| Hemerocallis hybrida is perennial root herbaceous flower,which is day lilies. Rhizomesof day lily show strong resistance to cold, drought, salt, disease, insect pests, etc. Day lily is arich germplasm resources in our country, can be grown in the north and south region, whichhas wide ornamental value and easy to cultivate and manage. It can clump planting in thelawn or the flower bed, along the road, which is a good garden green land perennial rootflowers. In today’s construction of ecological civilization, day lily has a broad marketprospect.However, the foreign varieties of day lily be grown widely, native species of China isdeficiency. It just has a few day lilies with different colors in China, which are difficult tomeet the needs of people to enjoy the beautiful scenery with Hemerocallis hybrida.Using agrobacterium-mediated genetic variations and tissue culture techniques as themethod, hemerocallis middendorfii which called” Sweet treasure” as the material, day liliesplant regeneration and genetic transformation have been studied. The plant regenerationsystem and genetic transformation system of” Sweet treasure” was established. Referencedata be provided for the increasing of the breeding intensity and the processing ofmarketization.1. High-frequency regeneration system of day lily is established.Using torus of Hemeroallis hybrid“Sweet treasure” as the exolant. Utilizing theorthogonal design to study the influence of the different hormone concentration to callusinduction, callus proliferation induced, adventitious bud induction and root induced ofHemerocallis in vitro.The results showed that disinfection conditions was soaking20s by70%alcohol and10min by0.1%HgCl2; starting rate of receptacle was68.24%,starting rate ofpeduncle was77.87%.6-BA is the main factor to callus induction of Sweet treasure. thesequence of influence of four hormones was6-BA＞2,4-D＞NAA>KT;among which theoptimal formula for callus induction listed as followed: MS+1mg/L BA+3mg/L2,4-D+0.3mg/L NAA; Callus induction rate is81.28%as the highest.6-BA was also the mainfactor to callus proliferation induced, the the sequence of influence of four phyto-hormoneswas6-BA＞NAA＞KT＞2,4-D; the best formula for a callus proliferation induced was:MS+1mg/L BA+1mg/L KT+0.2mg/L2,4-D+0.05mg/L NAA; callus induced proliferation rateis5.27times. GA was the main factor to adventitious bud induction, the sequence of influenceof three phyto-hormones was GA＞6-BA＞NAA; the best formula for adventitious budinduction was: MS+1mg/L BA+0.1mg/L NAA+0.5mg/L GA; adventitious bud induction rateis78.21%. The IAA was the main factor to root induced, the sequence of influence of threehormones was IAA＞MS＞sucrose＞NAA; the best formula for root induced was:1/2MS+0.3mg/L NAA+20g/L sucrose; induced rate is94.4%.The illumination was the main factor to acclimatization and transplant, the sequence of influence of three factors wasillumination＞temperature＞watering intervals; the best illumination was one bed shadecloths, shade rating60%~70%;watering intervals was four days;temperature was25℃;survive rate is98.3%.2The transfer conditions were studied and the genetic transformation system was optimized.Using the callus of Sweet treasure as explant,10d as one subculture period.Agrobacterium EHA105carried with PSN1301-CHS infected explant to tranfer CHS geneinto Sweet treasure. The main factors influencing the gene transformation of Sweet treasurewere studied: the best carbenicillin concentration was350mg/L as bacteriostatic agent;hygromycin was suitable to select transformed tissue, appropriate screening concentration was25mg/L; the time of preculture were2days for explant; appropriate concentration of Agroba-cterium OD6000.6; choose MS+3%sucrose as new medium to suspension bacteria; the mostappropriate infection time was15min.It was beneficial to add100μmol/L acetosyringone intococultivation medium for improving the transfer rate.2days were the best co-culture time forexplant. |
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