4. Transfer 2 g of the leaf pieces to 100 ml Erlenmeyer flasks containing 20 ml of filter-sterilized enzyme solution (0.5 % macerozyme, 0.8 % mannitol, and 1 % potassium dextran sulphate).

  • 5. Infiltrate the enzyme into the leaf tissue by briefly evacuating the flasks with a vacuum pump.

  • 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.

  • 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.

  • 8. Wash the cells twice with culture medium (Table 4.2) and culture.

    Table 4.2 Medium for suspension cultures of tobacco