| Eucalypts(Eucalyptus spp) is one group of the most fast-growing tree species in plantations in southern China and even all over the world. Ploidy breeding is a commonly used breeding technique to produce the new bred varieties to increase yield, produce a better product and enhance resistance through this breeding technique. The tetraploid plants may be produced with the induction of colchicine. Polyploid plants are necessary to breed the new improved varieties. In this study, the treatment material including stem segments of Eg5, a clone of E. grandis; DH32-29, a clone of E.urophylla×grandis; GL9, a clone of E.urophylla×grandis and DH201-2, a clone of E.grandis×tereticornis and callus tissue of DH201-2 clone(which was made from both neonatal leaf and tender stem cuttings) were treated with the traditional“soaking method” and the techniques developed by the author viz “coated method with absorbent cotton” and “insertion method in culture medium” at different strengths of colchicine; the softwares including SAS, Matlab, Lingo, Design Expert,DPS,SPSS,Sigmaplot and 1stOpt were used in this study.The main conclusions are as follows:(1) This study includes two parts in order to reach conclusions on the acquisition of the highest inductivity rate. When the “soaking method” was used: the first part was a “qualitative experiment” to find the significant influencing factors with the help of Plackett-Burman Design; the second part was a “quantitative test” to build a model to find the highest inductivity rate and the corresponding level of every significant factors.(2)Mathematical model was built to reflect the relationship between inductivity rate and every significantly influencing factor by using the Central Composite Design(CCD) functional module of the software package Design Expert which can find the highest inductivity rate and the corresponding level of every significant factor when the techniques of “coated method with absorbent cotton” and “insertion method in culture medium” are applied. In addition, the model can be used only if it is under the applicable conditions.(3) With the use of “soaking method”, stem segments of DH32-29 clone gained the highest inductivity rate of 34% under the condition of soaking concentration 0.22%, soaking time 25.2h; stem segments of GL9 clone gained the highest inductivity rate of 20% under the condition of soaking concentration 0.21%, soaking time 25.6h; stem segments of Eg5 clone gained the highest inductivity rate of 19% under the condition of soaking concentration 0.21%, soaking time 25.8h; stem segment of DH201-2 clone gained the highest inductivity rate of 25% under the condition of soaking concentration 0.28%, soaking time 15.5h and soaking temperature 28.4℃; The callus tissue of DH201-2 clone which is made from neonatal leaves gained the highest inductivity rate of 53% under the condition of soaking concentration 0.24%, soaking time 17.4h and soaking temperature 27.6℃; the callus tissue of DH201-2 clone which is made from tender stem cutting gained the highest inductivity rate of 37% under the condition of soaking concentration 0.27%, soaking time 16.4h and soaking temperature 28.6℃;(4) With the use of “insertion method in culture medium”, stem segments of DH32-29 clone gained the highest inductivity rate of 12% under the condition of colchicine concentration 0.43%, treatment time 39h; stem segments of GL9 clone gained the highest inductivity rate of 9% under the condition of colchicine concentration 0.32%, treatment time 41h; stem segments of Eg5 clone gained the highest inductivity rate of 8% under the condition of colchicine concentration 0.44%, treatment time 49h; the stem segment of DH201-2 gained the highest inductivity rate of 9% under the condition of colchicine concentration 0.54%, treatment time 30 h and treatment temperature 38℃; The callus tissue of DH201-2 clone which is made from neonatal leaves gained the highest inductivity rate of 15% under the condition of colchicine concentration 0.32%, treatment time 36 h and treatment temperature 32℃; the callus tissue of DH201-2 clone which is made from tender stem cutting gained the highest inductivity rate of 10% under the condition of colchicine concentration 0.45%, treatment time 29 h and treatment temperature 42℃.(5) With the use of “coated method with absorbent cotton”, stem segments of DH32-29 clone gained the highest inductivity rate of 20% under the condition of colchicine concentration 0.28%, treatment time 30h; stem segments of GL9 clone gained the highest inductivity rate of 17% under the condition of colchicine concentration 0.28%, treatment time 29h; stem segments of Eg5 clone gained the highest inductivity rate of 14% under the condition of colchicine concentration 0.26%, treatment time 33h; stem segments of DH201-2 clone gained the highest inductivity rate of 17% under the condition of colchicine concentration 0.45%, treatment time 18 h and treatment temperature 46℃; The callus tissue of DH201-2 clone which is made from neonatal leaves gained the highest inductivity rate of 33% under the condition of colchicine concentration 0.37%, treatment time 20 h and treatment temperature 33℃; the callus tissue of DH201-2 clone which is made from tender stem cutting gained the highest inductivity rate of 24% under the condition of colchicine concentration 0.38%, treatment time 20 h and treatment temperature 43℃.(6) Vitrified shoots of Eg5 clone of E.grandis commonly occur after the polyploid induction with colchicine so there has been a need to improve the existing culture medium; use of the special culture medium developed in this study has basically eliminated this vitrification phenomenon.(7) Tetraploid plants which were successfully developed in this study can develop triploid plants if sexual reproduction is used and triploid plants are more suitable for developing improved varieties. |
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