为了建立栓皮栎体胚遗传转化体系,以未成熟合子胚诱导的体胚为转化受体,采用带有pCAMBIA1301质粒的根癌农杆菌菌株EHA105,其中含有hptⅡ和GUS基因,研究不同因子对遗传转化的影响.结果表明:选择培养中适宜的抗生素浓度为300 mg/L头孢霉素和30 mg/L潮霉素.预培养时间为15 d时,转化率达91.7％;菌液浓度为OD600=0.5,乙酰丁香酮浓度为200μmol/L时,转化率可达100％.在添加潮霉素的选择培养基中培养4周后,组织化学染色和PCR检测证实,GUS报告基因已经转入到体胚中并正常表达.

选用幼龄、成龄、老龄栓皮栎在幼年期和成年期的叶片,研究成熟效应过程中蛋白质及组分变化规律.结果表明,蛋白质含量随成熟效应呈现由高到低的变化趋势为幼年期＞成年期,幼龄＞成龄＞老龄;不同蛋白质组分在成熟效应过程中变化动态和含量都存在差异,清蛋白含量逐渐增加,出现了27.0、61.8 kD与104.8 kD的清蛋白条带,球蛋白、醇溶蛋白含量逐渐减少,14.4、28.4、53.4 kD与66.0 kD的球蛋白条带和53.4 kD的醇溶蛋白条带消失,谷蛋白含量无明显变化,4种蛋白组分含量表现为清蛋白＞球蛋白＞醇溶蛋白＞谷蛋白.

The teaching process of breeding of ornamental plants was reformed by timely updating teaching content,creating teaching environment,improving teaching method,strengthening practical teaching,flexible assessment,so as to improve students' interest in learning and ability of solving problems,which has significance on training students' innovation ability.

植物体细胞胚胎发生是一个复杂的过程,为进一步揭示胚性愈伤组织诱导的潜在能力,以栓皮栎未成熟合子胚诱导产生的胚性愈伤组织和非胚性愈伤组织为材料,通过对可溶性糖、淀粉、可溶性蛋白、游离脯氨酸、总酚含量和蛋白酶活性的测定,研究体细胞胚胎发生过程中2种愈伤组织生化特性的差异.结果表明,胚性愈伤组织中可溶性糖、淀粉、可溶性蛋白质及脯氨酸含量分别为97.6、25.3、28.3 mg·g-1和202.8 mg·g-1,均高于非胚性愈伤组织;两者的蛋白酶活性无显著性差异;但非胚性愈伤组织中多酚类化合物的含量显著高于胚性愈伤组织.

A somatic embryogenesis protocol for plantlet regeneration of Cornus walteri from hypocotyl explants was established. Embryogenic calluses were induced on MS medium containing 3% sucrose, 0.3% gellan gum, 800 mg l(-1) casein hydrolysate, 400 mg l(-1) L-glutamine, 2,4-D, BA, and thidiazuron after 4 weeks of incubation in darkness. High induction frequency (>80%) of embryogenic calluses was achieved with 13.5 mu M 2,4-D. Somatic embryos subsequently developed on a medium with 0.44 mu M NAA and 0.44 mu M BA, yielding 47.1% induction rate. The optimum medium for somatic embryo maturation and secondary somatic embryo production was MS medium containing 0.22 mu M NAA and 0.44 mu M BA. Explant genotype had a significant impact on somatic embryos production and maturation. A typical mature somatic embryo consisted of two large cotyledons and a short embryo proper. Approximately 90% of selected mature somatic embryos germinated and 62.2% converted into plantlets on plant growth regulator-free germination medium that was similar to our induction medium, but eliminating casein hydrolysate and L-glutamine. Histological observation of explants at various developmental stages of somatic embryogenesis revealed that somatic embryos passed through early globular-, globular-, heart-, torpedo-, early cotyledonary-, and cotyledonary-stages, which was similar to the development of a zygotic embryo in vivo.

ISSR reaction conditions of Quercus variabilis were optimized systematically via single-factor experiment by using the tender leaves of Quercus variabilis as test materials,resulting in establishment of optimal ISSR reaction system and thermal cycling conditions of Quercus variabilis.The results showed that the optimal ISSR reaction system included a total volume 25μL containing 2.5μL 10×Buffer,30ng template DNA,1.5UTaq DNA polymerase,2.5mmol/L MgCl2,0.6μmol/L primer,0.3mmol/L dNTPs.Thermal cycling conditions were as follows:denaturation 1min at 94℃;35cycles of 30sat 94℃,60sat 51.5~59.0℃,2min at 72℃;and a final extension of 5min at 72℃at the end of the amplification.Based on the optimized reaction system,12primers with superior stability and polymorphism were selected from 100ISSR primers.166bands were amplified in 22individuals of the natural population in Shaanxi Province,among which 142were polymorphic loci,the percentage of which was 85.54%,Nei's index 0.2928and Shannon's information index was 0.4381,indicating the relatively higher genetic diversity within population of Quercus variabilis.

We studied the effect of types of explants and plant growth regulators on callus induction, the effect of plant growth regulators on adventitious shoot formation and rooting, and developed the protocols for plant regeneration via adventitious shoot organogenesis in Notholin hyacinthinum preliminarily.Different explants could induce callus formation.However, there was significant difference in induction percentage, in which the explant derived from scale leaves produced up to 90.5%. The optimal medium for callus induction was MS medium with 0.5 mg· L-1 NAA, and the medium supplemented with 0.1 mg· L-1 NAA was more suitable for callus proliferation.The highest frequencies of adventitious shoot formation (89.8%) was on the MS medium supplemented with 0.1 mg· L-1 TDZ, but the basal medium MS supplemented with 1.0 mg· L-1 6-BA and 0.1 mg· L-1 NAA was in favor of the shoot proliferation and growth.The highest rooting percentage was 91.4%, and the excellent development root systems were in 1/2 MS medium supplemented with 1.0 mg· L-1 NAA.

Taking the immature zygotic embryo and radicle of Platycladus orientalis as test materials,the effects of basic medium,different explants,plant hormones,zygotic embryo developmental stage in callus induction were studied.The results showed that immature zygotic embryo and radicle could induce on optimal medium.The optimal combination of medium for immature zygotic embryo was DCR+2.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.5 g/L glutamine,the inducing rate was 96.67%.The optimal combination of medium for radicle was DCR+1.0 mg/L 2,4-D+0.1 mg/L 6-BA+0.5 g/L glutamine,the inducing rate was 93.33%.The best period of immature zygotic embryo sampling was around July 3.

An investigation was carried out on the variations of the nitrate reductase,soluble protein,chlorophyll as well as soluble sugar of 5-year-old plus trees from open pollinated progenies of Quercus variabilis populations.The results showed that there were significant differences in the nitrate reductase activity,the content of soluble protein,the chlorophyll content and the content of soluble sugar among different populations(p0.05).Significant seasonal variations were observed within the same provenance.In July,several physiological indices of families reached their maximum values,and significant differences were found among different families.There existed significantly positive correlations between sapling tree height and other indices,such as the nitrate reductase activity,the content of soluble protein,the chlorophyll content and the content of soluble sugar,and the corresponded correlation coefficients were 0.547,0.643,and 0.492,respectively,which could be used as physiological indices in early selection of Q.variabilis.

Using leaves of Cornus walteri as material,ISSR reaction conditions of Cornus walteri were optimized systematically via single factor with two circular tests,and the optimal ISSR reaction system and thermal cycling conditions of Cornus walteri were established.The results showed that the optimal ISSR reaction system included a total volume 25μL containing 2.5μL 10×Buffer,50ng template DNA,1.5UTaq DNA polymerase,2.0mmol/L MgCl2, 0.6μmol/L primer,0.15mmol/L dNTPs.Thermal cycling conditions were as follows:denaturation 5min at 94℃; 38cycles of 50sat 94℃,60sat 48.8~59.1℃,1.5min at 72℃;and a final extension of 10min at 72℃at the end of the amplification.Based on the optimized reaction system,17primers with superior stability and polymorphism were screened out from 100ISSR primer candidates.Subsequently,annealing temperatures of selected primers were subjected to further optimization.As a consequence,the optimized system in this study may provide technological base and reference for identification of germplasm resources,genetic diversity and molecular breeding of Cornus walter.

Germplasm resources for the genus Juglans(Juglandaceae) are rich,and the genus contains many tree species,commonly called walnuts,that are valued for their high quality timber and nuts.The CTAB method was improved to extract genomic DNA from walnut buds,fresh and dried leaves.Using the obtained DNA as templates,the important parameters influencing the application of RAPD-PCR for walnut species were optimized.The results indicate that the modified CTAB method more effectively removed most of the polysaccharides in the cytoplasm and inhibited the formation of oxidized polyphenolic compounds,compared with the traditional CTAB method.The optimal RAPD-PCR reaction conditions for Juglans cinerea,Juglans ailantifolia,J.nigra,and species hybrids included,in a total volume 20 μL,1μL of 10 ng/μL DNA,2 μL of 10×Taq DNA polymerase reaction buffer(1.5 mmol/L Mg2+),2.5 μL of 200 mmol/L dNTPs(0.25 mmol/L),2 μL of 1mg/mL BSA(bovine serum albumin,0.1 mg/mL),2 μL of 25 mmol MgCl2(2.5 mmol),4 μL of 50 mmol RAPD decamer primer pair,0.5 units Taq polymerase,and 5.0 μL sterilized distilled water.Thermal cycling conditions were as follows: denaturation 3 min at 92℃;35 cycles of 1 min at 92℃,1 min at 35℃,2 min at 72℃;and a final extension of 10 min at 72℃ at the end of the amplification.

The shoots of Populus canescens and P.alba× P.glandulosa cv.84K in vitro were used as materials to study the effects of different cadmium levels on the growth of plantlets and the activity of antioxidant enzymes,and to reveal the difference of resistance between these two poplar varieties.The results showed that the growth of the plantlets was promoted at low Cd concentration(50 μmol·L-1) and was inhibited at high Cd concentration(200 μmol·L-1).The SOD activity and CAT increased at first and then decreased with the increase of Cd concentration,and the POD activity gradually increased.Compared with P.canescens,under Cd stress,the comprehensive growth indices and the activity of enzymes of poplar 84K were lower,indicating that P.canescens had a higher capacity of antipollution of cadmium than poplar 84K.

The objective of the paper was to establish an AFLP reaction system fitting for Quercus variabilis,after systematical study on several crucial aspects involving the process and time of the endonuclease digestion-ligation reaction,the preamplification and selective amplification.The results indicated that the extraction of the genomic DNA should choose the improved CTAB method from the tender leaves of Q.variabilis.One-step method,200 ng DNA dosage and digestion and ligation 4 hours were the best combination for the endonuclease digestion-ligation reaction system The preamplification reaction was carried out using 20 diluted digestion and ligation products.And the selective amplification was conducted with no diluted preamplification products immediately.The effectiveness test in each step and primer screening test demonstrated that the system was suitable for the AFLP analysis of Q.variabilis.

Effects of vitrification and cryopreservation on the preservation of embryogenic tissues in Quercus variabilis were investigated.Different factors involved in the preservation on cells viability were studied.The results showed that the viability of cryopreserved materials was affected by the concentration of cryoprotectant and the treatment time.The cryoprotectant treatment time and the melting methods were very important to the survival of cryopreserved materials.The materials were percultured for 3 days at 5 ℃ on the MS medium that contained 6% DMSO after subculturing for 10 days,used 60% PVS2 to treat 20 min and 100% PVS2 to treat 30 min at 0 ℃,then,put into liquid nitrogen rapidly after changing the solution with fresh PVS2,cultured on the MS medium after melting at 40 ℃ water bath,and the cells could resume growth.

【Objective】 The research was done to improve regeneration and proliferation abilities of somatic embryos of Quercus variabilis.【Method】 Factors affecting regeneration and proliferation abilities of somatic embryos of Q.variabilis were discussed by culturing embryogenic calluses at different development stages of somatic embryos,which were induced from immature zygotic embryos on the MS medium supplied with different phytohormone combinations in variable light conditions.【Result】 The embryoids regeneration ability of embryogenic callus presented downward trend with increasing times of subculture on medium,while match usage of 6-BA and NAA could delay this trend.The optimal medium for maintaining embryoids regeneration ability of embryogenic callus was MS medium containing 1.00 mg/L 6-BA and 0.25 mg/L NAA.Natural light was essential to maintain embryoids regeneration ability of embryogenic callus,while darkness and intensive light were inimical.Embryoids regeneration ability of embryogenic callus was also influenced by the development degree of zygotic embryos.Embryogenic callus from junior hart-shape or early cotyledonary zygotic embryos had higher embryoids regeneration ability than the one from mature cotyledonary zygotic embryos.The older was the development stage of somatic embryo,the weaker was the regeneration and proliferation ability.The regeneration and proliferation abilities of initial embryogenic calluses and globular-shape somatic embryos were higher than others.【Conclusion】 Regeneration and proliferation abilities of somatic embryos of Q.variabilis can be maintained effectively by selecting embryogenic calluses from junior zygotic embryos and culturing them on the MS medium containing 1.00 mg/L 6-BA and 0.25 mg/L NAA under the natural light.

The variations of the leaves and growth traits as well as peroxidase of 4-year-old plus tree open-pollinated progenies from six Quercus variabilis populations were investigated.The results showed that there were significant differences in seedling height,leaf length,leaf width,petiole length,and leaf area among different populations,and in seedling height,leaf length among different families.The peroxidase isozyme of Q.variabilis consisted of two locis.The mean of alleles was 2.479 and the expected heterozygosity was 0.282.Within the population,genetic variations accounted for 54% of the total genetic variations.With seedling height as the main character,10 superior families were selected preliminarily from 57 tested families.Their height growths increased by 31.7%~84.0% than the overall population average.

【Objective】 The study was to provide a basis to establish an efficient organ regeneration system for Quercus variabilis.【Method】 Selecting 4 strong Q.variabilis(30 years old),seedlings of 3-4 months from sand cultured nodal segments of the 4 trees,juvenile and mature nodal segments from the 4 trees were used as sources of initial explants.In vitro effects from nodal segments of different orientation and clones on the ability of elongation and rooting were compared,also the relationship between antioxidant enzymes and the ability of elongation and rooting were discussed.【Result】 No significant difference was found for the identical sources of nodal segments from different clones in axillary shoots number,length of tallest shoot and green leaves number were after the last elongation;The axillary shoots number,length of tallest shoot and green leaves number from juvenile nodal segment were more than those of seedlings and mature,which were significant(P<0.05) after the last elongation;The identical sources of nodal segments from different clones in rooting rate and roots number per explant were not significantly different after rooting;Juvenile nodal segment in rooting rate and roots number per explant,which were significant(P<0.05),were more than those of seedlings and mature rooting;The identical sources of nodal segments from different clones in antioxidant enzyme activities were not significantly different after the each stage;3 sources of nodal segment in antioxidant enzyme activities were more than that of another elongation stages after the last elongation and rooting,which were all significant(P<0.05).【Conclusion】 These results indicated that juvenile from the mother trees is the best orientation of expants for establishing an efficient organ regeneration system of cork oak(Q.variabilis);Certain relationship exists between antioxidant enzyme activity and the ability of proliferation and rooting.

Changes of several mainly storage substances as well as seed storage protein pattern during zygote embryos development of Quercus variabilis were studied.Similar variation trends were found among soluble sugar,reductant sugar,soluble starch and storage proteins content.The contents of these substances increased as the development of the embryo and decreased then.The storage protein band number also increased accordingly.

对栓皮栎开花习性,花器构造及花粉生活力和萌发率进行了分析.结果表明:栓皮栎在杨凌地区4月初开花,4月17日左右结束,雌花比雄花晚开2 d.雄花由苞片和雄蕊组成,雌花由花萼和雌蕊组成,花粉近椭圆形,黄色微透明.在冷藏3周内,花粉的生活力和萌发率均保持在90%左右,随后有所下降.不同个体花粉生活力和萌发率随时间的变化基本一致.