| Canna spp.is a perennial bulbous flower of the genus Canna in the Cannaceae family,with ornamental,ecological,and medicinal applications.However,Traditional methods of reproduction have drawbacks,for example,long-term asexual reproduction of field can easily lead to the accumulation of viruses in new plants,leading to variety degradation.As a result,the conventional seeding rate of seedable varieties is extremely low,and the breeding of new varieties is extremely slow.The development of transgenic engineering for Canna species requires an efficient regeneration system.Canna×generalis‘Mohong’,C.×generalis‘Grand Due’,C.edulis‘Xingyu-1’,C.×orchioides‘Lüyecheng’,‘Yinli’and‘Yellow Canna’were applied as materials to study different factors influencing the in vitro propagation and regeneration of Canna,by using the tissue culture technique and the uniform design method.The study was carried out to explore effective ways to improve the rapid propagation and efficient regeneration of Canna in vitro.Moreover,the study aimed to provide technical support for the conservation of Canna germplasm resources,large-scale production,selection,breeding excellent varieties and new species,and genetic transformation research.The main results are as follows:（1）Effects of different media on seed germination and efficient propagation of Canna.The results showed that the germination and survival rate of C.×generalis‘Mohong’seeds were above 80%.The germination rate under liquid state was above90%.The progeny of‘Mohong’and‘Grand Due’had a proliferation rate of 63.77%and 75.33%respectively,with proliferation coefficients above 3.Among the factors affecting the proliferation of adventitious buds in‘Mohong’,the overall effect of adventitious bud proliferation on MS medium was better than that on N6and B5.The best medium for adventitious bud proliferation is MS+6-BA 6 mg·L-1+NAA 1.5mg·L-1,and the adventitious bud proliferation rate was 88.89%,with a proliferation coefficient of 5.3.（2）Effects of different factors on rapid propagation of Canna with buds in vitro.The results showed that,the sterilization of C.×generalis‘Mohong’with 0.1%Hg Cl2+Tween 20 treatments was the best.The liquid state and the greenhouse substrate potted seedlings were beneficial for sterile line culture of rhizomes with buds.Moreover,among the factors affecting the proliferation of adventitious buds in‘Mohong’,the proliferation effect of adventitious buds in the liquid state+glass beads state was better than that in the semisolid medium.In addition,the suitable medium for the proliferation of seedlings in vitro was MS+6-BA 6 mg·L-1+TDZ0.2 mg·L-1+NAA 1 mg·L-1.The proliferation rate of adventitious buds was 85.71%,with a proliferation coefficient of 2.83.（3）The rooting medium suitable for sterile seedlings was1/2MS+NAA 0.5mg·L-1,the rooting rate was 100%.and the overall rooting condition was better than the rooting medium with 0 and 1 mg·L-1NAA.The rooting rate in the liquid+glass bead condition was 100%,better than in the semisolid medium.After 7 d domestication of the rooting seedlings,the transplanting survival rate was 100%,and the growth condition was good.（4）No obvious morphological variation occurred in the regenerated plants C.×generalis‘Mohong’.The primers P111 and P143 were used to amplify the field grown control and regenerated plants of C.×generalis‘Mohong’by SSR-PCR,and polymorphism was not found.Therefore,it indicated that no significant genetic variation was detected in the regenerated plants of C.×generalis‘Mohong’,and this tissue culture protocol was feasible.（5）The suitable materials for callus induction were budding rhizomes of asexual field seedlings,leaf sheath at stem base,corolla tube,and seed embryo of aseptic seedlings.MS medium was was beneficial to induce the callus of‘Mohong’seed embryo and N6medium is suitable for‘Lüyecheng’corolla tube induction.Among these three varieties,the callus induction of‘Lüyecheng’and‘Yinli’was higher than‘Yellow Canna’.Maltose was more effective than sucrose in callus induction of the corolla tube.14-days darkness treatment was favorable for corolla tube and leaf sheath callus induction.After optimization and verification,the best callus induction medium for the corolla tube was N6+6-BA 4.83 mg·L-1+IAA 0.5 mg·L-1,with an induction rate of 91.8%.The optimum medium for callus induction of leaf sheath at the stem base was MS+6-BA 6.62 mg·L-1+NAA 1.5 mg·L-1+TDZ 0.2 mg·L-1,with an induction rate of 80.77%.（6）In MS+6-BA 4 mg·L-1+NAA 1 mg·L-1+TDZ 0.5 mg·L-1medium,the callus proliferation rates of the embryo,leaf sheath at the base of the stem,and root tip were 54.76%,47.62%,and 16.67%,respectively.The proliferation rate of stem segments without bud,intact ovary,and corolla tube was 0.However,the callus induced from leaf sheaths at the base of stem and seed embryos proliferated into white and light green mass,and the proliferation effect was good.（7）In MS+6-BA 3 mg·L-1+TDZ 2 mg·L-1+IAA 1 mg·L-1medium,the root tips were proliferated into light brown loose callus without differentiation.However,the white and light green mass callus from embryo and the leaf sheath at the base of the stem developed differentiation,and the differentiation rates were 35.71%and42.86%,respectively.Moreover,white roots and young green shoots were induced. |
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