【作者】 王子成；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2003， 博士

【摘要】 植物遗传多样性在农业可持续发展中具有重要的作用。柑橘是世界上最重要的果树之一，具有重要的经济价值。中国是柑橘的主要发源地，在长期的自然进化与栽培过程中，形成了很多具有重要或潜在价值的柑橘类型或品种。保存这些柑橘类型与品种对世界柑橘业的可持续发展具有重要的战略意义。传统的柑橘种质资源保存方法是田间种植保存。由于病虫害，自然灾害以及经费短缺等原因，使中国柑橘种质资源的保存受到严重威胁，大量的柑橘种质资源面临或已经流失。为了克服传统保存种质资源所面临的问题，我们开始进行柑橘种质资源离体保存的研究。所取得的主要结果如下： （1）对23个品种的成熟果实未发育胚珠进行离体培养，获得红肉脐橙等21个品种的胚状体和10个品种的胚性愈伤组织，并诱导成苗，为进一步进行离体保存研究提供了材料。以黔阳无核大甜橙为试材对柑橘胚状体再生成苗的培养基进行了优化，得到一介高效、广谱的柑橘胚状体诱导成苗的培养基，其成分为：MT+BA 2.0 mg／L+IBA 0.2 mg／L+GA₃ 0.5 mg／L+葡萄糖30 g／L。 （2）对成年态柑橘茎段的离体培养进行了研究。根据真菌孢子萌发后对杀菌剂更敏感的原理，建立了一种新的外植体消毒方法，其基本程序为：先将材料进行初步消毒，放置2天后，再进行第2次消毒，然后将材料切割成0.5 cm长带一芽的纵切茎段，再进行培养。用此方法，红肉脐橙等4个柑橘品种成年态茎段初代培养平均污染率21.07％，成功率达到71.01％。这解决了成年态柑橘离体培养时污染严重的问题，为进一步建立成年态材料的离体培养体系扫清了障碍。对成年态柑橘茎段初代培养的培养基进行了优化，最佳培养基为MT+葡萄糖30 g／L+BA 2.0 mg／L+IBA 0.1 mg／L+GA₃ 0.5 mg／L+CH 500 mg／L。在成年态柑橘离体培养继代过程时，发现逐步去掉老组织可以避免愈伤化问题，从而初步建立起成年态柑橘材料的离体培养体系，并将其应用于成年态印度酸橘与飞龙枳的体细胞杂种及柑橘近缘属植物九里香和澳洲指橘茎段的离体培养，均获得了成功。 （3）对红肉脐橙等柑橘试管苗的常温保存（25±2℃）和低温（5±℃）保存进行了研究，分别将柑橘试管苗的继代时间延长至13个月和2 a，在进行低温保存时，发现棉塞封口易导致材料的污染。华中农业大学2002届博士论文 （4）对柑橘原生质体的超低温保存进行了研究，结果表明，柑橘原生质体超低温保存时，PvS处理时间以3 min为佳，玻璃化溶液中添加小牛血清蛋白对超低温保存有利，所试的3个品种超低温保存后成活率存在着差异，对伏令夏橙不同倍性的细胞进行超低温保存，其成活率与倍性成负相关。超低温后的原生质体可以进行分裂，并进一步形成细胞团。 （5）对柑橘茎尖玻璃化法超低温的基本条件进行了研究，初步建立起柑橘茎尖的玻璃化法超低温保存体系，并对其进行了改进，第一次将自由基清除剂GSH应用于茎尖的超低温保存实践，并在栽培柑橘品种茎尖超低温保存中获得了成功，12个品种平均成活率为84.05%，再生率平均达到77.93%，这在柑橘栽培品种茎尖的超低温保存中是首例报道。 （6）对几种柑橘类型的成年态茎尖的超低温保存进行了研究。红肉脐橙等4个柑橘栽培品种的成年态茎尖超低温保存平均成活率达到56.93%，再生率平均达到46.93%。成年态的印度酸橘与飞龙积的体细胞杂种茎尖超低温保存平均成活率达到95.0%，再生率达到92.0%。柑橘近缘属植物九里香和澳洲指橘茎尖超低温保存后，分别获得了91.43%和66.67%的成活率，有88.87%和40.35%再生植株。这在成年态柑橘栽培品种、体细胞杂种和近缘属植物茎尖超低温保存中均为首例报道。 （7）对来自于华农本地早同一株母树的成熟果实未发育胚珠进行离体培养，获得32棵试管苗，将再生植株与母株进行流式细胞分析和AFLP分析，发现1株为三倍体，其它31株再生植株与母株没有检测到变异情况的发生，说明用成熟果实未发育胚珠培养通过胚状体途径再生植株可以作为离体保存的材料来源。 （8）用AFLP标记对连续继代Za的来自于同一个胚状体的红肉脐橙60个试管苗的遗传变异情况进行了分析，发现了广泛的AFLP带型变异，其变异频率达到巧.0%（9/60）。对超低温保存后再生的柑橘与对照进行了遗传分析，发现经过玻璃化法超低温保存的积壳发生了DNA脱甲基化变异，验证了前人的结果，经过改良的方法超低温保存的439橘橙再生苗却没有检测到脱甲基化，只是发生了甲基化模式变化。更多还原

【Abstract】 It is the need for the sustainable development to keep the biodiversity. Plant genetic resource plays an important role in the sustainable development of agriculture. Citrus is one of the most important fruit trees in the world and has important economic values. China is the major origin place of citrus. During the natural evolvement and culture, many types or cultivars with important or potential values have been formed. It is strategic for the world citrus industry sustainable development to keep those citrus types and cultivars. Planting in the open field is the traditional method for the conservation of citrus germplasm. Citrus germplasms conservation in China is imperi led because of diseases and insect pests, natural disasters and shortage of fund. Many citrus germplasms are facing loss or have been lost. In order to settle those problems, we began to study citrus germplasm conservation in vitro. The major results are as follows:(1) Embryoids of 21 cultivars or types of citrus such as cara cara and so on, and embryonic calli of 10 cultivars have been obtained through culture in vitro of undeveloped ovules of 23 cultivars, as provided materials for the conservation in KJtmThe medium for citrus embryoids forming plantlets has been optimized. An effective and broad-used recipe for regeneration has been established. The optimal medium is: MT + BA 2. 0 mg /L + IBA 0.2 mg/L + GA30.5 mg/L + glucose 30g/L.(2) Approaches for in vitro culture of mature citrus stems were conducted. A high efficient method based on the principle of the resistance of fungi spores to disinfectant becoming weak after germination to obtain disinfected mature explants has been developed. The explants were kept for 2 d in culture room after the primary disinfection and were disinfected for the second time. Then the materials were cut into stems of about 0. 5 cm long with one bud and inoculated in the medium for germination. Four citrus cultivars had been tested and the average contamination rate and success rate were 21. 07% and 71. 01% respectively. The medium for primary culture of mature citrus had been optimized, and the best medium was MT + glucose 30 g/L + BA 2. 0 mg/L + IBA 0. 1 mg/L + GA3 0. 5 + CH 500 mg/L. Getting rid of the oldtissue gradually during subculture could avoid forming of callus.(3) We have studied the conservation of citrus plantlets at normal temperature (25 2 and with a 14 h photoperiod provided by daylight-type fluorescent lamps) and low temperature (5 1 with a 16 h/d photoperiod, 11 mol/m2 s) conditions. The result showed that the sub-culture times of Cara cara navel orange could be prolonged to 13 months and 2 years respectively and the materials conserved at low temperature were easy to be contaminated when cotton plugs were used to seal the tubes.(4) Cryopreservation of citrus protoplasts has been studied. Results showed that the best treatment time of PVS was 3 min and bovine serum albumin added in PVS was favorable to cryopreservation of protoplasts. Protoplasts derived from different cultivars embryogenic calli have different survival rate after cryopreservation. The survival rates of protoplasts of different ploidy citrus sinensis (L.) cv. Valencia varied, the diploid cells was the highest and the hexaploid the lowest. Protoplasts after cryopreservation could divide in BH3 medium and could form colonies.(5) We studied and improved the basic conditions for citrus shoot-tips cryopreservation by vitrifiction. The role of additive GSH (reduced form of glutathione) in cryopreservation of citrus shoot-tips using the vitrification procedure was investigated for the first time. The results showed that GSH was useful in cryopreservation citrus shoot-tips. The average survival and regeneration rate after cryopreservation of 12 cultivars were 84.05% and 77.93% respectively.(6) Mature shoot-tips of four citrus cultivars have been cryopreserved and the average survival and regeneration rate were 56. 93% and 46. 93% respectively. Mature explants of somatic hybrid of Citrus reticulata +Ponci更多还原