地被菊是一种兼具绿化、美化、彩化和香化功能的菊花新品种群，株型低矮或匍匐，但匍匐型品种奇缺。本课题组近年来选育出了一批株型完全匍匐、茎节部着生气生根、生长旺盛的新品种，具有较高的园林应用价值。大量研究表明植物枝条的生长方向是光和重力调控茎生长的结果，大多数匍匐生长突变体都与其地上部的重力丧失或钝感有关。然而，目前关于地被菊匍匐生长特性形成的机理尚未见研究报道。本研究以匍匐型地被菊‘雨花金华’为试材，采用形态解剖学和生理学等研究手段，探讨了匍匐茎的向重性反应及内源激素和Ca2+在其匍匐性形成中的作用。主要结果如下： 1．地被菊匍匐生长性状是茎（顶芽下第3节间）响应重力刺激的结果。‘雨花金华’在生长初期呈直立生长，与其它类型品种没有区别。当茎生长到6-7个节位大小时，顶芽下1-2节开始向下弯曲生长，植株表现出匍匐性状。将植株放置在黑暗条件并进行90°重力处理，3h后就可以观察到匍匐茎开始弯曲生长，表明匍匐茎能够感受重力方向的改变而自动调整生长方向来响应重力刺激，其匍匐生长特性的形成不是由于向重性反应的丧失或钝感，而是一种响应重力诱导的结果，是横向重力性。 2．形态解剖学研究表明，‘雨花金华’匍匐茎与直立型菊花品种茎的解剖结构相似，由表皮、皮层、内皮层、维管束和髓组成，每个内皮层细胞中含有4-8粒淀粉体；匍匐茎负向重力侧各组织（髓除外）在茎中的比例显著高于正向重力侧，茎的负向重力侧和正向重力侧呈现不对称性生长；匍匐茎内皮层细胞中92.5%淀粉体位于细胞的物理学中部，而直立茎中的淀粉体则主要位于内皮层细胞的物理学底部；重力处理2 min时，内皮层细胞中的淀粉体开始发生向重力方向沉降，5 min时，88.5%的淀粉体沉降到细胞新的物理学底部，表明内皮层中的淀粉体在感受重力过程中发挥着重要作用。 3．采用ELISA法测定了植株匍匐生长前后各节间茎及横向重性弯曲茎中内源激素的含量和分布，采用IAA免疫组化定位方法对IAA在横向重性弯曲茎中的分布进行了研究；同时测定了重力刺激后匍匐茎负向重力侧和正向重力侧的生长变化，并结合适宜浓度的外源生长调节剂（NAA、TIBA和GA3）处理，观察其对匍匐茎向重性反应的影响。结果表明：内源激素在‘雨花金华’茎负向重力侧和正向重力侧中的分布呈不均匀性，而且这种分布梯度在植株匍匐生长前后发生改变。匍匐生长前，第3节间（发生弯曲生长的部位）负向重力侧中IAA的含量显著高于正向重力侧，为正向重力侧的1.6倍，而匍匐生长后，二者无显著差异，其比值接近于1.0；GA3在匍匐生长前茎的负向重力侧和正向重力侧中的含量无显著差异，匍匐生长后在茎正向重力侧中的含量显著高于负向重力侧；iPAs的分布在茎匍匐生长前后无明显变化。重力处理促进了匍匐茎负向重力侧的生长，处理24 h后，匍匐茎负向重力侧相对生长率为正向重力侧的3.9倍。对茎的解剖结构研究发现，负向重力侧表皮细胞长度是正向重力侧的1.5倍，说明匍匐茎感受重力刺激后，负向重力侧生长快于正向重力侧而导致其弯曲生长。在重力刺激3 h、匍匐茎发生明显向重性弯曲之初，IAA、GA3和iPAs已向弯曲茎的负向重力侧积累，含量分别是正向重力侧的1.8倍、1.3倍和1.8倍，分布梯度均显著高于重力处理之前。IAA免疫组织化学定位结果也直观地表明重力处理后更多的IAA分布在横向重性弯曲茎负向重力侧的表皮和皮层组织中。 NAA和GA3涂抹在茎的负向重力侧时，促进了匍匐茎的横向重性弯曲；涂抹在正向重力侧则抑制了横向重性弯曲，但GA3的效果不如NAA显著。TIBA作用与NAA相反。由此推测，内源激素在‘雨花金华’茎中的不对称分布与其匍匐性状有着密切关系，很可能是茎生长到6-7个节位大小、感受重力刺激后，内源激素在茎的向重性反应部位形成了一定的分布梯度，更多的IAA和GA3分布在茎的负向重力侧，从而使茎负向重力侧生长快于正向重力侧，导致茎发生横向重性弯曲生长；弯曲生长后，茎的负向重力侧和正向重力侧中的内源激素分布差异减小，使得茎得以保持匍匐生长。 4．采用ICP-AES法和焦锑酸盐沉淀的细胞化学方法，研究了匍匐茎向重性弯曲过程Ca2+在负向重力侧和正向重力侧中的分布及其在细胞超微结构中的定位与分布；研究了外源Ca2+和EGTA处理对匍匐茎向重性反应的影响，并采用ELISA法测定了外源Ca2+和EGTA处理后的向重性弯曲茎中内源激素的含量和分布。结果表明：匍匐茎的负向重力侧和正向重力侧中总Ca2+含量无显著差异，不存在不均匀分布，重力处理对Ca2+在向重性弯曲茎负向重力侧和正向重力侧的分布也无显著影响。Ca2+在茎细胞中主要定位于细胞间隙、细胞壁和液泡。重力处理前，向重性弯曲茎负向重力侧和正向重力侧表皮细胞胞壁中Ca2+分布无明显差异；重力处理60 min后，Ca2+在负向重力侧表皮细胞细胞壁中的分布明显低于正向重力侧。此外，重力处理之前，内皮层细胞胞质中Ca2+分布很少，重力处理5 min后开始有所增加，15-30 min时分布非常丰富，60 min后恢复到最初的水平。表明在表皮细胞和内皮层细胞中Ca2+在细胞超微结构的分布表现出对重力刺激的响应。 外源Ca2+处理促进了匍匐茎的横向重性弯曲而外源EGTA处理起到了抑制作用。进一步研究发现，外源Ca2+处理使IAA和GA3在横向重性弯曲茎负向重力侧和正向重力侧中的分布梯度加大，但降低了iPAs的分布梯度；外源EGTA处理抑制了横向重性弯曲茎负向重力侧和正向重力侧中IAA和GA3的分布梯度加大，对iPAs的分布梯度影响不大。我们认为，Ca2+参与了匍匐茎的向重性反应，Ca2+很可能是通过改变其在细胞超微结构的定位与分布来影响细胞壁的可塑性与细胞的伸长，引起负向重力侧和正向重力侧的不均匀生长；同时作为第二信使传递重力信号，调控内源激素在茎负向重力侧和正向重力侧的不均匀分布，与内源激素协同作用参与了匍匐茎的向重性反应。

Ground-cover chrysanthemum, a new variety population, is especially fitted for beautifing garden, including two types of low and creeping architecture. The recently developed creeping type is well accepted as a ground-cover garden species, thanks to its stolons similar to those of the strawberry. Abundant studies showed that the shoot growth direction are regulated by illumination and gravity. Many mutants with creeping habit have been identified to be related to loss or reduced gravitropism. But now there is few investigation to discover the creeping mechanism of the creeping ground-cover chrysanthemum. The cultivar ‘Yuhuajinhua’ was selected as experimental material to explore the gravitropical response of the stolon, the role of phytohormone and Ca2+ in the creeping habit formation via anatomical and physiological means. Results showed as below: (1) The creeping habit was resulted from gravity stimuli. shoots of ‘Yuhuajinhua’ grew erectly at the shoot early growth, with no difference to the other uncreeping chrysanthemum. But approx. 1-2 nodes below the apex began to curve and exhibit creeping habit after vegetative growth of 6-7 nodes. After the plants were placed horizontally, the stems began to bend downward within 3h. The result showed that the creeping stem can also percept and respond to the change of gravity vector. The creeping growth is a kind of gravitropism- horizontal gravitropism. (2) The anatomical analysis showed that ‘Yuhuajinhua’ stolons were similar to those cultivars without stolons, consisted of epidermis, cortex, endodermis, vascular bundle and pith, 4-8 amyloplasts contained in per endodermal cell. Except for the pith, the proportion of the various tissues present in the upper side of stem was higher than that in the lower side. Most of the amyloplasts (92.5%) lied in the centre of the endodermal cells of the stolon, rather than at the bottom, as is typical for non-creeping cultivars. When ‘Yuhuajinhua’ plants were oriented horizontally and kept in the dark, the endodermis amyloplasts sedimented in 2 min according to the gravitational direction, and 88.5% of them sedimented to the bottom of the cell. The results indicated that amyloplasts located at the endodermis play an important role in gravity perception. (3) The content and distribution of phytohormone in the different internodes were detected by ELISA before and after shoot creeping, also in the gravitropical bending stem. Immunocytochemical localization technique was carried to further analyze the distribution of IAA in the gravitropical bending stem. The elongation of the upper and lower side was also measured during gravistimulation. Exogenous growth regulators (such as NAA, TIBA and GA3) were layed on the upper and lower side of the crreping stem, respectively, to investigate its effects of the response of gravity stimuli. All results showed that there was asymmetric distribution of phytohormone in the upper and lower side of the stem, which changed before and after stem creeping. Before creeping, the content of IAA in upper side of the third internode,where the stem curved, was 1.6 folds to that in the lower side; but after creeping, there was no significant difference between them. GA3 was the same as IAA before stem creeping; but the content in the upper side became lower than the lower side after creeping. There was no different in the content and distribution of iPAs before and after creeping. The elongation of the upper side of the gravistimulated stem was increased, with the RGR 3.9 folds to the lower side after 24 h gravistimulation. And the length of the epidermal cell of the upper side was 1.5 folds to that of the lower side. It showed the gravistimulated bending growth was leaded by the asymmetric elongth of the two sides of the stem. Also, it was discovered that more IAA, GA3 and iPAs redisbuted to the upper side of the gravitropical bending stem after the 3 h gravistimulation. The gradient of them were promoted by gravistimulation. The Immunohistochemical localization of IAA was also observed that more distribution in the epidermis and cortex of the upper side. In addition, NAA applied to the upper side of the stem increased the gravitropical curvature, whereas applied to the lower side decreased the gravitropical curve. So did the GA3. The action of TIBA was on the contrary to the IAA and GA3. So it was supposed that there was close relation between the phytohormone gradient and the creeping growth of the stem. It should well be that the asymmetric distribution of IAA and GA3 leaded asymmetrical growth of the upper and lower side of the stem after percepted the gravistimulation, which caused the bending growth. When the stem become growing in creeping, the distribution gradient was shorten so as to keep the creeping growth. (4) ICP-AES technique and pyroantimonate precipitati reaction have been used to study the distribution of Ca2+ in the tissues and ultrastructure of cells of the upper and lower side of the bending stem upon gravistimulation. Gravistimulated curvature was measured after the creeping stems were treated with exogenous Ca2+ and EGTA, so did the content and distribution of phytohormone. Results showed that there was no significant difference in the total Ca2+ between the upper and lower side of the curved stem, which was unaltered by gravistimulation. Ca2+ mostly located in the intercellular space, cell wall and vacuole. Before gravity stimuli, the distribution of Ca2+ in the cell wall of the upper side was similar with that of the lower side of the bending stem. After 60 min gravistimulation, lower calcium pyroantimonate precipitation was observed in the cell wall of the upper side of stem. The distribution of cytoplasmic Ca2+ in the endodermal cell depended on the gravity stimuli, with an increase in the cytoplasmic Ca2+ occured after 5 min, keeping for 15-30 min. Exogenous Ca2+ promoted stolon gravitropic bending, whereas EGTA inhibited. Further evidence showed that exogenous enhanced the IAA gradient in the bending stem, whereas EGTA weakened. Exogenous Ca2+ promoted stolon gravitropic bending, whereas EGTA inhibited. Further evidence showed that exogenous enhanced the IAA gradient in the bending stem, whereas EGTA weakened. Taken together, it was proposed that Ca2+ was concerned with the asymmetric elongation of the upper and lower side of the stem upon gravistimulation, by changing the localization in the cell wall, which plays a crucial role in determining the structural rigidity of the cell wall. Ca2+ also acted as an second messenger to regulate the asymmetric distribution of phytohormone in the upper and lower side of the stem. It was evident that Ca2+ and phytohormone had cooperated actions in gravitropical response.