Embryogenic callus was developed from young leaves of sugarcane (
Saccharum spp.
hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l
-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l
-1) 2,4-
d and 500 mg l
-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l
-1 sucrose, 500 mg l
-1 CH and 2.26 mol (0.5 mg l
-1) 2,4-
d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l
-1) and sucrose (60 g l
-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10
7 to 1.0×10
8 ml
-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10
5 m l
-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-
d (2 mg l
-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l
-1) +5.37 mol NAA (1.0 mg l
-1) + activated charcoal (200 mg l
-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS
salts of Murashige & Skoog (1962) basal medium
- AA
salts of Muller & Grafe (1978) basal medium
- N6
saits of Chu
et al. (1975) basal medium
- 2,4-
d
2,4-dichlorophenoxyacetic acid
- CH
casein hydrolysate
- KM8P
protoplast culture medium of Kao & Michayluk (1975)
- KPR
protoplast culture medium of Kao (1977)
- P9
protoplast culture medium (Chen & Shih, 1983)
- BA
Benzyladenine
- Picloram
4-amino-3,5,6-trichloropicolinic acid
- NAA
Naphthalene acetic acid
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