Autotetraploid Induction With Oryzalin In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.,2n=2x=18), an annual and biennial in Raphanus genus of cruciferae family natives to China, is an important vegetable crop worldwide. The polyploid breeding can not only promote crop’s output and quality, but also plays an important role in the plant genetic evolution. The mechanism of the induction by dinitroaniline herbicides Oryzalin is the same as colchcine:the inducers can rapidly inhibit the formation of spindle fibers so that the doubling of chromosomes within one cell is achieved. Meanwhile, Oryzalin can also inhibit some of the shortcomings of colchicine. As we all know the efficient induction system of tetraploid radish has not been established. In particular, Oryzalin as a novel microtubule inhibitor has not been reported to induce radish tetraploid. In this paper, we compared the effects of colchicine and Oryzalin in inducing chromosome doubling of Nau-DY and Nau-ZQH in vivo (soaking seeds and apical meristem appling). Morphological and cytological characters were used to eliminate much of the diploid population, and the ploidy level of induced plant was ultimately determined by chromosome counts. This study provides a theory basis for tetraploid radish induction and identification. The abnormal behaviors in the meiotic of PMC was observed using light microscope and DDRT-PCR was applied to isolate of genes differentially expressed in diploid and tetraploid ’Nau-XBC radish, which provide a theoretical basis on the molecular genetic mechanisms of ploidy breeding. The results are as follows:Apical meristems of the diploid radish were treated with Oryzalin at concentration of0.01,0.02,0.04,0.06mM Oryzalin and0.1%colchicine for the control. The treatment was conducted at9:00am and4:00pm each day for3days. The results show that the most effective induction was30%in Nau-DY and26.67%in Nau-ZQH which were treated after0.04mM Oryzalin for6times. Meanwhile, autotetraploids were also obtained in other treatments with different frequency. The seeds with shorter and thicker root-tips were observed by germinated seeds-immersion method; unfortunately, no plants were developed into plantlets for the inducers’toxic. The tetraploid plants had deep green leaves, longer and wider stomata, et al. We found that in all diploid to tetraploid, the number of the chloroplast in guard cell of stomata was significantly increased. Chromosome counts cells of root tip and pollen mother cell combined with the chloroplast of guard cells were used to confirm the ploidy level of induced plant.The fertility reduction of autotetraloid has been attributed to irregularities at meiosis. The results in this study showed that:tetraploid radish had low quantity of pollens, with big difference in the pollen size and abnormal pollen shape. The rate of tetraploid pollen germination is quite lower than the diploid. Some abnormal behaviors were observed in the meiotic process of PMC of tetraploid radish,such as univalent and ring-of-four at diakisesis, chromosome lagging, chromosome bridge in anaphase I and anaphase II, micronucleus, abnormal ditrad and tetrad,predivision at anaphase II.In autopolyploid plants, the allele dose-effect present in the expression of some genes, including the genes related to plants’growth and resistance. In this study, Differential-display reverse transcription-PCR (DDRT-PCR) was applied to isolate of genes differentially expressed in diploid and tetraploid’Nau-ZQH’radish. A total of4000cDNA fragments were obtained by using60random primers, however, most of the transcripts were similar and expressed in both diploid and tetraploid radish.80differentially expressed fragments were observed in tetraploid to diploid’Nau-ZQH’radish, and only20TDFs were cloned, sequenced, of which17TDFs showed high sequence similarity to the genes of known or putative function in Arabidopsis or other plants. These genes were transcriptional factors, expression regulators, stress responding and transport facilitation genes, photosynthesis genes as well as genes involved in cellular metabolism and organization; moreover, two genes were directly related to fertility and methylation of tetraploid. The results showed that plants rapidly and simultaneously change the expression of a set of genes to cope with the polyploidy.