【作者】 肖祖飞；

【导师】 许雪峰； 张新忠； 韩振海；

【作者基本信息】 中国农业大学 ， 果树学， 2014， 博士

【摘要】 利用优良的矮化砧木密植是实现集约化生产和经营的重要保障。与压条相比,绿枝扦插繁殖系数高、繁育周期短、生产成本低,是苹果矮化砧木快速繁殖的高效途径,而其中诱导茎发生不定根是扦插成功的关键。为了建立快速、高效的苹果矮化砧木绿枝扦插快繁技术,本文研究童性、IBA、H2O2对苹果矮化砧木绿枝扦插生根的影响。试验中以小金海棠(Malus xiaojinensis, MX)、SH1、SH6、M9、M26等苹果砧木为材料,通过绿枝扦插技术研究不同苹果砧木绿枝扦插生根能力及影响苹果砧木绿枝扦插生根的因子；选取童期小金海棠(J)、成年期小金海棠(A)、M9和M26茎尖分生组织作为外植体,研究连续茎段继代诱导苹果砧木返童提高绿枝扦插生根能力,得出如下结果：1、苹果砧木绿枝扦插生根率存在极显著的品种间差异,多数砧木不易生根。取自童期小金海棠(J)实生苗的插穗扦插获得最好的移栽率(94.00%),其次是B9(62.17%)、p22(52.17%)、MM106(36.67%)和CG80(32.50%),生根能力最差的是GM256、M7、M26和CG24。2、童性是苹果矮化砧木不定根形成的重要影响因子。取自童期小金海棠(J)实生苗的插穗和成年期小金海棠萌蘖(S)上的插穗绿枝扦插移栽率高达94.00%、93.35%,显著高于成年期小金海棠(A)(15.01%), SH1(16.00%)和SH6(34.67%)。3、氧化还原平衡态与苹果砧木绿枝扦插生根有关。不同浓度的H2O2处理未能提高插穗生根率,而IBA(3,000mg·L-1)+H2O2(50mM)处理显著高于其它处理,表明H2O2对IBA有增效作用。0.5mM DPI(二苯基碘,活性氧产生的抑制剂)能完全消除H2O2对插穗生根的促进作用,而DPI对IBA没有影响。4、通过连续茎段继代培养可成功诱导苹果矮化砧木返童。随着茎段继代次数的增加,小金海棠(A)、M9和M26叶片中内源IAA的含量、生根相关基因CKI1, ARRO-1、ARF7, ARF19和器官形成相关基因KNAT1的表达量显著提高,而内源ABA的含量、DNA甲基转移酶基因DRM2的表达量和DNA总甲基化水平逐渐下降。小金海棠(A)、M9和M26茎段继代到15代,小金海棠(A)叶片出现裂刻,表明小金海棠(A)、M9和M26在第15次继代成功返童,但小金海棠(A),M9和M26继代到第18和21代才出现不定根,生根率分别为42.48%,22.92%和74.28%。组培苗叶片IAA含量增加到～130(ng·g-1,鲜重)是小金海棠(A)、M9和M26生根的临界值。5、取自组培返童的田间苗(R)的插穗扦插移栽率显著提高。小金海棠(R)、M26(R)移栽率分别达到96.75%和81.82%,显著高于小金海棠(A)、M26(A)(F=35.18,F0.05=3.48)。6、5月份扦插生根效果最佳,好于6月和7月(F=552.85, F0.05=5.14)。7、河沙+草炭(v/v,1：1)为苹果砧木绿枝扦插适宜基质。结论：失去童性是使苹果砧木绿枝扦插不易生根的重要因子,通过茎段继代培养返童可使苹果砧木易生根。更多还原

【Abstract】 The application of apple dwarfing rootstocks is great guarantee to realize orchard garden’s intensive production and management. For perennial woody plants, cutting propagation is an efficient technique of rapidly propagating apple dwarfing rootstocks with a higher coefficient, shorter cycle and lower cost than layering, but inducing stem occur adventitious roots is the key to success for cutting. In order to establishing an efficient and rapid propagation system of apple dwarfing rootstocks cutting, in the paper, juvenility, IBA and H2O2were studied about the influence of rooting ability of apple dwarfing rootstocks.In the experiment, Malus xiaojinensis (MX), SH1, SH6, M9, M26etc. were used as material to study rooting ability of different apple rootstocks and influence factors of leafy cuttings rooting. Shoot apical meristem in adult reproductive phase MX, shoot tip meristems taken from apomictic seedlings of MX and adult phase M9, M26were used as explants. Research on serial subcultures induced rejuvenation of apple dwarfing rootstocks, and improve rooting ability of softwood leafy. The results are as follows:1. There are significant difference between varieties, the majority of apple rootstocks is not asy to root. The highest transplanting rates (94.00%) were observed in Malus xiaojinensis from apomictic seedlings (juvenile phase, J), followed by B9(62.17%), p22(52.17%), MM106(36.67%) and CG80(32.50%), and the worst is GM256, M7, M26and CG24.2. A lack of juvenility is the most important limiting factor governing adventitious rooting of leafy cuttings in apple rootstocks. Rooting rates of juvenile MX (J) from seedlings and juvenile like MX (S) from suckers were dramatically higher (94.00%,93.35%, respectively) than of adult materials MX (A)(15.01%), SH1(16.00%), SH6(34.67%) cuttings.3. Redox homeostasis was relate to rooting of apple dwarfing rootstock cuttings. The rooting ability cuttings were significantly higher in the treatment of3,000mg·L-1IBA+50mM H2O2than the other treatments. Treatments with H2O2alone, careless of concentrations, did not affect transplanting rates than water. Exogenous H2O2enhanced the effects of IBA on adventitious rooting and the enhanced portion of effect was abolished by H2O2inhibitor DPI (0.5mM). However, DPI didn’t diminish the effect of IBA on adventitious rooting.4. Serial subcultures in vitro caused successful rejuvenation of reproductively mature shoots. With the passages of subculture, the indole-3-acetic acid (IAA) contents, the expressions of rooting related genes CKI1, ARRO-1, ARF7, ARF19and the expressions of organ formation related genes KNAT1increased significantly in leaves of in vitro shoots derived from MX (J), MX (A), M9and M26. In contrary, the abscisic acid (ABA) contents, the expressions of DRM2gene and total DNA methylation levels decreased. After15passages of in vitro subculture, the micro-shoots of MX (A), M9and M26from adult phase explants were rejuvenated successfully, and the lobe on leaflets of MX (A) appeared. But the rooting ability of rejuvenated micro-shoots was recovered delayed at the18th or21st passage of subculture(42.48%,22.92%,74.28%respectively). The minimum IAA content threshold would be～130(ng·g-1fresh weight), at which level, the plantlets from MX (J), MX (A), M9and M26rooted well.5. Transplanting rates of leafy cuttings from tissue culture rejuvenated were significantly higher than from adult donor plants. Transplanting rates of leafy cuttings from MX (R) and M26(96.75%,81.82%, respectively) were significantly higher than from adult donor plants MX (A) and M26(A)(F=35.18, F0.05=3.48).6. The transplanting rates was also significantly affected by the cutting seasons. The transplanting rates of cuttings were much higher in May than in June or July (F=552.85, F0.05=5.14).7. Sand+peat (1:1, v/v) substrate is an suitable cutting substrate.Conclusively, a lack of juvenility is the most important limiting factor governing adventitious rooting of leafy cuttings in apple rootstocks. Serial subcultures in vitro caused successful rejuvenation of reproductively mature shoots and hence recover the ability of adventitious rooting ability of leafy cuttings.更多还原