【作者】 牟会斌；

【导师】 张效平；

【作者基本信息】 南京农业大学 ， 园林植物与观赏园艺， 2001， 硕士

【摘要】 本文在综述国内外有关微型月季（Rosa chinensis. Minima或Rosa.roelletti）研究进展的基础上，以几个花色具代表性的微型月季品种为试材，研究了培养基成分及培养条件对微型月季组织培养中无菌系的建立、增殖与壮苗，瓶内生根与驯化及瓶外生根技术进行了系统的研究，同时还对微型月季的组培生根机理进行了初步探讨。 试验表明：以开花枝下具完整叶、带腋芽的茎段为外植体（外植体长度/直径组合为9.0～10.0mm/3.0-3.5mm，茎节上下各带5.0mm亲本组织）有利于腋芽诱导，将外植体用100mg/L庆大霉素溶液浸泡30分钟可有效防止内生菌污染。灭菌后接种于100ml三角瓶，每瓶接种4-9个外植体。诱导培养基为MS+6-BAlmg/L+NAA0.1～0.2mg/L+KT0.5mg/L，接种后先暗处理2天，然后转入1600—2000Lux的光强下培养，光照16小时。培养温度为20℃。 将腋芽再生芽转入MS+6-BA3～5mg/L+NAA0.1～0.2mg/L+蔗糖（生产上可用白砂糖）30～35g/L+琼脂6～6.5g/L的培养基上，培养基的pH值调至5.9。培养温度为22℃，光照800～1600Lux，光照16小时。在以后的继代增殖中以3芽为一转接单位有利于增殖。 将增殖和壮苗培养后的苗（苗高约20～25mm）转入1/2MS(将Ca²⁺浓度提高到1.5倍)+NAA0.2mg/L（或NAA0.1mg/L+IBA0.1mg/L）+蔗糖20～25g/L+琼脂7g/L+活性炭0.4g/L的生根培养基中，培养基的pH值调至6.0。培养温度在转接后的第一周为22℃，然后逐渐降到18℃。培养容器以试管最佳，生产上可用广口瓶。其它培养条件同上。 将培养6～8周、20～25mm高的苗取出，洗净基部琼脂后浸入50g/L的IBA溶液中5分钟，然后插入由蛭石和粗沙按1：1（v/v）混合而成的基质中，于自然散射光下培养，培养温度22℃。21天后将驯化成活的苗移入由园土、蛭石和珍珠岩按！：1：1（V／v／v）混合而成的基质中，其它栽培条件同驯化条件，并逐渐过渡到自然条件。 在瓶内、瓶外生根苗的比较研究中发现，不同的生根方式对植株生长发育的影响较大。经两种生根方式处理ZI 天后，瓶外所生的根较长、数量较多、横截面积较大，其中维管柬所占的比重较大，且维管束发育早，根的韧性好、白色、有侧根；而瓶内所生的根较细、易碎、黑色、没有侧根。除‘小红’外，所有瓶外生根的植株其根（横截面积和干重）和茎（高度、横截面积和干重）的生长量均比瓶内生根的植株多。所有瓶外生根的植株产花量增加，花期提前。 0 通过对微型月季组培苗生根过程中内含物的阶段性测定发现，生根部位的IAA含量在根原基出现前增加，而根原基出现后下降；iPAs和ZRs在根原基出现前略有下降，而在根原基出现后的分化过程中急剧上升；可溶性糖和淀粉在根原基出现前积累，而根原基出现后因消耗含量下降；可溶性蛋白在根原基发生和分化的过程中增多，而在根原基分化后根的伸长过程中含量下降；POD活性在根原基发生和分化的过程中增强，而在根原基分化后根的伸长过程中活性降低：外源IAA处理会加强以上指标的变化强度，从而有利于生根。因此，在组培苗的生根过程中，根原基的形成为生长素所刺激，而在根原基的分化和根的伸长过程中需低浓度的细胞分裂素与生长素配合，可溶性糖和淀粉等碳水化合物为根原基的分化和根的生长提供能源，在根原基的发生和分化过程中还伴随着一系列的生理生化变化，使蛋白质含量和酶活出现阶段性变化。更多还原

【Abstract】 By summarizing the research about Miniature Rose (Rosa chinensis. Minima or Rosa roelletti), this study aimed to select optimum culture medium and conditions for Miniature Rose with several representative varieties in flower colour, especially focused on the techniques of the establishment of aseptic cultures, proliferation, strengthening and the rooting in vitro and ex vitro of the plantlet. Further more, this article discussed the mechanism of the rooting during the tissue culture. At last, the tissue culture system of Miniature Rose was established as follows: 1. Establishment of aseptic cultures: the explant (9.0?0.0mm in length/3.O?.Smm in diameter, 5.0mm above/under the node) was node segment with one axillary bud under the intact leaf from the flowering stem. After being disinfected with 75% alcohol and 0. 1% HgCJ2 , the explants were dipped in to lOOmg/L gentamicin solution for 30 mm to eliminate the germs in, then were inoculated in 100m1 vessels, each with 4? explants. The inducing medium was: MS+6桞A lmg/L+NAA 0. 1?. 2mg/L +KT O.5mg/L. After inoculation, 1600?000Lux light intensity, 16梙s light period and temperature in 200C was succeeded with treatment of 2梔ay darkness. 2. Proliferation and Strengthening: The regenerated bud was transferred to the medium of MS+6桞A3?mg/L+NAA 0. 1?. 2 mg/L +Sucrose 30?5g/L+agar 6?.5g/L. PH5.9, Th22XD, LI (Light intensity) 800?600 Lux, LP(Light Period>l6hs. The proliferation of Miniature Rose has the quantity effect Three buds as a transferring unit resulted in the highest proliferating rate. 3. Rooting in vitro: The strengthened plantlet (height of 20?5mm) was transferred to the rooting medium of l/2MS(the concentration of Ca2~ was increased to 1.5 times)+ NAA 0. 2mg/L (or NM 0. lmg/L +IBA 0. lmg/L) +Sucrose 20?5g/L+agar 7g/L+AC 0. 4g/L. after being transferred, the temperature was 220C in the first week, than decreased to l8~C. Tube was the best container for rooting. Other culturing conditions were the same as above. 4. Rooting ex vitro: Cultured 6? week, the plantlet (with the height of 20?5mm) was taken out from the vessel, after washed off the agar round the end, the plantlet was dipped into JBA solution(50g/L) for 5mm and then was inserted to the medium mixed of peat and sand(l :1, V/V), growing under natural light with T=22 慍 . 2ldays later, transferred the acclimated plant to the medium mixed of loam, peat and perlite (l:i:i,V/V/V), slowly developed to the natural condition. By comparing in vitro and ex vitro rooted plant, we found that different rooting method affects the plant growth and development. 2ldays after these two rooting treatments, the ex vitro root was white, longer, larger in quantity and diameter, larger proportion of vascular, which developed earlier and had better flexible, and had branch root while the in vitro root was dark, thinner, brittle and without branch root. Except for the variety of 憀ittle red? the growth of root (diameter and dry weight) and stem (height, diameter and dry weight) of the ex vitro rooted plant were larger than those of in vitro rooted plant. 5. Discussion of the mechanism of rooting during the tissue culture: By determining the endogenous substance periodically during the rooting of Miniature Rose tissue cultured course, it was found that the content of IAA in rhizogenic zone increased before the formation of primordium and decreased after formation. While iPAs and ZRs slightly decreased before更多还原