| In this paper, Different stages of Peony embryo were used to directly induce somatic embryos. peony leaves, stems, anthers, petals were used to induce the callus,then through callus proliferation and differentiation to achieve small plants. Based on the induction of small plant, The roots were induced in this experiment. At last acclimation and transplanting were succeed. At the same time using paraffin sections and scanning electron microscopy methods observed the callus structural changes during development.The main results were as follows:Somatic embryo induction of peony. The seed embryo of peony after flower 110d of 'Feng Dan'were used to induce the somatic embryo,the induction rate was higher than other stages. Embryo, cotyledon, hypocotyl explants were all able to induce somatic embryos,but the highest induction rate were appeared while using embryo as explants. Sucrose pretreatment to embryo were helpful to somatic embryos induction.The experiment showed that using 90g/L sucrose dealing with embryo 2h were best. The best medium of somatic embryos induction were MS+2,4-D 0.2mg/L+6-BA 0.2mg/L.With this medium, the rate of embryo induction was highest of all. Peony proliferation of somatic embryos and plant regeneration. The results showed that the most suitable culture medium of peony'Feng Dan'the proliferation were MS+6-BA0.1 mg/L+sucrose 60.0 g/L+CH0.4 g/L. Activated charcoal played significant role in somatic embryo maturation and different culture media on somatic embryo maturation effect was not significant.6-BA can promote the germination of somatic embryos, but the excess of it will make somatic embryos more callus and thus can not be achieved plant regeneration. GA3 were good for break dormancy and promote germination. IAA were good at promoting rooting of somatic embryos.Callus induction of peony. The best medium for callus induction of peony leaves was Modified MS medium.Without adding any plant growth regulator medium can not induce callus. Adding auxin or cytokinin alone can induce callus organization, but there were differences in induction rates. The best auxin was 2,4-D 0.2 mg/L and the best cytokinin was TDZ 2.0 mg/L. Using 2,4-D, NAA, TDZ for orthogonal experiment, the optimal medium were modified MS+2,4-D 0.2 mg/L+NAA 0.2 mg/L+TDZ 3.0 mg/L and the induction rate of callus was 87.8%.The tender stems of peony were used as explants for callus induction,The best medium were MS+NAA0.05 mg/L+6-BA2.0 mg/L+2,4-D1.0 mg/L.In this medium, the highest induction rate of callus was 87.8%. The order of sucrose concentrations to peony stem callus induction were 30.0 g/L> 40.0 g/L> 20.0g/L> 10.0 g/L, so the best sucrose concentration was 30.0 g/L. Using the lower and middle stem were better than other oposition.With this stems, callus induction rate will be higher and callus will be more.The results showed that the best period was the middle develop stage. The best growth regulators were MS+2,4-D2.0 mg/L+6-BA1.5 mg/L+NAA1.0 mg/L. The best sucrose concentration was 6%, After 8 days cold pre-treatment in 4℃, the callus grew well, the induction efficiency reached to 50.8%.The petals were used as explants in this experiment, The best medium wereMS+2,4-D 2.0 mg/L+6-BA 1.5 mg/L+NAA 0.3 mg/L,The induction rate reached to 98.9%.In this experiment,The roots of peony were used as explants,but there were no callus appeared, the reason of it probably due to the root fungi are more difficult to sterilization, It was likely to cause pollution. After inoculated root to the medium for a period of time, The root got brown black and there were no callus appeared.The most appropriate medium for callus proliferation of leaves were MS+NAA0.2 mg/L +6-BA2.0 mg/L+KT2.0 mg/L.30.0 g/L of sucrose as a suitable carbon source for callus proliferation. CH with 0.4 g/L was good for callus proliferation. The results showed that the growth curve of leaf callus was the S-curve, The grow process can be divided into slow growth phase, exponential phase and stationary phase, The growth period of leaf callus was about 30d.The callus proliferation medium of Stems and petals were same, They were MS+ NAA0.2 mg/L+6-BA3.0 mg/L+sucrose 30.0 g/L+CH0.4 g/L+Agar 7.0 g/L. The medium of anther proliferation were MS+NAA0.2 mg/L+6-BA2.0 mg/L+sucrose 30.0 g/L+CH0.4 g/L+Agar 7.0 g/L.12h light was good for stem proliferation.The effects of light intensity, anti-oxidants and adsorbents to callus browning were studied in this experiment. The results showed that after dark conditions for 1 week and then cultured in light intensity 19umol/m2/s may be good choice, In this condition,not only ensure the callus proliferation but also greatly reduce the callus browning. Several antioxidants had good effect to callus proliferation and reduced the rate of callus browning. AgNO3 was good for inhibiting the browning. 1.0g/LPVP and 2.0g/L activated carbon were good for inhibiting browning. Callus structure observation of peony. The result showed that bud primordia are not from the surface of callus but occurred near the surface, and then began to break through the surface cell to grow. Different developmental stages were found in different shapes.Using scanning electron microscopy, different callus culture occurs at different times different changes can be found, some callus can develop into embryoids, while others are unable to complete normal development. embryogenic callus and non-embryogenic callus may exist on one callus.15-20d after subculture ofth was the fastest time, after this period callus began to grow slow,so after the 15-20d of subculture is more good subculture period.Callus differentiation of peony. The most suitable medium for callus differentiation of leaves were MS+6-BA2.0 mg/L+NAA0.2 mg/L+TDZ0.3 mg/L+sucrose 30.0 g/L+Agar 7.0 g/L. CH had little effect on the callus differentiation., The highest callus differentiation rate achieved when the sucrose concentration of 30g/L. Activated carbon were negative to callus differentiation, but good for the growth of adventitious buds.The most appropriate callus differentiation medium of stem were MS+6-BA3.0 mg/L+ NAA0.2 mg/L. That of anther were MS+6-BA2.0 mg/L+NAA0.2 mg/L+KT0.3 mg/L, After dried 3d with filter paper,the anther callus formation quickly. The suitable medium of petal callus differentiation were MS+ZT 0.5 mg/L+6-BA2.0 mg/L.Root induction and transplanting of peony. Four mediums were used in rooting induction,The results showed that 1/2MSand WPM were appropriate medium.The suitable sucrose concentration was 30.0g/L. The best medium were 1/2MS+NAA0.1mg/L+ IBA0.2mg/L+sucrose 30.0 g/L. Small plants can be transplanted after the domestication of survival, the survival rate was 40.0% or so. |
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