| Teak (Tectona grandis L. f.) is a famous hardwood species and its wood is widely used forshipbuilding, bridge construction, furniture making, carving and etc. Teak is broadly cultivatedin tropical and subtropical region of China, including nine provinces and more than sixtycounties. However, there is no teak distribution in China and germplasm for cultivation is poor.Teak cultivation in China encounters lots of problems, such as soil acidity, frost bite, typhoon,rust disease, bollworm and etc. Genetic transformation technique is useful for transgenicbreeding of cold resistance, acidity resistance, disease resistance and insect resistance, whichhas great significance for further dissemination of teak cultivation in China.This study deals with Agrobacterium-mediated transformation of teak callus andprimordium, dissection of primordium, selection and identification of transformants. Theresults are summarized as follows.1) The optimized parameters of callus transformation by EHA105 are: Pre-culture time, 11～13 days; OD value of stock solution, 0.4～0.5; co-culture time, 7～9 days; co-culture pH,6.0. For LBA4404 transformation, pre-culture time is 11～15 days, OD value of stocksolution is 0.5～0.8, co-culture time is 13 days, co-culture pH is 5.6～6.2. Re-suspensionof bacterium solution improves transformation efficiency. The addition of acetosyringonedoesn't enhance transformation efficiency. The suitable inoculation time is 25 minutes.There are genotype difference of Agrobacterium susceptivity for callus transformation. Thecalli of clone 7813, 108 and 7531 have high infection susceptivity.2) Lateral shoot primordia are located on the surface of stem in axillae. Its vertical position isright above the node. Incision near node upside has the highest chance to wound theprimordia. Primorium wounding encourages the adsorption and transformation ofAgrobacterium. The strong recovery ability of lateral shoot primordia from woundingguarantees that a few transformed cells can replace untransformed cells and make up wholeprimordium.3) Parameters for LBA4404's transformation of lateral shoot primordia are as follows:pre-culture time, 3～5 days; OD value of stock solution, 0.5; co-culture time, 5～9 days; co-culture pH, 5.6～6.0; co-culture temperature, 27℃, susceptible clone, 108; suitableexplant, middle stem sections from shoots with 26～36 propagation culture. For EHA105transformation, pre-culture time is 3～5 days, OD value of stock solution is 0.5, co-culturetime is 3～6 days., co-culture pH is 5.2～5.4, co-culture temperature is 27℃, susceptibleclone is 108, middle stem sections from shoots with 36 propagation culture are bestexplants.4) Teak callus is sensitive to kanamycin as low concentration can inhibit the germination ofcallus from stem explants. The suitable concentration for clone 108 is 20～25mg/L and thatfor clone 7531 is 15～20mg/L. When callus clump is formed, its kanamycin resistanceincreases. A concentration of 75mg/L is needed to inhibit the growth of 108's callus clump.5) There are eminent genotype difference in kanamycin resistance of lateral shoot primordia.The suitable concentration for clone 7663 is 50mg/L, that for clone 8003 is 50～100mg/L,for clone 7557 and 108, 100～150mg/L. It is necessary to change the selection pressureduring the selection process. The optimized selection procedure is eighteen days' selectionand disinfection by 150mg/L kanamycin+250mg/L cefotaxime+250mg/L carbenicillin→eighteen days' selection and disinfection by 50mg/L kanamycin+250mg/L cefotaxime+250mg/L carbenicillin→eighteen days' selection and disinfection by 100mg/L kanamycin+250mg/L cefotaxime+250mg/L carbenicillin→selection and propagation by 100mg/Lkanamycin.6) GUS staining showed that primordia transformation was practicable. Counted by explants,transformation rate by LBA4404 and EHA105 is 0.185%. Counted by T0 plants, thetransformation rate of LBA4404 is 11.11% and that of EHA105 is 18.18%. PCR resultsshowed that four plantlets with positive GUS staining were transgenic.Teak primordia was genetically transformed in this study and the advantages ofprimordium transformation method are as follows: simple transformation operation, abundanceof explants, genetic stabilization in the development of transgenic plants, less regenerationdifficulties and easy combination with normal selection breeding. |
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