Protoplast isolation, culture and plant regeneration on wheat (Triticum aestium L.)

Successful application of biotechnology in new wheat cultivars improvement is depend on most suitable cultured callus, cell suspension, protoplast and plant regeneration capacity. In this investigation we have assessed the capacity of immature and mature embryos of five wheat cultivars (Seri 82, Panda, Shafir, Gemini, Çukurova 86) to form embryogenic callus, cell suspension, protoplast isolation and plant regeneration on a range of media. Initially, immature embryos (15 days after pollination) were cultured on MS (MURASHIGE and SKOOG, 1962); LS (LINSMAIER and SKOOG, 1965), B5 (GAMBORG B5, 1975), N6 (MOROCZ et al., 1990) with different concentration 2,4-D (lmgA, 2mgA, 3mg/l, 5mg/l) and mature embryos were cultured on MS, LS, B5, N6 with different concentration of 2,4-D (lmg/1, 3mg/I, 5mg/l, 8mg/l). Immature and mature embryos formed callus on all media but immature embryos of various cultivars showed significant differences in callus formation in response to all media tested. However, callus formation from immature embryos with 2mg/l 2,4-D and mature embryos on MS and B5 media with 5mg/l 2,4-D concentrations were higher than on other media and concentrations. Callus was initiated from immature and mature embryos on MS medium with potato extract, wheat starch, casein hidrolisate and asparagine. Most cultivars formed calli on media augmented with organic substrates. Consequently, MS medium containing 2mg/l 2,4-D, 100mg/l casein hydrolisate, 500mgfl glutamine was used as the maintenance medium for callus subcultures. The embryogenic cell suspension cultures were established from immature embryos derived embryogenic callus of Seri 82 wheat cultivars which well dispersed, off-white, rapid growing. Embryogenic cell suspension of Seri 82122 were cultured in liquid MS media with casein hidrolisate, glutamine and asparagine. Increases in PCV% were highest in MS media supplemented with 100mg/l casein hidrolisate, 500mg/l glutamine, 150mg/l asparagine. Fast growing cell suspension of Seri 82 cultivar were used for the protoplast isolation. 2xl07 purified protoplast per one gram initial fresh weight could be obtained in average at the and of a 6 h incubation period, using the enzyme solution of VASIL et. al., (1990). A comparison of the division and plating efficiency on different days following subculture, and different density, and in in different liquid or agarose media. Indeed, protoplasts from isolated on the third of fifth days of subculture, and lxlO6 protoplast/ml density, and 1.2% agarose concentrations in MS media supplemented with 0.5mg/l 2,4-D highest division and plating efficiency. For further somatic embiyo and plant regeneration containing 4-5 week old microcalli transferred on MS regeneration media supplemented with various auxins (0.1-0.5mg/l 2,4-D; O.lmg/1 IAA; 0.1mg/l NAA) and cytokinins (lmg/1 BAP; lmg/1 kinetin). Plant regeneration was obtained from microcallus after 16- 20 weeks at the rate of 38.7 percent on MS media supplemented with O.lmg/1 IAA, lmg/1 BAP, lmg/1 kinetin. In cytological studies with cell suspension cultures and regenerated plants from protoplasts. To observe the chromosomes lost at the rate of 52 percent, extra chromosomes at the rate of 2 percent in cell suspension cultures, normal chromosomes number (2n=42) at the rate of 46 percent. Regenerated plants from protoplasts observed normal chromosomes number (2n=42), but some plants coincided abnormal seems such as large leaf lamina, extreme tillering, and albino plants.123 Seedling leaves were used for mesophyll protoplast isolation. To obtain seedlings, seeds of Sen 82 wheat cultivar were surface sterilized and cultured in vitro. The leaves were cut with razor blades at 1-2 mm from the base of the leaf blade from 7-8 days old seedlings. The leaf segments were transferred to enzyme solution using of WATANABE et.al.,(1988). The leaf pieces were immersed for 3 min under partial vacuum. They were incubated at 25 °C and a protoplast were isolated per one gram leaf tissue. Mesophyll protoplasts were cultured at the different culture medium, but it wasn't coincide with the cell division in four weeks.