【作者】 范永梅；

【导师】 甘霖； 邓秀新；

【作者基本信息】 湖南农业大学 ， 果树学， 2003， 硕士

【摘要】 冰糖橙（Citrus sinensis Osbeck）是20世纪70年代湖南省选育出的优良甜橙品种，它以肉质脆嫩化渣、风味浓甜、少核或无核而深受消费者喜爱，在湖南省黔阳县和永兴县等地重点推广。进入20世纪90年代后，在部分产区由于柑橘碎叶病毒（Citrus Tatter Leaf Virus，CTLV）的感染等原因，使冰糖橙的单位产量下降，果实品质变劣，失去了原有品种的优良特性，市场竞争力下降。目前，由于冰糖橙组织培养方面的基础性研究的缺乏，很大程度上限制了生物技术在其品种改良中的应用。因此，建立冰糖橙胚性愈伤组织诱导及再生体系，并通过胚性愈伤组织培育出无病毒苗木，或进行冰糖橙品种改良，已成为目前冰糖橙生物技术研究中亟待解决的关键问题。 本研究以冰糖橙成熟果实中未发育胚珠为外植体，进行胚性愈伤组织的诱导和植株再生，并对再生植株进行了指示植物与A蛋白酶联免疫吸附法（PAS-ELISA）检测CTLV。建立了冰糖橙未发育胚珠培养获得无CTLV苗木的培养体系。主要的结果如下： 1．在不同的培养基和不同的培养条件下，冰糖橙胚性愈伤组织和胚状体的发生频率不同，麦芽提取物和GA₃有利于胚性愈伤组织的发生，暗培养有利于胚性愈伤组织的诱导。 2．与以往的研究结果不同的是，在诱导培养基中添加AgNO₃并不能促进冰糖橙胚性愈伤组织的形成，反而抑制了胚状体的发生；然而，在继代培养基中添加5mg／L的AgNO₃有利于小而弱的胚状体基部形成胚性愈伤组织，形成胚性愈伤组织的百分率达28％，经过多次继代处理建立了冰糖橙的胚性细胞系。 3．冰糖橙胚状体在EME500（MT+ME500mg／L）、EME1000（NT+ME1000mg／L）与MKBN（MT+KT 0.5mg／L+BA0.5mg／L+NAA 0.1mg／L）三种培养基上再生芽的比率不同，MKBN培养基分化的芽最多（达88.2％），EME1000次之（80.9％），第三是EME500（78.7％）。 4．对冰糖橙再生芽比较了试管生根和试管嫁接两种成苗方法。在1／2MT培养基上单独添加NAA（0.5mg／L）有利于再生芽生根，生根率达76.7％；以酸橙（Citrus aurantium）作砧木进行试管嫁接，其嫁接成活率可达88.9％。 5．对再生苗进行了CTLV检测：同时采用了指示植物腊斯克枳橙（Rusk Citrange）法与PAS-ELISA法检测，两种方法检测结果一致，均未检测到CTLV，可初步确定所获得的再生苗为无CTLV的苗木。 对利用未发育胚珠诱导胚性愈伤组织的优势和利用未发育胚珠培养培育无病毒苗木体系与热处理、茎尖培养、茎尖微芽嫁接获得无病毒苗木体系之间的优缺点、CTLV的检测技术以及无病毒苗木在柑橘生产中的应用价值进行了讨论。更多还原

【Abstract】 Bingtang orange (Citrus sinensis Osbeck) ( BT ), selected out from Hongjiang of Hunan in 1970s, was a very seedless sweet orange cultivar, and had been widely spreaded in the south of China. The yield and quality were getting down since 1990 because of infecting of citrus tatter leaf virus and the other reasons. It was very important to find a way by using biotechnology to overcome these problems. Present, as the lack of the fundamental research about the tissue culture, the application of biotechnology on the improvement of BT had been limited. Therefore, establishing the system of induction of embryogenic calli of BT and regeneration of plantlets, then obtaining the citrus tatter leaf virus-free plantlets or improving this cultivar are significant the sustainable development of the citrus industry.In this study, the undeveloped ovules of mature fruit of BT were used as explants to induce embryogenic calli and plantlets regeneration. What’s more, we identified the regenerated plantlets whether there were citrus tatter leaf virus in them by indicator (Rusk Citrange) and PAS-ELISA. The system of production of citrus tatter leaf virus-free plantlets from undeveloped ovules of BT was established. The main results are as follows:1. The regenerateratio of embryogenic calli and embryoids of BT varied on different medium and culture conditions (light or darkness). Malt extract, GAs and darkness were beneficial for induction of embryogenic callus.2. Silver nitrate was not beneficial to the induction of embryogenic callus, and it controlled the formation of embryoids. During the subculture, silver nitrate (5mg/L) was suitable for embryogenic callus from the embryoids, and 28 percent could be obtained. The embryogenic cell line of BT was established by several subcultures.3. The regeneration rate of plantlets from embryoids varied on different medium. The rate was higher (88.2%) on the MKBN (MT+ KT 0.5mg/L + BA0.5mg/L + NAA0.1mg/L) medium than on the EME1000 (MT+ ME1000mg/L)(80.9%) and EME500 (MT+ ME500mg/L)(78.7%) media.4. The rooting rate was 76.7 percent on the medium with NAA (0.5mg/L). Grafted on sour orange in vitro, the survival rate was 88.9 percent.5. The regenerated plantlets were identified whether there were citrus tatter leaf virus in them by using Rusk citrange as indicator and by PAS-ELISA. The results showed that the citrus tatter leaf virus-free plantlets of BT were obtained. Of course, it needs further study to examine citrustatter leaf virus by RT-PCR.The advantage and disadvantage of this system of virus-free plantlets from undeveloped ovules of citrus, the examining technique of citrus tatter leaf virus and the application of the virus-free plantlets in citrus industry were also discussed at the end of this paper.更多还原