| Peach is China’s important economic tree species. Traditional breeding of peach was constrained by long-period and heavy workload. Modern biological technology, especially the development of plant tissue culture and genetic engineering provided possibility for improving peach characters. High efficiency and stable regeneration system is the basis of transgenic technology, while the peach belongs to Prunus(Prunus SPP.) is difficult to regenerate in vitro. The regeneration of peach in vitro was affected by several factors, including genotype, plant growth regulator, basic culture medium, culture conditions, etc. In this study, a procedure for efficient regeneration of ‘Huangshuimi’, ‘Qiumihong’ and Maotao which could be laid the foundation for the research of peach genetic transformation were developed by using leaf of primary cultivation and stem segments, leaf, cotyledon, epicotyl from seedlings as explants, respectively. The main results as follows:1.Peach tissue culture and the leaf regeneration in vitro(1)Primary cultureFour factors affecting the primary cultivation were analyzed, including the time of gathering the materials, the state of the buds, the sterilization method and culture medium. The results showed that late April is suitable for gathering the bud. The stem segments with axillary bud(5~7 leaf) of ‘Huangshuimi’ which were soaked in the mercuric chloride 6 min were inoculated into MS + 6-BA 1.5 mg/L + NAA 0.1 mg/L + Ag NO3 0.5 mg/L. The lowest pollution rate and the highest germination rate were 11.1% and 58.7%, respectively. The stem segments that just gave birth to tender tip of ‘Qiumihong’ which were soaked in the mercuric chloride 7 min were inoculated into MS + 6-BA 1.5 mg/L + NAA 0.1 mg/L + Ag NO3 0.5 mg/L. The lowest pollution rate and the highest germination rate were 16.7% and 24.3%, respectively. The stem segments with axillary bud(5~7 leaf) of Maotao which were soaked in the mercuric chloride 7 min were inoculated into MS + 6-BA 1.5 mg/L + NAA 0.5 mg/L + Ag NO3 0.5 mg/L. The lowest pollution rate and the highest germination rate were 19.4% and 55.7%, respectively.(2)Regeneration of leaf in vitroThe effect of different plant growth regulators on the rate of callus formation f-rom leaf in ‘Huangshuimi’, ‘Qiumihong’ and Maotao were studied. The results indicated that the suitable medium for callus induction is MS + ZT 3.0 mg/L+ 2, 4-D 0.6 mg/L + Ag NO3 0.5 mg/L. The highest callus formation rate of ‘Huangshuimi’, ‘Qiumihong’ and Maotao could be up to 93.3%, 70.0% and 47.7%respectively. The lowest browning rate were 37.3%, 37.3% and 48.0%, respectivel-y. 2.Peach embryo culture and the research of regeneration of different explant in vitro(1)Regeneration of stem segments in vitroThe effects of different plant growth regulators on frequency of callus formation and frequency of adventitious shoot regeneration were conducted by using stem segments of ‘Huangshuimi’ seedlings as explants. Moreover, the influence of different types of plant growth regulator on the induction of adventitious root was analyzed by using two-step rooting method. The results indicated that the optimal medium for callus induction from stem segments was MS + 6-BA 2.0 mg/L + NAA 0.5 mg/L + Ag NO30.5 mg/L, while for adventitious shoot induction was MS + 6-BA 3.0 mg/L + NAA 0.1 mg/L + Ag NO3 0.5 mg/L. The frequency of callus formation and frequency of adventitious shoot regeneration were 85% and 47%, respectively. The adventitious shoots were cultured in WPM + ZT 2.0 mg/L + NAA 0.1 mg/L + Ag NO3 0.5 mg/L, then transferred to the MS + NAA 0.5 mg/L + GA3 0.5 mg/L + Ag NO3 0.5 mg/L. The rate of adventitious root regeneration and the average number of roots were 50% and 5.00, respectively. An efficient regeneration system from stem segments of ‘Huangshuimi’ seedlings which lays the foundation for the genetic transformation of peach was established in this study.(2)Regeneration of leaf in vitroThe effect of different plant growth regulators on the rate of callus formation and the state of callus from leaf in ‘Huangshuimi’, ‘Qiumihong’ and Maotao were studied. The results indicated that the rate of callus formation is 100% and the suitable medium for callus induction is MS + ZT 4.0 mg/L + 2, 4-D 0.8 mg/L + Ag NO3 0.5 mg/L.(3)Regeneration of cotyledon in vitroTake cotyledon of ‘Huangshuimi’, ‘Qiumihong’ and Maotao from embryo culture seedlings that training about 45 d, crosscutting 2~3 knives, inoculated to MS + NAA 0.1 mg/L + TDZ 2.0 mg/L + Ag NO3 0.5 mg/L with abaxial side contact medium. After 45 d found that only part cotyledon incision of ‘Qiumihong’ produce adventitious bud.(4)Regeneration of epicotyl in vitroTake embryo culture seedlings of ‘Huangshuimi’, ‘Qiumihong’ and Maotao that training about 45 d, each embryo culture seedlings for 1~2 epicotyl, cut into 1~2 cm long, inoculate to MS + NAA 0.1 mg/L + 6-BA 2.0 mg/L + Ag NO3 0.5 mg/L, culture around 30 d found that part of the buds grow into adventitious bud of ‘Qiumihong’. |
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