Abstract
The culture procedure was examined to improve the shoot regeneration rate from apple leaf segments, using the culture growth period, composition of plant growth regulators in the medium, leaf area, and vitrification rate as indexes. The apple cultivar ‘Fuji’ was used as the plant material. Each ‘Fuji’ apple had a different culture growth period. The culture growth period is the number of days from the date the plants were placed in the culture system to the date they were used in the experiment. Three types of shoot regeneration media with different concentrations of plant growth regulators were used. The shoot regeneration rates were lower in all three media with a 6-month culture growth period. Therefore, the 6-month treatment was considered unsuitable for experiments of shoot regeneration from leaf segments. The addition of high concentrations of cytokinin was effective in improving the shoot regeneration rate with the 6-month treatment; however, no significant effect was observed in the 1-year or 7.5-month treatments. In the 6-month treatment, there was a significant correlation between leaf area in the first month after the beginning of incubation and the regeneration rate in the third month for all three types of media. Therefore, it was considered that an increase in leaf area was necessary to improve the regeneration rate. The tendency to require a larger leaf area to increase the regeneration rate on all media decreased with an increase in the culture growth period. The leaf area increased with increasing concentrations of thidiazuron and an increased cytokinin/auxin ratio. It was also found that a higher thidiazuron concentration or cytokinin/auxin ratio in the regeneration medium led to more vitrification. The shorter the culture growth period, the higher the rate of vitrification. The shoot regeneration rates exceeded 90% in some petri dishes with MBNZ511 medium after culture growth periods of one year or 7.5 months. In these cases, the average leaf area in the first month after the beginning of incubation was more than 30 mm2. Therefore, it is inferred that transformants using the Agrobacterium method could be efficiently obtained by selecting leaf segments larger than 30 mm2 in the first month after the beginning of incubation. The shoot regeneration potential from leaf segments is an inherent character of individuals.
