Primery Study On Mutation Induced By ⁶⁰Coγ-ray Radiation In Breeding Of Butterfly Orchid (Phalaenopsis Sp.)

Phalaenopsis amabilis (Linnaeus) C. L. Blume, called Butterfly orchid in English, belongs to Family Orchidaceae, Genus Phalaenopsis (Blume, 1825), and is a kind of perennial epiphytes. The Butterfly orchid, whose flower is like colorful, flying butterfly, is international and expensive popular flowers with high ornamental and economic value. It becomes the most widely cultivated varieties in the world, and also is called "Empress Orchid." The characteristics of cultivars could be degraded during the long term of vegetative propagation, so improvement and innovation of its germplasm resources was great significance. As an effective means of promoting plant variation, radiation-induced mutation had displayed an extremely important effect and great prospects in flower variety improvement. Currently many research works had focused on the field of mutation breeding in the world, where the ⁶⁰Coγ-ray radiation was the means most widely used.The total of 160 plants from four cultivars of orchid, propagated by tissue culture, were used as material, after treated with four different doses of ⁶⁰Coγ-ray, that are 15Gy, 20 Gy, 25 Gy and 30 Gy respectively, their biological properties were observed and recorded. The genomic DNA was extracted by CTAB method to test the extent of genetic variation in the different doses of ⁶⁰Coγ-ray treatment. The parameters of RAPD-PCR including the concentration of Taq polymerase, template DNA, magnesium ion, dNTPs and arbitrary primer were optimized gradually to establish the optimal RAPD-PCR system. The total reaction volume was 25μL, including 2.0 unit of Taq polymerase, 2.5 mmol·L-1 MgCl2, 0.2 mmol·L-1 dNTPs and 0.8μmol·L-1 arbitrary primers. The schedule of RAPD-PCR program was 3 min initial denaturation at 94℃, then 45 s denaturation at 94℃, 1 min anneal at 38℃, 1.5 min extension at 72℃, 35 cycles amplification, and 10 min final extension at 72℃.Suitable random primers, by which stable and clear bands could be amplified, were screened out from 72 ones for different varieties, and 30, 21, 20 and 24 primers was suitable for Variety A, B, C and D, respectively. According to the amplified bands of RAPD-PCR coded in 0 (absent) or 1 (present), and the cluster analysis was done by the Between-groups Linkage method and the coefficient of Euclidean Distance Square (CEDS) in the software of SPSS 11.0 for Windows. The results of the cluster analysis showed that the genetic distance between different treated plants in Variety A were larger, that clustered in accordance with treated doses basically and the individuals fell into the same cluster as they were treated with similar radiation doses. In compare with the control, plants treated with 30 Gy had the farthest genetic distance accounting for the largest degree of variation happened. The range of variation was the most wide in treatments of 25 Gy, of which the genetic distance between plant were farthest in the cluster analysis, for example, the treatment of 25-4 (non-variance) and 25-6 (mutant) were fell into the fourth and fifth cluster respectively.Eight mutants with variant flower shape were found in Variety A treated with 25 Gy or 30 Gy. The stunted pedicel was more obvious as the increases of the dose of radiation which had provided a possible way to change the traditional group-potted-growing into the single-stock-potted-growing in the cultivation of butterfly orchids.There were many specific bands amplified in template g-DNA with variant flowers and by using different primes in RAPD-PCR. No specific band could be found only in one plant in the Variety D treated with 25Gy at molecular level, which had morphologically variations in flower character, which might be false variation caused by the environmental factor. It was postulated that the optimal doses of radiation by ⁶⁰Coγ-ray for Variety A and D was 30 Gy, for Variety B was 15 Gy, and for Variety C was 25 Gy, respectively.