【作者】 王茜龄；

【导师】 余茂德；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 桑树是重要的经济林木树种,桑叶是唯一能使家蚕正常新陈代谢的饲料:桑果,即桑椹,具有较高营养价值和保健作用,发展前景广阔。桑树多倍体特别是三倍体植株具有高产,优质,抗逆性强的特点,受到广大育种科技工作者的重视。三倍体桑树品种一般是由优良的四倍体与优良的二倍体杂交后,经选择、培育而获得。自然界的野生四倍体桑树极少,且野生性状强,经济性状较差,不能满足人工三倍体新桑品种选育亲本的要求,人工选配的二倍体杂种F₁植株,经秋水仙碱诱导获得优良四倍体桑植株,因有杂交优势及多倍体效应,具有较高的产叶量,可直接应用于生产,也可用作培育三倍体新桑品种的亲本。因此,四倍体育种亲本材料多由人工诱变创制获取。人工创造四倍体桑育种亲本材料,目前国内外主要采用物理的辐射诱变处理;化学的秋水仙碱诱导处理和生物的杂交技术获得,这三条途径主要在大田进行,受天气,季节影响,周期长。因此本论文主要进行两方面研究,一是采用桑树组织培养与秋水仙碱诱导相结合,在试管内诱导获取人工四倍体桑育种材料,拟克服大田实验的不足之处:二是在大田通过秋水仙碱诱导经杂交选育而成的二倍体桑品种优良单株的萌动冬芽,从中选育出果叶兼用的四倍体桑优系,拟解决农民栽桑经济效益单一的问题。并分析了该四倍体新桑品系与二倍体亲本间,在形态性状、解剖结构及生理生化上的变异,其主要研究结果如下:1.桑树离体高效再生组培体系的研究本研究以提高桑树离体再生植株频率,建立稳定的桑树组织培养技术体系为研究目的,以桑树种胚为材料,自来水冲洗,用0.1%的HgCl₂消毒8 min,无菌水洗4-5次,每次约1 min。消毒后种子放在摇床上或者平摊在灭菌过的培养皿中发芽。用解剖刀切割下胚轴和子叶,并且把下胚轴分成约1.5mm长的上、中、下三段作为外植体,接种在MS培养基附加3 mg·L^-1BA+0.3 mg·L^-1 IAA+30 g·L^-1葡萄糖+7 g·L^-1琼脂粉的培养基中,进行愈伤组织和不定芽诱导。实验结果表明,三段下胚轴之间的不定芽诱导率存在显著差异,下胚轴上段,即靠近胚芽的一段诱导率最高,获得了78.7±2.1%的不定芽诱导率:中段次之,不定芽诱导率为39.5±7.996；下段,即靠近胚根的一段褐化严重,基本上诱导不出不定芽。再以下胚轴上段为外植体,接种在MS培养基添加不同的生长素IAA、2,4-D、NAA、以及不同浓度激素组合中,进行愈伤组织和不定芽的诱导。实验结果表明,在桑树组织培养中,细胞分裂素3.0m g·L^-1 BA与生长素0.3 mg·L^-1 IAA组合对桑树不定芽诱导是最有效的,不定芽的诱导率达到77.6±5.9%；2,4-D诱导的愈伤组织生长快,白色疏松,较难分化出不定芽:AgNO₃对不定芽的诱导有促进作用。适当降低生长素的浓度有利于提高增殖培养的增殖系数。在生根培养中,无机盐的浓度和生长素对生根影响较大,较高的无机盐浓度降低生根率并且延长生根时间,无机盐浓度太低,根系柔弱,影响移栽成活率;在1/2MS+0.5 mg·L^-1 IBA的固体培养基中生根率可以达到99%。移栽成活率也可达到95%以上。该实验结果将为桑树离体条件下诱导多倍体育种材料奠定了基础,也为桑树遗传转化提供了良好的转化受体技术体系。2.桑树组织培养诱导多倍体育种材料的研究本研究以在桑树离体的条件诱导多倍体育种材料为目的,以桑种子、下胚轴、子叶、不定芽为材料,设计了4个实验:（1）用0.15%、0.2%、0.25%的秋水仙碱溶液处理中桑5801与纳溪桑的杂交F₁种子24h、48h、96h,然后接种子叶、胚轴到诱导培养基中。（2）用0.05%、0.1%、0.2%的秋水仙碱溶液处理子叶20min、40min、60min以后接种到诱导培养基中。（3）以2-4 cm长的不定芽分别在0.1%、0.15%、0.2%的秋水仙碱溶液中浸泡2天和3天,然后,用无菌水洗涤4-5次,转接入到新鲜的培养基中,无菌水浸泡作为对照。（4）以0.15%、0.2%、0.25%滴定不定芽的顶芽5天,早晚各一次,两周以后调查不定芽的变异率。实验结果表明,秋水仙碱溶液处理种子及子叶以后经过离体培养,都未获得再生植株；经过秋水仙碱处理过的不定芽生长缓慢,叶片发生畸变,经过体细胞染色体倍数性鉴定,进一步确定桑树四倍体及二倍体。另外,秋水仙碱的浓度是诱导桑树四倍体成功的关键,以0.2%的秋水仙碱浸泡2天后获得桑四倍体的诱导率为14%,以0.25%滴液5天获得了20%的诱导率。经过生根培养,移栽获得再生四倍体植株。该四倍体植株表现出叶片增大,锯齿加深,叶肉变厚,叶色深绿色等性状。既可以作为杂交亲本,也可以在生产上直接利用。这是国内首次在离体条件下,诱导获得的桑树多倍体植株,为桑树多倍体育种材料创制提供了一条新的途径。3.人工四倍体果叶兼用桑新品系的选育研究本研究以满足农民种桑需求,增加农民的亩桑产值,解决蚕农种桑养蚕收效单一,发展我国蚕桑产业为目的,以既产果,又产叶为主要育种目标,采用桑树细胞染色体工程,以我校保存的中桑5801号（二倍体,2n=2x=28）为材料,选育果叶兼用的新型人工多倍体桑树品种。经过观察、调查,从我校保存的中桑5801号中选出发芽早,结果多,生长势旺盛的优良单株,进行了繁殖。2005年2月22日至3月1日,分别用0.2%秋水仙碱溶液:2ppm6-BA+0.2%秋水仙碱溶液;2ppm6-BA+0.25%秋水仙碱溶液:2ppm6-BA+0.22%秋水仙碱溶液,每天早上8:00时,在桑树体细胞进行有丝分裂前一个小时,采用注射器,对11株,277个脱苞冬芽进行化学诱导处理,连续诱导处理8天,然后对诱导处理的冬芽进行培护管理。2005年5-6月,采用植物酶解去壁低渗法,对277个化学诱导处理冬芽的萌发新梢芽叶,逐个进行体细胞染色体倍数性鉴定。2005年12月20日-26日,我们把一个纯合四倍体（2n=4x=56）,2个嵌合体的V1代枝条上的冬芽分别进行了冬季芽接:2006年3月进行嫁接成活率调查,嵌合体的嫁接成活率略高于纯合体株系。2006年5-6月,我们对纯合四倍体（2n=4x=56）V1代嫁接成活的植株,采用植物酶解去壁低渗法,逐一进行体细胞染色体倍数性复查,V1代嫁接成活的15个植株,全为纯合四倍体（2n=4x=56）,命名为嘉陵30号。四倍体果叶兼用桑新品系嘉陵30号桑叶叶片增大,叶肉增厚,叶色加深,节间密,叶序紊乱,产叶量高:桑椹果粒大,紫黑色,果肉肥厚,味道鲜美可口,产果量也高。2006年12-2007年1月嫁接V2代冬芽,经过2007年培护管理,2008年重庆市桑树品种区域实验,桑椹产量达到777.5kg/667m²·年,桑叶产量达到2136kg/667m²·年。这一品种的育成推广,将增加农民收入,提高蚕农亩桑效益,增加蚕农收入,促使蚕桑产业可持续发展。4.桑树果叶兼用四倍体与无性系二倍体亲本形态性状及生化成分的比较分析本研究以了解桑树二倍体经过秋水仙碱诱导成四倍体以后,桑树形态性状,生化成分发生的变化为目的,以诱导获得的人工四倍体果叶兼用桑新品系及其无性系二倍体亲本为材料,从外形特征,桑叶产量,桑椹产量,桑叶蛋白质含量、可溶性糖含量、桑椹的氨基酸含量,抗氧化物酶活性作了比较实验。结果表明,四倍体新桑品系染色体数目增加一倍以后,桑叶的海绵组织增厚,桑叶单株产量提高19.05%,桑椹产量提高16.1%;桑叶蛋白质含量提高10.2%,可溶性糖含量提高10.7%;桑椹的氨基酸含量也明显提高,但不同的氨基酸含量增加的量不同,维生素C含量提高12.9%,多酚含量提高43.3%,总花青素含量提高34.1%,黄酮含量提高47.6%。四倍体桑叶可溶性蛋白质含量和抗氧化物酶活性都高于无性系二倍体亲本。据此推测,二倍体桑树染色体加倍以后,植株抗逆性有可能增强。其研究结果,为该人工四倍体材料在桑树多倍体育种中的应用,提供了较好的参考价值。5.桑树果叶兼用四倍体与无性系二倍体亲本光合生理特性及光合结构的研究光合速率是决定作物产量的重要因素,本研究拟从光合生理特性及光合结构阐明该多倍体桑表现出产叶量和产果量高于无性系二倍体亲本的原因为目的。以前面实验获得的人工四倍体新品系及其无性系二倍体亲本为材料,用便携式Li-6400光合测定仪测定光合速率日变化、光响应曲线及CO₂-光合曲线;石蜡切片,显微镜观察输导组织即导管和筛管:通过电镜观察气孔结构及叶绿体内部结构等方面进行研究。实验结果表明,四倍体桑在4月中旬的日变化情况与二倍体相似,均呈双峰曲线,峰值都出现在上午11:00和下午16:00左右,而四倍体桑的Pn全天都高于无性系二倍体亲本。四倍体桑的表观量子效率较无性系二倍体亲本高,分别为0.069和0.05;四倍体桑的光饱和点略小于无性系二倍体亲本,分别为329μmol·m^-2·s^-1和339μmol·m^-2·s^-1;饱和光下四倍体桑的光合速率高于无性系二倍体亲本,分别为21.4μmol·m^-2CO₂·s^-1和15.7μmol·m^-2CO₂·s^-1;四倍体桑的呼吸速率较无性系二倍体亲本高,分别为1.26μmol·m^-2CO₂·s^-1和1.16μmol·m^-2CO₂·s^-1;四倍体桑的CO₂补偿点较无性系二倍体亲本低分别为60.56μmol·m^-2CO₂·s^-1和67.41μmol·m^-2CO₂·s^-1;四倍体桑的CO₂饱和点比无性系二倍体亲本的CO₂饱和点低,分别为800μmol·m^-2CO₂·s^-1和900μmol·m^-2CO₂·s^-1,在饱和CO₂下其同化速率分别为31.57μmol·m^-2CO₂·s^-1,和31.78μmol·m^-2CO₂·s^-1;四倍体桑的羧化效率（CE）较无性系二倍体亲本高,分别为0.096和0.067。这些实验结果表明,四倍体桑的光合性能较无性系二倍体亲本具有明显的优势。四倍体桑叶绿素含量显著高了二无性系二倍体亲本,四倍体桑的叶绿素a,叶绿素b两种色素的含量均较无性系二倍体亲本高,充分表明四倍体桑较二倍体桑能更好地适应或利用不同的光质成分,其光合能力也相应增强。四倍体桑的RuBP羧化活性极显著高于无性系二倍体亲本,这可能是四倍体桑的净光合速率高于二倍体桑的重要原因。四倍体桑与其无性系二倍体亲本的这些生理差异初步阐明了四倍体产叶量及产果量较无性系二倍体亲本高的生理机制。从桑叶叶片和茎段的解剖结构来看,人工四倍体新桑品系的气孔变大,叶绿体的长度和宽度都明显高于无性系二倍体亲本,表现出巨大性。叶绿体内部淀粉粒增大,数量减少,基粒片层和基质片层都很丰富,基粒片层厚,并且垛叠疏松,排列有序,有利于光合速率的提高。无性系二倍体亲本的淀粉粒多,基粒片层薄,垛叠紧密,不利于光合速率。因而,无性系二倍体亲本的Pn偏低,这可能与叶绿体的结构有关。另外,四倍体桑的输导组织也有一定的变异,导管增粗,导管数增加。这些结构变异构成了人工四倍体新桑品系产叶量高、产果量高的结构基础。更多还原

【Abstract】 Mulberry is an important economical woody plant. Its leaf is the main diet for silkworm. Mulerry fruits are eaten fresh and are also used in marmalades, juices, liquors natural dyes and in the cosmetics industry. The plant has also been used medicinally. The black and red mulberry fruits are healthier for human body because of high nutrient value and medical function. So the mulberry variety for fruit shows a good prosperity in productional application. Mulberry polyploid, especially triploids is welcomed because they have high yield, high-quality, disease-resistant, resists the going against strong, fine properties that adaptability is wide,etc. Breeding of artificial triploid in mulberry has become an important approach at home and abroad. Desirable triploids are being developed using induced superior tetraploids for hybridization with diploids. Most of natural tetriplods are unsuitable for superior tetraploid parents due to poor nutritional value and low propagation efficiency. So we have to get by artificial creation. At present, the main methods artificial tetraploid are Co-γradiation-induced mutation, Colchicine-induced mutation, crossbreeding in the field and are affected by environment easily. Most of the tetraploids by artificial diploid hybrid doubled have high yield and applied to the production. It is still an important way for polyploid breeding in mulberry through crossing the tetraploid with diploid and breeding triploid hybrid combinations by using induced tetraploid. So this study contains two sections: one section is in vitro induction of tetraploid in mulberry, the other section is selective breeding of artificial tetraploid with high leaf-yields and high fruit-yieds by Colchicine-induced in mulberry and a comparative study on morphological, anatomical Structure of stem and leaf, physiological and biochemical mechanism difference between tetraploid and diploid of mulberry. The main results were briefly summarized as follows:1. Establishment of in Vitro and Plant Regeneration System of MulberryThe aim of this study is to establish a rapid and efficient plant regeneration system ofmulberry through mulberry seed embryo. The seeds were collected during April-May every year from mature tree of Zhongsang5801×Naxi （Moms multicaulis Perr.） and preserved in 4℃refrigerator for providing explants at any time throughout the year. Seeds were washed and soaked for one day, and then their surfaces were sterilized in 70% alcohol for 1 min followed by 0.1% mercuric chloride for 8 min. Finally, the seeds were rinsed for 4-5 times in sterile distilled water for 1 min . The sterilized seeds were cultured in sterile distilled water on the rocking bed or placed on the moist filter paper in sterile Petri dishes. Seeds were germinated at 25±2℃using a 12 h photoperiod with a light intensity of 50μmol m-2 s-1 with fluorescent lights （40 W GE Deluxe Daylight bulbs）. After 5 days, under aseptic conditions, germinated seedlings were cut with sharp scissors to produce segments of hypocotyls and cotyledons, the hypocotyl and cotyledonof every seedling was cut into three segments with 1.5mm long as explants. The result as follows: the optimum shoot inducing medium is MS+6-BA 3.0mg/L+IAA 0.3 mg/L+ dextrose 30 g/L + agar powder 0.4%. the optimum root inducing medium is MS+IBA0.5 mg/L. Addition of AgNO₃ to the medium promoted the differentiation percentage of （adventitious） shoots. shoot induction frequences of three segments of hypocotyl showed significant difference in the same media. The highest was the segment near the embryo bud 78. 7±2.1%, and the lowest was the segment near the embryo root 16±2.2%. The middle is 39.5±7.9%. the rooting rate was above 99.0% and the transplanting survival rate was above 95%.2. In Vitro Induction of Tetraploid in MulberryThe aim of this study is induction of tetraploid in mulberry in vitro. Seed, hypocotyl, cotyledon and multiple shoot was tested as explants. This paper designed 4 experiments: （1） seeds of crossbreed variety were treated with three different concentrations （0.15, 0.2 and 0.25%） of colchicine for 24h, 48h, 96h. （2） cotyledons were treated with three different concentrations （0.05, 0.1 and 0.2%） of colchicine for 20min, 40min, 60min. （3） multiple shoots（2-4cm） were treated with three different concentrations （0.1, 0.15 and 0.2%） of colchicine for 2 days or 3 days by impregnation （4） By using dropping liquid, multiple shoots（2-4cm） were treated with three different concentrations （0.5, 0.2and 0.25%） of colchicine for 5 days, twice every day. The result shows: tetraploidy plantlet induced only in multiple shoots; The combination of concentration of colchicine and explants might be the key factor. Tetraploidy at a frequency of 14% was obtained using 0.2% colchicine for 2d by impregnation, And the frequency of tetraploidy was 20% when 0.2% colchicine for 5d by using dropping liquid. The leaves of tetraploid that developed from colchicine treated buds were distorted while the upper newly formed leaves were large, thick and dark green, with stronger venation and deeper serration. The length and breadth of the leaves were also greater in colchicine-induced plants （20.4±0.9 cm and 17.1±0.1 cm） compared with that of untreated plants （16.7±1.1 cm and 12.8±1.7 cm）. The tetraploids can be used not only as tetraploid parents but also as practical variety. This is the first report on in vitro induction of tetraploid in mulberry in China. Thus, it provides a new and efficient path for mulberry polyploid breeding.3. Induction of Tetraploid in Mulberry for Fruit and LeafThis study is in order to meet peasant’s demand, increase income per hm² in economic benefit to develop secriculture industry. The objective is to select and breed new variety with not only high leaf-yields but also high fruit-yields in mulberry. Based on long term surveying on the local mulberry resources. Zhongsang 5801 is the better diploidy parent, Its leaf is bigger and good quality and its fruit has a nice taste. We injected colchicine solution into winter bud in mulberry for 8d by the concentration of 2ppm6-BA+0.2%,2ppm6-BA+0.22%,2ppm6-BA+0.25% on Feb 22,2005 After 1 month, we identified and separated 1 tetraploid bud from 277 injected buds and propagated the tetraploid by grafting and matched cultivation managemen in Dec,2005. The tetraploid plant of muberry showed better economic properties than the diploidy parents. The mean leaf yield reached a higher level to 2088kg/667m² and fruit yield reached 777. 5kg/667m² in Chongqing regional test from in 2008, 34.7% in leaf and 46.57% in fruit higher than that of the control variety "Hongguo 2" in 2008; So this way will increase the income of farmers and strengthen their enthusiasm of planting mulberry to raise silkworm. It leads to sustainable development of sericulture.4. Comparison of Growth and Physiological Indices between Diploid and Tetraploid Mulberry PlantsThe comparative study of growth and physiological indices between diploid and tetraploid mulberry plants were carried out. The difference were evaluated for leaf character, leaf yield, fruit yield, chemical composition, soluble protein content, total sugar content; Amino acid, Vc, polyphenol, anthocyanin and flavone of fruit and the activity of POD, SOD. The results have shown statistically significant diferences between tetraploid and diploids for all the parameters. mulberry leaf yield increased 19.05% and the mulberry fruit yield increased 16.1% of titraploid than of diploid ; the content of prtain and soluble sugar in the mulberry leaves was 10.2% and 10.7% higher than diploid; The anti-oxidative substance such as Vc, polyphenol, anthocyanin and flavone in tetraploid were 12.9%, 43.3%, 34.1%, 47.6% higher than in diploid. The activity of POD, SOD in tetaploid were significantly higher than that in diploid . So we speculated that Tetraploids induced from hybrid seedling diploid were much stronger growing and stronger stress resistance. The researches provided some useful information of polyploid breeding.5. Photosynthetic Physiological Characteristics and Photosynthetic Structure of Tetraploid and DiploidThe aim of this study is to elucidate the physiological mechanism of stronger growing in tetraploid than that in diploid. Under field cultivation conditions, Photosynthetic pigment （chlorophll-a, chlorophll-b） contents per unit area leaf of mulberry increased with increasing ploidy. There was significant difference between tetraploid and diploid. Net photosynthetic rate （Pn） of different ploidy watermelon showed（?）midday depression’ phenomenon in April. The Pn diurnal changes varied as atwo-peaked curve, its values reached at 11:00 am. and 16:00 pm.; The Pn of first peak was higher than the second. The Pn was 4x＞2x . The critical points of light saturation and maximum photosynthetic rate, apparent quantum yield （AQY）, carboxylation efficiency is 329umol·m^-2·s^-1, 21.4μmol·m^-2·s^-1, 0.069, 0.096 respectively in tetraploid and 339 umol·m^-2·s^-1, 15.7μmol·m^-2·s^-1, 0.05, 0.067 respectively in diploid. The tetraploid shows significant superior photosynthetic character. The abilitly of carboxylation of tetraploid was one time higher than diploid, which may be closely related to net photosynthetic rate.Observation of the morphology and anatomy of tetraploid showed that leaf palisade and sponge tissue thickened, the stoma become larger, the number of chloroplast in per guard cell increased. Convex lens observed that the number of starch grain in each cell reduced in tetraploid and the number of basal granules and basal granule layers of leaf of grape in greenhouse were higher than diploid. These basal granule layers are highly ordered and uniform to improve the photosynthetic rate. Forthermore, structure of conducting tissue was studied and the number of vessel increased and the vessel in tetraploid was bigger than diploid. These structural change constitute the physiological mechanism of improvement of yield in tetraploid.更多还原