| Chrysanthemum is one of ten traditional famous flowers in China and four cut flowers in the world, it play an important position on gardens and greening. However, chrysanthemum is seriously infested by pests from the time of its seedling establishment to its flowering stage. Aphid infestation usually hurt tender bud tip, inhibiting the plant growth and development, results in dwarf plant, yellowing leaf and yield loss, effectting the quality and ornamental value seriously, it as a result of their direct jeoprridize chrysanthemum itself, and their indirect action as vectors of TAV, PVP, CMV viruses. Currently, chemical pesticide was the major method to control aphid during horticultural practices, which not only consumed higher agrochemical costs, caused environmental pollutions but also resulted in pesticide residue. Thus, the control of aphid had become a major problem on industrial production of chrysanthemum. Development of the genetic resistance to aphid infestation has become a priority in chrysanthemum breeding programmes. However, there was a few reports of studies on the aphid resistance of chrysanthemum. So, the purpures of this study was to explore the identification of the method of aphid-resistance in chrysanthemum cultivars, describe the aphid resistance mechanisms and develope the new aphid-resist germplasm of chrysanthemum via genetic engineering. We carried a series studies on artificial aphid inoculation test, compared different aphid resistant varieties of leaf epidermal surface morphology, antioxidant and defense enzyme activity in response to aphid infestation and try to transformation of chrysanthemum with LLA gene. The main results are follows:1. Chrysanthemum aphid resistance grade and different aphid-resistance to different cultivars.In order to discover aphid resistant germplasm, aphid number and aphid number ratio were investigated in 35 cut chrysanthemum cultivars via artificial aphid inoculation test. The results indicated that different chrysanthemum cultivars had different levels of aphid resistance. By 21 days after inoculation, the number of aphid varied upon to cultivars ranging from 0 to 267. The aphid resistance of chrysanthemum can be divided into five grades in terms of aphid number ratio, i.e., high resistance with an aphid number ratio of 0-0.25, moderate resistance (0.26-0.50), resistant grade (0.51-0.75), low resistance (0.76-1.25) and non-resistant grade (>1.25), respectively. Among 35 tested cultivars,15 cultivars represented by'Jinba','Fengcheju 1', 'Nannonggongfen' are non-resistant cultivars with aphid number ratio above 1.25. Four cultivars represented by'Xiaoli','Han 1', 'Nannongchaxiang' are cultivars of low resistance to aphid with aphid number ratio of 0.76-1.25. Three cultivars, 'Yangguang' , 'Mantouju' and 'Youxiang' , are aphid resistant cultivars (aphid number ratio,0.51-0.75). Two cultivars,'Han 5' and' Han 6', are moderately resistant to aphid (aphid number ratio,0.26-0.50), while 11 cultivars represented by'Han 4', 'Nannongzixuanzhang','Keiun' are highly resistant to aphid (aphid number ratio,0-0.25).2. Study on leaf epidermal micro-morphology of different aphid resistance cultivars, activities of antioxidant and defense enzymes in chrysanthemum cultivars of different aphid resistance in response to aphid inoculation.Using high-resistant cultivar'Keiun', morderate-resistant cultivar'Han 6'and susceptible cultivar 'Jinba' as material, a comparative analysis of structural characteristics of leaf surface (trichomes, glands, wax, etc.) and antioxidant and defense enzyme activity in response to aphid infestation, we found that high resistant cultivar'Keiun' had thicker, higher and denser trichomes, bigger and full glands, and more waxiness on the lower leaf epidermis than that on moderately resistant'Han 6'and susceptible'Jinba'. Preliminary inferred that aphid resistance in chrysanthemum had related to leaf trichomes, glands, wax content. After aphid inoculation, the activity of antioxidant and defense enzymes levels were significantly different. In resistant cultivars, superoxide dismutase (SOD), peroxidase (POD), ascorbic acid enzyme (APX) activity levels rising faster than susceptible cultivar and peaking earlier. APX activity has been significantly changed in resistant cultivars. Preliminary inferred that aphid resistance in chrysanthemum had related to leaf trichomes, glands, wax content. After aphid inoculation, the activity of antioxidant enzymes and defense levels were significantly different. In resistant cultivars, superoxide dismutase (SOD), peroxidase (POD), ascorbic acid enzyme (APX) activity levels rising faster than susceptible cultivar and peaking earlier. APX activity has been significantly changed in resistant cultivars and the activity of SOD, POD during 3 d to 7 d were observed in resistant cultivars of 'Keiun' and 'Han6' than in 'Jinba'. Polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) activity were enhanced in aphid inoculated plants compared with the control. It responsed increased fastly and peak earlier, and maintain high activity levels post-inoculation. We suggest that antioxidant and defense enzymes both contribute to aphid resistance of these chrysanthemum cultivars, it can be used as one of biochemical marker for aphid resistance of seedlings of cut flower in chrysanthemum.3. Transgenic Chrysanthemum Plants Expressing a LLA Gene via Agrobacterium-mediated.Using leaf disks of cut-flower chrysanthemum 'Jinba' as explants, the concentration of the appropriate antibiotic selection has been studied, The results showed that the leaf was very sensitive to hygromycin (Hyg) and carbenicillin (Carb),10 mg·L-1 of Hyg concentration had been completely inhibited differentiation and rooting of adventitious buds could be completely restricted with 20 mg·L-1. In actual research, concentration of Hyg for selecting was a little lower than critical concentration, so we chose 8-10 mg·L-1 as the optimal concentration of Hyg for Leaf disc regeneration and 20 mg·L-1 for root selecting. Also, the leaf was very also sensitive to Carb. The Carb concentration had a certain degree inhibition of chrysanthemum. As the concentration elevated, the effect of inhibition increased, but the rate of bud differentiation had declined. Carb concentration 500 mg·L-1 could completely control the growth of bacterium but inhibit the shoot regeneration severely. While the concentration declined with 300 mg·L-1, it could be restrain the bacterium completely but keep higher regeneration rate of 50%. So we carried with gradual-reducing bacterium controlling method, using 500 mg·L-1 of Carb concentration in the early stage, then declined to 250-300 mg·L-1.The LLA gene was successfully transferred into chrysanthemum via Agrobacterium-mediated leaf transformation, One hundred and five resistance bud were obtained. Fifty-four putative transformants of chrysanthemum were obtain after root screening. Polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) analysis showed that the LLA gene was successfully integrated into the chrysanthemum genome and expressed in seven transgenic lines. The artificial aphid inoculation test showed that the aphid resistance of different transgenic plants was different, and the rate of aphid population inhibition ranged from 12.2% to 76.8% with an average inhibition rate of 41.8% in transformed chrysanthemum. |
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