【作者】 薛建平；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2004， 博士

【摘要】 地黄（Rehmannia glutinosa L.）为玄参科多年生药用草本植物，其新鲜块根或块根的加工品作为鲜地黄、生地黄和熟地黄入药，是我国著名的“四大怀药”之一。由于在生产中长期采用块根营养繁殖，地黄易受多种病害的侵袭，其中以病毒侵害最为严重，通常田间感染率达100％，致使地黄品种退化，直接影响药农的经济效益，利用茎尖脱毒培养技术是解决地黄病毒病的最根本途径。然而脱毒苗繁殖系数小、成活率低、育苗周期长，运输极不便利，从而大大限制了其在农业生产上的推广。近年来，人们先后在试管中诱导出了半夏、马铃薯、芋等，试图尝试来替代试管脱毒苗，其中试管马铃薯的培养技术日趋成熟，完全可以替代试管苗作为种用材料，但试管地黄的研究国内外尚未见报道。另外，地黄属异花授粉植物，种子不能用来保种，生产上采用块根来留种，但同样易受气候、栽培条件及病虫害的影响，造成种质退化，甚至丢失。玻璃化冻存法是近年来发展起来的一种植物资源超低温保存方法，但地黄的低温保存研究尚未见报道。因此，本研究选用地黄“85-5”为试验材料，开展了试管地黄的诱导及茎尖玻璃化法超低温保存的初步研究。所取得的主要研究结果如下： 1．试管地黄的形成包括不定根的形成及块根膨大两个主要时期。块根的形成前提必须首先有不定根的发生，低浓度的6-BA和NAA配合使用可诱导植株产生不定根，以MS+6-BA2mg／L+NAA0.1mg／L效果较好。试验发现，在上述基本培养基中添加蔗糖能促进不定根膨大形成块根，和3％蔗糖浓度相比，在含5％和8％蔗糖的高糖培养基中，幼苗叶片深绿平展，苗不伸长，处于生长抑制状态，植株上形成白色短粗的不定根均可膨大形成试管地黄，且发育良好，说明高浓度的蔗糖对不定根膨大形成试管地黄起着至关重要的作用，本试验以5％为宜。在试管地黄的诱导过程中，不宜添加GA₃和活性炭。试管地黄诱导的最适培养条件为培养温度25℃，光照强度2000—3000lx，光照时间12h／d。 2．试管地黄的形态建成与内源生长物质的变化有关。IAA在不定根膨大初期含量呈现下降趋势，20d时开始缓慢上升。GA₃在转苗初期呈现较平稳的趋势，在25-30d时GA₃的含量迅速增长，而后则呈下降趋势，但依然处于较高水平。ABA的含量在10—20d块根膨大时呈缓慢上升趋势，20d—30d块根迅速膨大期迅速增长，30d以后含量较为平稳，较高的内源ABA含量有利于地黄块根的膨大。JA在不定根膨大初期处于较低水平，以后则一直呈缓慢的上升趋势，在试管地黄迅速膨大时期，JA在根中的含量最高，JA在地黄离体块根的形成过程中有可能起着极为重要的作用。 3．以无菌苗叶片为外植体，地黄在组织培养条件下的形态建成受内源生长物质的调控。试验观察到，在MS+6-BA2.0mg／L培养基中可直接诱导小植株的再生，这一过程中，培养初期内源IAA的含量略下降，7d后急剧增长，当不定芽形成后，I从的含量则呈现下降趋势;GA3的含量在培养1一14d呈增高趋势，14d后则急剧下降;JA的含量整体变化幅度不明显。在MS+6一BA 1.omg/L+ NAAo.smg/L培养基中可诱导形成试管地黄，此间IAA含量的变化与诱导植株再生的情况相反，呈快速上升趋势;G凡的含量先明显下降，而14d后上升至刚离体的水平;JA的含量在培养初期l一7d期间急速下降，当不定根形成时，又恢复到原先水平，14d后在不定根膨大形成试管地黄的过程中其含量迅速增长，21d时JA含量是叶片刚离体时的近5倍。两种培养基中叶片在l一14d培养期间，ABA的含量均下降，14d后再上升，但是叶片在诱导植株直接再生的过程中，ABA上升和下降的幅度明显大于叶片诱导形成试管块根时。试验结果说明，内源IAA和G凡对植株的再生起关键作用，而JA和ABA参与了不定根的诱导及其进一步膨大形成试管块根，并且有可能起着极为重要的作用。 4.试管地黄在形态解剖方面与自然条件下生长的地黄具有一致性，两者的成熟结构基本上相同。在横切面上，二者都分为周皮和维管组织两部分，周皮的构成（木栓层、栓内层、木栓形成层）基本一致，维管柱中各种组织细胞在结构或数量上均相似。因此，试管地黄在诱导过程中其结构上未发生明显变化。。 5.建立了地黄茎尖超低温保存的技术体系。切取10mlll长的地黄脱毒苗茎尖接种于含0.2一0.6mol/L蔗糖的MS培养基上，并添加l%一5%乙酞胺、5%DMSO;在黑暗、10℃条件下预培养l一5d后，切取0.5一3.snun长的茎尖，用60%P VS:在25℃下装载25min，然后用100护VSZ于0℃脱水处理10一50min，换入新鲜的PVSZ，迅速投入液氮（LN）中，24h后取出于40℃水浴中化冻，立即用1.Zmol/L蔗糖的液体培养基洗涤2次，每次10min，然后用氯化三苯基四氮哇（TTC）法检测成活率，或接种于MS+6一A o.3m目1+N AA 0.02m乡飞+3%蔗糖的固体培养基上，于25℃暗培养一周转至光下培养。结果表明，地黄茎尖超低温冻存后用TTC法检测茎尖最高成活率达75%以上，植株再生率达50%，再生后的苗与对照没有形态上的变异。更多还原

【Abstract】 Rehmannia glutinosa Libosch is perennial herbaceous medicine plant of scrophulariaceae family and known as one of four famous Huai Chinese traditional medicine. Its fresh tuberous root named fresh R. glutinosa and finished products named crude R. glutinosa or prepared R. glutinosa were used as medicine. R. glutinosa is easy to suffered by many diseases especially viruses that usually infect plants by 100% and lead to serious variety degeneration and decrease economic benefit of the farmers. Shoot-tip culture in vitro is the most efficient approach to produce virus-free plants. However, low propagation rate, long procedure, low survival ratio and difficulties in plantlets handling prevent this strategy from its popularization. In recent years, the microtubers ofPinellia ternate and potato, corm of dasheen have been induced in vitro aiming at to substitute virus-free plantlets. Especially virus-free potato micro tubers has proved a possible alternative in seed potato production. Up to now, there is no reports on tuberous root of R. glutinosa in vitro. In addition, it is unsuitable to conserve R. glutininosa with true seeds because it belongs to cross-pollilated plants. Vegetable propagation by tuberous roots is, therefore, commonly used for the germplasm conservation and commercial production. However, conservation through tuberous roots can be easily influenced by climate, cultivation conditions, insect pests and diseases, which leads to degenerationand even loss of the germplasm. Cryopreservation by vitrification is a hopeful method for conservation of plant gemplasm developed in recent years, but little attention has been payed on R. glutininosa. In the present research, the induction and formation of the tuberous root of R. glutinosa in vitro and cryopreservation of shoot tips were conducted with variety "85-5". The main results gained as following:1. Formation of tuberous root of R. glutinosa in vitro included two main phases: adventitious root initiation and tuberous root swelling. The formation of tuberous root occuredafter the adventitious root initiated that favoured by addition of low concentration of 6-BA and NAA and the best medium combination was MS + 2mg/L6-BA + 0.lmg/L NAA. With this medium, it was found that sucrose has functions of enhancing swelling of the adventitious roots to form root tubers. Comparing with 3% sucrose, When grew on the medium added with5% and 8% sucrose, the leaves of plantlets showed deep green and flat, shoot growth was partially suppressed, and short and white adventitious roots formed that became tuberous roots later. The results revealed that high concentration of sucrose played important role in adventitious roots initiation and tuberous root formation, and 5% sucrose was the best in the present research. The results also showed that 12 h daylength,2000-3000 Ix light intensity and 25 癈 were favourable for tuberous root induction while GA3 and active carbon were not necessary substances of the medium.2. The endogenous growth regulators related to morphogenesis of tuberous roots in vitro. Hereafter endogenous growth regulators were isolated and purified from roots of different growth periods, the results indicated that the contents of GA3, IAA, ABA and JA fluctuated during tuberous root development of R. glutinosa. The levels of IAA decreased in first 20 days followed by a slow increase. GA3 was stable in early growth stage but rose quickly from 25 to 30 days, then declined gradually but still maintain a relative high level. The level of ABA increased slightly in the first 10 to 20 days and then went up rapidly in the bulking phase of tuberous roots between 20 to 30 days and henceforth trend to stability. This demonstrated that high level of ABA was helpful to tuberous root swelling of R, glutinosa. The results also showed that JA might play a very important role in tuberous root formation since its level was low in the initial stages but increased to the highest level during tuberous root bulking.3. Taking the leaves of R. glutininosa plantlets as explants, en更多还原