| Ginger(Zingiber officinale roscoe) belongs to family of Zingiberaceae and perennial herb, and its edible organ is fat rhizome underground. According to statistics by FAO(Food and Agriculture Organization of the United Nations), C hina is the largest ginger producers to the world market. Currently, with increase of ginger quantum of international trade, higher demand to commercial character of ginger variety is proposed, therefore it is important for breeding new ginger variety.Due to poor flowering and seed set, conventional crossbreeding is inapplicable, nature variation selection, ploidy breeding and biology breeding provide some promising strategies for the crop improvement of ginger. Ploidy level of genetic resources is fundamental and essential to develop an efficient breeding program. However, information on ploidy of the ginger germplasm is scarce. The object of this study was to determine DNA ploidy levels and chromosome numbers of the C hinese ginger germplasm resources, on this basis, to set up shoot propagation system in vitro of different ploidy ginger, to study ginger ploidy breeding technique, and to develop ginger cell suspension cultures method, and these knowledge will offer further insights into ginger germplasm creation and breeding new variety. The main results are as follows:1 Sixty-three ginger varieties representing diverse geographic locations in C hina were analyzed for ploidy by flow cytometry. O ur results indicated that 16 ginger varieties(25.4% of the accessions) were mixploids with diploid and tetraploid cells, which tetraploid cell percentage was higher than 40%, and 47 ginger varieties(74.6% of the accessions) were diploid. No tetraploids or other polyploidy occurred in nature and it is the first time to report natural occurrence of mixploid ginger. C hromosome numbers were counted in actively growing root tips using conventional rapid squash techniques. The results showed he basic chromosome number of ginger in C hina is x = 11. Phenotypic alterations occurred in response to changes in ginger ploidy level. Mixploid ginger was typed as a ?giant ginger? with stronger stems, fatter fingers, bigger rhizomes, fewer shoots, and the diploid was typed as ?small ginger? with thinner stems, more shoots, and a smaller single finger and rhizomes. Based on clustering analysis of each discrepant trait, it was found that the single finger mass of ginger was the most important and key morphological character distinguishing the mixploid from the diploid.2 Plant growth regulator combinations had significant effect on in vitro shoot survival rate, shoot initiation, shoot multiplication and plant height of plantles. With regulator combinations of 0.4 mg·L-1 NAA and 2.5 mg·L-1 KT, higher shoot survival rate of mixploid ginger “No.1 shannong” was 73.5%, and bigger shoot multiplication in subculture is 5.8. For diploid ginger “Guangxi small ginger”, the combination of plant growth regulator suitable for shoot initial was same as shoot multiplication, which NAA was 0.2 mg·L-1and KT was 1.5 mg·L-1. The genetic stability of micropropagated clones of different ploidy ginger was evaluated at different subculture times using nter-simple sequence repeat(ISSR). The results showed primers utilized in this study gave rise to a total of 60 scorable bands in “No.1 shanong” and 43 scorable bands in “Guangxi small ginger”, ranging from 200 bp to 2000 bp in size. ISSR analysis of micpropagated plants of different ploid ginger showed a profile smiliar to that of the field plants indicating that no genetic variation had occurred in vitro. FCM analysis showed no significant variation in nuclear DN A contents and ploidy levels in long-term in vitro maintained thorough different ploidy ginger shoot. There was also no phetotypic significance between accamalited plantl and field plant, for example, fresh weight of rhizome with filed plant of “No.1 shannong” was 1452.0g, similar to that of accamalited plant, 1453.3g, which showed no significant difference.3 Taking mixploid ginger “No.1 shannong” as material, different colchicines concentrations and time duration of treatment were combined to induce diploid and tetraploid ginger. Survival rate of stem cutting in vitro is higher than 80%, and mutation rate is more than 25%, when colchicine concentration increased to 0.15% for 12h~16h. Compared to mixploid ginger(control), diploid ginger was a plant with shorter plant height, thinner stem and leaf. Plant height, stem diameter and leaf width of diploid ginger was lower than these of CK by 30.3%, 10.5% and 27.3%. Two tetraploid phenotypes was induced from mixploid by colchine, which type Ⅰ?s plant height, stem diameter, leaf length, leaf width and leaf thickness were lower than those of CK by 47.0%, 42.1%, 28.6%, 26.7% and 40.5%, these indexes of type Ⅱwere higher than those of CK by 7.6%, 13.2%, 34.3%, 60.0% and 35.7%.4 Mixploid giner(CK) and induced diploid had higher plant survival rates, more stable ploidy in subculture. With increase of subculture times, survival rate of tetraploid reduced. At 10 th subculture, plant survival rate of type Ⅰhad reduced to 0, and typeⅡis only 1.5%. ploidy level of typeⅡwas inconstantly, which 17.9% diploid ginger was generated from typeⅡat 7th subculture. When different ploidy ginger plantlet in vitro was acclimated and transplanted, their survival rate and growth vigor were different. Transplanting survival rate of CK and diploid ginger was 100%, which growed quickly, and tetraploid ginger had lower transplanting survival rate, weaker growth vigor. With plant growing, transplanted tetraploid plant died successively. After 120 d transplanted, plant surviva l rates of type Ⅰ and Ⅱwere 44.0% and 28.0%, but their survival rates all were 0 after 180 d transplanted. Therefore, tetraploid may not be stable ploidy level ginger.5 “Laiwu” giant ginger was taken the material to study induction of leaf-derived callus, establishment of cell suspension and plant generation in vitro ginger. The results showed that young leaves of in vitro ginger as explant were cultured on revised Murashige and Skoog medium supplemented with 2,4-D 1.0 mg·L-1, 6-BA 0.5 mg·L-1 and sucrose 30 g·L-1, and it resulted in callus with loose structure rapid growth. Obtained callus were cultured in modified liquid MS medium supplemented with 2,4-D 0.15 mg·L-1, 6-BA 6.0 mg·L-1 and sucrose 30 g·L-1, and shook at 25 ℃ under dark condition for 25-30 d, then good-diversity and rapid-growing cell suspension cultures were established. The optimal parameter of cell-suspension cultures were: 1.0g-1.5g initial inoculum, a sub-culture every 15 d, and 3/4 volume of updating liquid medium during subculture. Plantlet formation occurred when suspension cell were transferred to solid MS medium containing 2,4-D 1.0 mg·L-1, 6-BA 0.5 mg·L-1 and sucrose 30 g·L-1. |
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