Rapid Propagation Of Ancient Trees And The Relationship Between Tree Age And Telomere Length

Old trees are the precious legacy of the long history, leaving to humanity with important scientific, cultural and economic values. In order to inherit the historical culture and reserve the valuable plant genetic resources of the old national garden plants, in vitro and seed-germinating propagation of the ancient trees from Beijing area were studied. Optimized determination of trees telomere length was finished, relationship between telomere length and age of different trees was compared. The main findings are as follows:1. Rapid propagation of Ancient trees1.1Tissue CultureNodal explants derived from ancient mulberry, which has been living in the Summer Palace for more than100years, were used to initiate shoot cultures. Bud formation on the nodal explants was induced on an optimal medium MS+6-BA0.5mg/L+NAA0.1mg/L+PVP0.2g/L. To get a high propagation ratio, the seedlings were transferred to the medium of MS+6-BA0.8mg/L+NAA0.1mg/L+PVP0.2g/L. The optimal medium for adventitious root inducing was1/2MS+NAA0.5mg/L+PVP0.2g/L, and the rooting rate was100%. The plantlets were transplanted to pots with vermiculite-perlite mixture-humus soil (1:1:1) mixture and acclimated for several weeks. The acclimated plantlets were transferred outdoors with95%transplantation success.An efficient and reproducible method for the regeneration of multiple shoots of ancient Diospyros kaki L. f. from the Summer Palace has been developed in flasks containing primary shoots as the explants. Explants derived from in vitro grown seedlings were cultured on1/2MS supplemented with0.8mgl’6-benzylaminopurine(6-BA),0.1mgl-11-naphthlcetic acid(NAA), and0.5gl-1polyvinylpyrrolidone(PVP).1/2MS supplemented with5.0mgl-16-BA and0.2mgl-1NAA was found to be best for adventitious shoot induction and for the multiplication of individual shoots.However, the growth rate of the shoots was very low on the above medium.To mediate the growth and propagation of the shoots, selected shoots were transferred to the medium of1/2MS+2.0mg/LZT+0.1mg/LIAA+0.5g/L PVP. In-vitro-produced shoots were rooted using a two-step method. Firstly, shoots were cultured on1/2MS with0.6mgl-1NAA,0.3mgl-1IBA and0.5gl-1PVP. Secondly, the shoots were transferred to1/2MS with0.5gl-1PVP. The most effective first-step treatment was found to be dark training for9～10days, which initiated rooting at a frequency of30%. If the shoots were immersed in3-indolebutyric acid (IBA) or indole-3-acetic acid (IAA) at250mg1’for2min, and then transferred to1/2MS with0.5gl-1PVP, the initiated rooting was improved.1.2Propagation through seed germination Seeds of Chinese pine, catalpa tree, oriental arborvitae and ginkgo-were collected from the Summer Palace and the Botanical Gardens Seedlings were obtained by the following methods:Chinese pine seeds were kept on the cold storage for a period of time then directly sowed in the matrix, and the matrix surface was covered with a layer of thin sand; Seedings of P.orientalis can be obtained by sowing and keeping the environment slightly damp; Ginkgo seeds were on the low-temperature refrigerator and refrigerated for some time, and then soaked in water until germination, planting seedlings are available in the matrix.2. The detection of telomere length for different old treesTelomeres are related to cell growth, differentiation, aging and death. With cell division, telomeres (the physical ends of linear chromosomes) progressively shorten until they reach a critical length, at which point the cells enter replicative senescence. Tree telomere length will shorten in each round of cell cycle, so telomere length can be used as a predictor of tree life-spans.Telomere shortening can be used as both an in vitro and in vivo marker of cell aging. By adjusting the concentration of PVP, CTAB protocol was optimized for extracting the total DNA of trees; and the purer genomic DNA are extracted by improved methods. Catalpa tree was, used as a model to establish the method of telomere length measuring.30μg genomic DNA was digested by Hinf I or Taq I for24to36h, then telomere lengths were measured by the method of southern blotting, which is a most popular method to measure the telomere length. It is found that the regularity for relationship of telomere length with age of trees is species-specific, being not the same. The telomere lengths of Ginkgo, Catalpa tree, Sophora japonica changed according to ages, while the trends were different. But the telomere length in Chinese pines did not change with the tree ages.