Pinto G, Loureiro J, Lopes T, Santos C
Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal.
Theor Appl Genet. 2004 Aug;109(3):580-7. doi: 10.1007/s00122-004-1655-3. Epub 2004 Apr 14.
Flow cytometry was used to measure the nuclear DNA content of Eucalyptus globulus Labill. somatic and zygotic embryos and leaves in order to determine if somatic embryogenesis induces DNA content and ploidy changes in this species. Mature zygotic embryos derived from open-pollination orchard families were collected from a location in the centre of Portugal. One group was kept for nuclear DNA content and ploidy analysis, and the other group was used for establishing embryogenic cultures. Mature zygotic embryos were grown on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 3 mg l(-1) alpha-naphthaleneacetic acid (NAA) for 3 weeks and then transferred to MS medium without growth regulators. Globular somatic embryos from approximately 8-month-old embryogenic cultures were used in the assay. DNA ploidy levels and the nuclear DNA content of mature zygotic embryos, somatic embryos and leaves from the mother field tree were determined using flow cytometry combined with propidium iodide staining. Zygotic embryos had a nuclear DNA content of 1.32 pg/2C, somatic embryos had a nuclear DNA content of 1.39 pg/2C and leaves from the field tree had a nuclear DNA content of 1.40 pg/2C. The values estimated for the somatic embryos and mother plant did not differ statistically from each other (P < or = 0.05), but both differed from those of the zygotic embryos (P < or = 0.05). These results clearly indicate that no changes were induced during the embryogenic process. However, the differences found between the field plants and zygotic embryos did suggest that some aspects must be evaluated carefully, as propidium iodide fluorescence may potentially be influenced by the presence of secondary compounds (e.g. anthocyanins, tannins) in E. globulus somatic embryos and mature leaves. Therefore we believe that the somatic embryogenesis methodology used did not induce major genetic changes in the somatic embryos and that our primary goal of "true-to-type" propagation was assured.
采用流式细胞术测量蓝桉体细胞胚、合子胚及叶片的核DNA含量，以确定体细胞胚胎发生是否会引起该物种DNA含量及倍性变化。从葡萄牙中部某地收集来自开放授粉果园家系的成熟合子胚。一组用于核DNA含量及倍性分析，另一组用于建立胚性培养物。成熟合子胚在添加3%（w/v）蔗糖和3 mg l(-1)α-萘乙酸（NAA）的Murashige和Skoog（MS）培养基上培养3周，然后转移至不含生长调节剂的MS培养基上。使用约8个月大的胚性培养物中的球形体细胞胚进行测定。采用流式细胞术结合碘化丙啶染色法测定成熟合子胚、体细胞胚及母本田间树叶片的DNA倍性水平和核DNA含量。合子胚的核DNA含量为1.32 pg/2C，体细胞胚的核DNA含量为1.39 pg/2C，田间树叶片的核DNA含量为1.40 pg/2C。体细胞胚和母本植株的估计值在统计学上彼此无差异（P≤0.05），但两者均与合子胚的估计值不同（P≤0.05）。这些结果清楚地表明，胚胎发生过程中未诱导变化。然而，田间植株与合子胚之间的差异确实表明，必须仔细评估某些方面，因为碘化丙啶荧光可能会受到蓝桉体细胞胚和成熟叶片中次生化合物（如花色苷、单宁）的影响。因此，我们认为所采用的体细胞胚胎发生方法未在体细胞胚中诱导重大遗传变化，并且我们“保持原种特性”繁殖的主要目标得以确保。
