| Citrus fruits is one of the most important tree crops in the world. Seedless is a desirable horticultural attribute in Citrus and is positively associated with triploidy. Most pummelo and sweet orange cultivas are very seedy. Therefore, to select and breed seedless varieties would be the best way of keep their competitiveness.At present, the use of tetraploids crossed with diploids to produce triploid seedlings is a proven method of producing seedless or nearly seedless citrus varieties, while, the absence of a range of suitable tetraploid germplasm has greatly hindered cross breeding between tetraploids and diploids in Citrus.In this study, there are two strategies to obtain polyploids. Screening seedlings and 2n gametes breeding were carried out on Citrus grandis Osb.cv.Shatianyou and Citrus sinensis Osb.cv.Hongjiang.In order to obtain polyploids, screening seedlings resulted from seeds of open-pollinated fruits by chromosome counting, the results are as follow: the rates of triploids and tetraploids of cv.Shatianyou were 0.08% and 0.15% respectively. While, the frequency of tetraploids of cv.Hongjiang was 2.50%. The proportion of aneuploid cell is very low. Meantime, 8 unexpected diploids with obvious leaf variation. This method of obtaining polyploids can overcome the problem of aneuploidy which can result from irregular meiosis in tetraploids. Though the occurrence of polyploids was at a low rate, it may be an efficient pathway of polyploidy breeding in Citrus.Plants morphology observation on different ploidy seedlings with similar age, growth stage and environment background suggest that polyploids grow slower than comparable diploids. In general, tetraploids have darker green leaves and larger leaf area than corresponding diploids, but with smaller leaf index. Stomata of polyploids are larger and spaced father apart on the undersurface than diploids. Our observation and data indicate that leaf thickness and stoma density are useful in the diagnosis between tetraploids and diploids in Citrus.It is significant to culture lots of polyploids for enhancing breeding efficiency by tissue culture during short stage. The epicotyl, hypocotyl, cotyledon, and stem segment of 20d3seedlings could form adventitious bud, leaf fraction was an exception. The optimum medium of multiplication was MS+BA1.0mg/L+NAA0.3-0.5mg/L+sucrose 3.0%, the induction rates of roots were 95.7% at the medium MS+IBA1 .Omg/L+ sucrose 3%.Karyotype analysis led us to the conclusion that these spontaneous polyploids of cv. Shatianyou resulted from sexual polyploidization. Meanwhile, whether these tetraploids of cv. Hongjiang originated from apomictic seedlings was not clear. Exact origin of these Citrus polyploids can be determinated by molecular maker and GISH.Experiments of observation of spontaneous occurrence and induction of 2n pollen were made. About 0-3% large pollen grains (unreduced pollens) from spontaneous pollens of 12 species in Citrus were observed in consecutive two years, which proved existence of unreduced pollens. Low proportion of 2n pollens made it difficult to be collected. Studies on pollen chromosome doubling were performed by colchicine-treated with different concentration and various continuous time. Best results for inducing 2n polllen grains were obtained when flowers were treated with 0.3% or 0.4% colchicine for 4d, the frequency was 9.2% or 8.3% respectively. In vitro germination of these different ploidy pollen grains were examined, 79.6% 2n pollens have normal fertility, though they started to germinate slower than normal male gametes. Then, we made 2n pollen pollinate with diploid pummelo. As a result, 1 triploid and 2 tetraploids were obtained from crosses.Pummelo is typically mono-embryonic, these tetraploids are potential progenitors in programmed crosses. We believe that these polyploids are very useful for citrus polyploid breeding. What's more, 2n gametes breeding will possess charming perspectives.
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