【作者】 张晓曼；

【导师】 王志刚； 杨建民；

【作者基本信息】 河北农业大学 ， 森林培育， 2011， 博士

【摘要】 本研究以我国特有种小报春（Primula forbesii）为试验材料,系统研究其生物学特性;对小报春、胭脂花（Primula maximowiczii）、齿叶灯台报春（Primula serratifolia）、藏报春（Primula sinensis）、偏花报春（Primula secundiflora Franch）5种报春花属植物进行染色体核型分析;建立小报春组培快繁体系,并利用秋水仙素对小报春进行多倍体诱导,采用形态学、解剖学、细胞学、DNA含量、叶绿素荧光分析等途径进行多倍体鉴定,以诱导出多倍体小报春。对充分发挥和利用我国野生植物资源优势,保护、开发我国栽培名花和丰富园林花卉种类有重要意义。主要结果如下:1通过对小报春生物学特性研究结果表明:种子的最适宜储存方式为低温干燥储藏,室温储藏1a后多数种子失去生命力。赤霉素对提高种子萌芽率作用不大,但经赤霉素处理的种子发芽时间短,发芽整齐。种子适宜发芽温度为20²5℃。小报春喜弱酸性水分和土壤环境,土壤和水质酸碱度（pH=6.8）,利于种子萌发。以腐殖土∶泥炭藓∶细沙=2∶1∶1作为培养基质有利于种子萌发。小报春的花为异型花,是典型的异花授粉植物,花期在云南原生地为11月15日至3月26日,夜间无开花。晴天时小报春日开花高峰期均在11:15时～11:45时之间。阴天时小报春的花时较晴天时的花时更分散,而且开花高峰期向后延迟。小报春花粉属于3-拟孔沟花粉。长、短花柱花的花粉粒大小差异较大,短花柱的花粉粒较大,而长花柱的花粉粒较小,长柱型花有较长的花柱和乳突细胞,长花柱乳突约是短花柱的1.5倍,长花柱细胞的长度明显长于短花柱的细胞。长花柱花（♀）×短花柱花（♂）杂交组合或短花柱（♀）×长花柱花（♂）杂交组合均有较高的杂交结实率,长花柱花自交结实率为0。2 5种报春花属植物染色体核型研究结果表明:小报春染色体核型公式:K（2n）=2x=18m,臂指数51.65%,核型为“1A”型。偏花报春染色体核型公式:K（2n）=2x=20m+2sm,臂指数52.00%,核型为“1A”型。藏报春染色体核型公式:K（2n）=2x=20m+2M,臂指数53.42%,核型属“1B”型。胭脂花染色体核型公式:K（2n）=2x=22m,臂指数51.07%,核型属于“1A”型。齿叶灯台报春染色体核型公式:K（2n）=2x=22m,臂指数52.59%,核型属于“1A”型。3小报春组培快繁再生体系与多倍体诱导体系建立研究结果表明:用叶片作为外植体诱导愈伤组织形成快,质地紧密。激素浓度配比以NAA浓度为0.5mg·L^-1,6-BA浓度为3.0mg·L^-1配比时愈伤组织诱导率最高,为85.0%,并且愈伤组织形成较快,质地紧密呈嫩绿色,具有分化能力。最适合小报春丛生芽诱导的培养基激素配方为MS+NAA0.10mg·L^-1+6-BA1.0mg·L^-1（pH=5.8）。以1/2MS为基本培养基,NAA浓度为1.00mg·L^-1时能在最短的时间培育出最多的根,生根率为80.0%,根粗壮。利用秋水仙素处理组织培养过程中产生的丛生芽是诱导多倍体的有效方法。用0.10g·L^-1的秋水仙素处理小报春丛生芽48h,诱导效果最好,死亡率为15.0%,变异率为70.0%,为最佳处理组合。对变异植株进行细胞染色体鉴定最终的到了同源四倍体株系19株,其中13株来自秋水仙素对丛生芽诱导,6株是来源于秋水仙素对愈伤组织诱导而得到的。秋水仙素结合组织培养技术进行多倍体诱导获得多倍体植株的机率要比田间活体诱导效果显著。田间诱导没有得到四倍体植株。4二倍体与四倍体小报春形态及细胞学研究结果表明:四倍体小报春比二倍体株高变矮,叶柄变短变粗,叶片明显变宽、变厚、颜色深绿,叶片背面的绒毛变多变长。四倍体植株单花冠直径明显增大,花的萼片、黄色的花眼都较对照增大。果实较对照增大,差异极显著。四倍体植株的气孔保卫细胞长度和宽度明显大于二倍体,气孔、保卫细胞的大小和叶绿体数与倍性成显著正相关性。四倍体小报春体细胞中期染色体由36条中部着丝点染色体组成。臂指数为（51.32±0.76）%,核型公式为K（2n）=4x=36m,核型不对称性属1A型。采用流式细胞仪和PartecDPAC软件对二倍体和部分经染色体数鉴定2n=4x=36的植株进行了细胞DNA含量测定和分析,结果表明,染色体加倍的植株其叶片细胞中的DNA含量也相应增加了一倍。与染色体数的鉴定结果一致。5二倍体与四倍体小报春叶绿素荧光特性研究结果表明:四倍体小报春的叶绿素a、叶绿素b、叶绿素总含量及类胡萝卜素都高于二倍体小报春。在二倍体和四倍体中,四倍体的Fo小于二倍体,Fm、Fv、Fv/Fm、Fv/Fo和PI均大于二倍体。说明四倍体表现出较好的光合性能。更多还原

【Abstract】 This study uses P. forbesii, which is endemic to China as test materials, and holds a systematic research of its characteristics of introducing cultivation, and its biological characteristics of flowing, pollination, fruiting. And the research also analysis the karyotype of five primroses genera plants P.forbesii, P.maximowiczii, P.serratifolia, P.sinensis, P.secundiflora Franch.Establish the rapid propagation system of the group growing P.forbesii and use the colchicine to polyploidy induce the P.forbesii. Using morphological and anatomical,cytology,DNA content,chlorophyll fluorescence analysis and such kind of means to conduct polyploid appraisal the purpose is to induce a polyploid P. forbesii to improve its appreciation,enhance its resistance.With its domesticated success and cultivating new polyploid primroses varieties, to make full use of our country’s wild resources advantage, and have important meanings in the protection of resources, develop cultivation in China. Main results are as follows:1、The results of the Biological characteristics of P forbesii study indicate that: The fruit of P.forbesii is capsule, spherical, after dehydration the epicarp will craze membranously when mature, outside have 1-nerved calyx. Normally mature seeds are tiny, irregular hexahedral, their epidermis have mesh shape structure. The seeds of 1000-grain weight (0.118±0.0023) g. The seeds of the P.forbesii should be stored for dry at low temperature, if storage at the room temperature, after a year most seeds lose their vitality. GA3 in improving seed germination rate effect is not big, but the GA3 treatment of P.forbesii seed germination time is short, sprout neatly. The seeds of P.forbesii is suitable to make a spring at a germination temperature 20 ~ 25℃. P.forbesii is lean to weakly acidic water and soil environment. So in domesticated P.forbesii, the soil and the water should be improved to pH = 6.8 in order to favor the seed germination, make it normally grow up. In A. budding soil corrosion: peat moss: sand = 2:1:1 as train matrix helpful for P.forbesii’s seed germination, germinating capacities and sprout potential is obviously higher than other matrix. So in planting early introduction cultivation matrix should choose (budding soil corrosion: peat moss: sand = 2:1:1) as reproductive growth of soil matrix.P.forbesii are typical different flowers pollination plants.The bloom days of P.forbesii in Yunnan are from around November 15 to March 26, without night blossom. During Sunny days, the blossom peak ranges between 11:15~11:45. During the Cloudy days, the blossom hours are more scattered than during the sunny days, and flowering peak backward delayed. Through the determination of the pollen life force of P.forbesii, the pollen could be cultivated in 25℃, which has the highest rate, within 30min have pollen germination, cultivate 120min after, pollen germination rate can reach 48.92%, after 180min observation shows pollen germination rate can reach 62.87%, the influence of temperature and sucrose for P.forbesii pollen germination shows that the optimum temperature conditions is 25℃, sucrose concentration of 10%.When boric acid concentration is 10mg/L, P.forbesii pollen germination rate is the highest. P.forbesii has the best storage result when stored in low-temperature -20℃add desiccant. From the body pollen germination method is suitable for the life force test of P.forbesii. P.forbesii belongs to 3-fitting hole groove pollen. The long and short flower of P.forbesii has very different the pollen grains sizes, short styles of the pollen grains is larger, but long styles of the pollen grains is lesser, long styles have longer stigma and papillary cells, long styles papilloma covenant is of 1.5 times of the length of short styles, the long styles cells obviously longer than short styles cells. The Hybrid seed setting of Long styles flowers (♀)×short styles flowers (♂) or short styles (♀)×long styles flowers (♂) combinations are higher.2、The study of 5 kinds of primroses genus karyotype indicates: P.forbesii,Somatic chromosome number is 2n=2x=18, Brachial index is 51.65%, Karyotype formula is K(2n)=2x=18m, The karyotype asymmetry genera 1A. Chromosome of P.secundiflora Franch is larger, Centromere is more clear. Somatic chromosome number is 2n=2x=22, Brachial index is 52.00%, Karyotype formula is K(2n)=2x=20m+2sm, The karyotype asymmetry genera 1A. P.sinensis Somatic chromosome number is 2n=2x=22, Brachial index is 53.42%, Karyotype formula is K(2n)=2x=20m+2M, The karyotype asymmetry genera 1B. P.maximowiczii,Somatic chromosome number is 2n=2x=22, Brachial index is 51.07%, Karyotype formula is K(2n)=2x=22m, The karyotype asymmetry genera 1A.P.serratifolia Brachial index is 52.59%, Karyotype formula is K(2n)=2x=22m, The karyotype asymmetry genera 1A. except the P.secundiflora Franch, the karyotypes of other four plants are first reported.3、The result of the building of P.forbesii tissue regeneration system and rapid propagation autopolyploid induction system shows that use blades of callus induction formed fast, texture closely. Alexipharmic disinfection mode selection method of dealing with Numbers is 8, namely: detergent cleaning 20min, tap water flushing 30min, ShengGong soak, 7min 0.1% sterile water flushing 4~6 times. Hormone concentration ratio in olumes is 0.5 mg·L-1, 6-BA concentration ratio is 3.0 mg·L-1 when callus induction 85.0%, and the highest for the callus forms faster, texture closely in peak green, which has differentiated ability. The most suitable hormone formula of multiple shoot clumps induction medium for P.forbesii is MS + NAA0.10 mg·L-1 + 6-BA1.0 mg·L-1 (pH = 5.8). With 1/2MS for basic medium, NAA concentration would be 1.00 mg·L-1, which can produced the most root in the shortest possible time, rooting rate is 80.0%, and roots are very strong.It is an effective method in induction of polyploidy ornamental plants to use colchicine processing the clump sprouts during the process of tissue culture produces.With the 0.10% colchicine to process the clump sprouts of P. forbesiifor 48 h, which has the best inducing effect, the mortality rate is 15.0%, mutation rate is 70.0%, which is the best treatment combinations. Cell chromosome identification of the variant plants eventually results in strains of autotetraploid strains, with 13 strains from colchicine on clumps shoot induction and 6 strains are from colchicine on callus induction. The combination of colchicine and tissue growing skills will get much more polyploidy plants than in the field when using the polyploidy skills. In the field, there could not have induced tetraploid plants4、The study of morphology and cytology on P.forbesiidiploid and tetraploid shows that: tetraploid P.forbesii’s heights are more shorter, petioles are more shorter and thicker, leaves are more wide and thick, its color is dark green, and more longer hair in the back of the leaves than diploid P.forbesii. Tetraploid plants, the average leaf index is 1.09±0.07 in contrast with 1.38±0.24 of the diploid average leaf index, tetraploid single corolla diameter increased significantly with an average of (2.27±0.03)cm, in contrast with(1.24±0.55)cm of the average corolla diameter, and the flower sepals, Yellow eye increased as compared with the control. The fruits are Larger than the ones in control, the fruit of tetraploid has an average diameter of(0.89±0.07)cm, whereas the fruit diploid has an average diameter of(0.41±0.15)cm, the difference was extremely significant. Stomatal guard cells of tetraploid plants were significantly greater than the diploid in the length and width, diploid guard cell chloroplasts is an average of 18, the average number of tetraploid guard cell chloroplasts is 55, reached a significant difference. Stomatal, Guard cell size and chloroplast’s number and ploidy is a significant positive correlation. Diploid stomatal density is(94.20±8.56)/mm2, tetraploid stomatal density is(78.91±6.97)/mm2, the stomatal density on the existence of diploid and tetraploid are significantly different. By chromosome analysis of colchicine processed cell regeneration plants, we found that some of the regenerated plants are tetraploid (2n = 4x = 36). By karyotype analysis, tetraploid somatic of metaphase chromosomes consist of 36 metacentric Centromere chromosomes. Brachial index is (51.32±0.76)%, karyotype formula is K (2n) = 4x = 36m, the karyotype asymmetry is 1A. To further determine the doubling plants homozygous of ploidy, using flow cytometry and PartecDPAC to conduct diploid and part of plants which were chromosomes identified 2n = 4x = 36, measured and analyzed cellular DNA content. he results show that the leaves of the chromosome doubling plant, whose DNA content in cells in a corresponding increase doubled, which is consistent with the identification chromosome numbers.5、The preliminary study results of the chlorophyll fluorescence of P.forbesii diploid and tetraploid show that: Tetraploid P.forbesii’s chlorophyll a, chlorophyll b, total chlorophyll and carotenoids are higher than diploid’s. In diploid and tetraploid, the tetraploid’s Fo is smaller than diploid, but Fm, Fv, Fv / Fm, Fv / Fo and PI is higher than diploid, which indicates that tetraploid have better photosynthetic performance.更多还原