Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究

青杄（Ｐｉｃｅａ ｗ ｉｌｓｏｎｉｉＭａｓｔ．）胚性愈伤组织在改良５９ 附加２, ４－Ｄ及Ｋｔ各１ ｐｐｍ 的培养基上继代３ 年（每月继代１ 次）,仍具有旺盛的增殖能力。在胚性愈伤组织转入１／２改良５９ 并附加ＡＢＡ １ ｐｐｍ 的分化培养基上,约３ 个月左右可分化出大量体细胞胚。体细胞胚分化率达９０％ 以上。经继代３ 年的胚性愈伤组织细胞的染色体倍性十分稳定,其染色体数及核型为２ｎ＝ ２４＝ １６ｍ （６ｓｃ）＋ ８ｓｍ ＋ ２Ｂ。这一结果与由实生苗根尖压片所得结果基本一致     

Differentiation Potential and Chromosome Stability of Pollen Callus of Maize in Subcultures

Pollen calli were subcultured for 20 months. According to the degree of totipotency observed during this period they can be divided into the following four types: 1) The potential of differentiation of the calli was lacking. 2) The calli were able to differentiate into a few root-like structure and green spots. 3). The calli were able to grow into not only a few green spots but also a few shoots. But they could not develop into normal plants. 4). This type of callus possessed a great ability of differentiation and regeneration. They could develop into embryoids as well as shoots in the same medium. After transferring on to the regeneration medium, they readily regenerated into green plantlets with profuse roots. When chromosome counts were made on the ealli, it was found that variations in ploidy were not great. From 1539 randomly chosen cells at metaphase stage, 90% were haploid. As the counts were made on the No. 1 callus type and the root-tip cells of the 35 pants regenerated from this same type 89.7% of the former 427 cells and 87.4% of the latter 645 cells were determined to be haploid. The calli of the Type No. 1 possess unlimited totipotency demonstrated by regeneration. These stability and totipoteney may play an important role in practical application and theoretical research of maize.     

青杄PsbO基因cDNA序列克隆及生物信息学分析

利用RACE技术从已有的青杄均一化cDNA文库中克隆得到青杄(Picea wilsonii)PsbO基因cDNA的全长,并用生物信息学的方法对该cDNA的核苷酸序列、氨基酸序列的相似性,及编码蛋白PwPsbO的理化性质、亲水性/疏水性、二级和三级结构,PwPsbO基因进化树等进行了预测和分析.采用实时荧光定量PCR(RT-qPCR)技术,测定了青杄不同组织中的PsbO基因的相对表达水平.结果发现:青杄PsbO基因编码346个氨基酸,预测相对分子质量为36.6 kD,理论pI值为5.29,属于可溶性蛋白.二级和三级结构预测表明其含有较多的α螺旋、β折叠和随机卷曲结构.RT-qPCR发现该基因在青杄针叶和茎中表达量最高.这些结果为青杄中PsbO基因的初步功能研究奠定了基础.     

青杄均一化cDNA文库构建及EST序列分析

以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。     

青杄PSAK的克隆及生物信息学分析

目的:获取青杄的PSAK全长cDNA序列,对其进行生物信息学分析,并鉴定其在青杄各组织中相对表达量。方法:以多年生青杄(Picea wilsonii)的cDNA文库为模板,通过RACE PCR的方法获取PSAK的末端序列,经过与EST序列拼接得到PSAK基因的cDNA全长序列。通过特异引物将其克隆在pEASY-T1载体上,基于其氨基酸序列,利用DNAMAN、ExPASy、SWISS-MODEL等工具对其进行生物信息学分析,进行了半定量RT-PCR与RT-qPCR实验来检测其在mRNA水平的组织表达情况。结果:青杄PSAK基因编码140个氨基酸,蛋白分子量为14.23kDa,理论等电点为10.29。蛋白的C端有较为保守的结构域,与拟南芥、烟草等物种具有较高的相似性。PwPSAK主要在青杄的针叶与茎中进行表达。结论:成功获取了PwPSAK的单克隆并进行了生物信息学分析,为青杄中PSAK后续的功能研究提供理论依据。     

青杆PwUSP1基因的克隆及表达模式分析

广泛逆境胁迫蛋白（universalstressprotein,USP）在非生物胁迫响应中起重要作用,但在植物中其功能还大部分未知。本研究通过BLAST分析青杆EST文库,得到职zP基因的EST序列,通过RACEPCR方法获取USP基因的末端序列,经过与EST序列拼接得到USP基因的cDNA全长序列,命名为PwUSP1。分析发现PwUSP1全长cDNA为1167bp,编码区为519bp,编码172个氨基酸残基。生物信息学分析显示,PwUSP1编码的蛋白相对分子质量为19．07kDa,理论等电点为6．38,为非跨膜的亲水性蛋白。PwUSP1具有USP家族典型的UspA结构域和ATP结合位点G-（2x）-G-（9x）-G（S／T）。半定量RT-PCR与RT-qPCR分析表明,PwUSP1在青杆的根、茎、针叶、花粉、种子中均有表达,在根和花粉中表达量较高。同时,PwUSP1受干旱和盐胁迫的诱导表达上调,均在处理6h后表达量较高,推测该基因可能在青杆逆境胁迫响应中发挥作用。     

Mitochondrial dynamics and its responds to proteasome defection during Picea wilsonii pollen tube development

Tip growth of pollen tubes is essential for higher plant sexual reproduction and has been proposed to be highly regulated by the ubiquitin/proteasome pathway (UPP). The dynamics of mitochondria and the functions of the UPP on mitochondrial dynamics during pollen tube development are still poorly understood. In the present study, using real‐time laser scanning and transmission electron microscope, it was revealed that mitochondria in Picea wilsonii, are either ellipsoid or filamentous with various lengths. Time‐lapse images indicated that the two forms of mitochondria interconvert frequently through opposite process of fusion and fission. Examination of mitochondrial morphology during four key stages of in vitro pollen tube development revealed a link between mitochondrial remodeling and the process of pollen tube elongation. We also report that MG132, a specific proteasome inhibitor, not only strongly disturbed the mitochondrial remodeling but also significantly reduced mitochondrial membrane potential during pollen tube development. This finding provides new insight into the function of the proteasome in tip growth of pollen tubes. Copyright © 2010 John Wiley & Sons, Ltd.     

青杄FKBP12基因原核表达和多克隆抗体的制备

目的:制备青杄FKBP12基因的多克隆抗体,为进一步分析FKBP12的蛋白定位、表达等提供基础。方法:采用PCR方法扩增FKBP12基因得到其全长cDNA序列,并将其克隆至原核表达载体pET-48b中,转化入BL21菌株。经IPTG诱导,表达了分子量约为33kD的重组蛋白,SDS-PAGE和Western blotting检测鉴定表达产物。此蛋白经亲和纯化后,作为抗原注射新西兰兔,进行抗体制备。结果:成功获取了多克隆抗体,制备的FKBP12兔抗血清效价在1∶729 000以上,ELISA结果表明融合蛋白具良好的免疫原性。间接ELISA法检测纯化后抗体效价,表明纯化后抗FKBP蛋白兔多克隆抗体效价高,检测灵敏度为16ng/mL。结论:所制备的抗体能满足后续试验要求的效价值,为进一步研究提供基础。     

粗枝云杉胚性愈伤组织增殖后期的体细胞胚发生方式转变

粗枝云杉愈伤组织在增殖后期体细胞胚的分化能力显著降低,转变愈伤组织增殖方式和体细胞胚分化培养方式有利于体细胞胚发生能力的提高。采用液体悬浮增殖取代半固体增殖更有利于胚性的保持。在增殖后期,首选的体细胞胚发生方式为＂液体增殖-滤纸分化＂,其次为＂块状增殖-块状分化＂,最后是＂块状增殖-滤纸分化＂。     

Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis

Picea koraiensis, called Korean spruce, is an evergreen tree and found mostly in northeast Asia. In this study, plant regeneration via somatic embryogenesis from open-pollinated immature zygotic embryos of nine genotypes of elite trees was established. Immature zygotic embryos were cultured onto RJW medium modified from 505 medium with 21.48 μM NAA, 2.22 μM BA, and 2.32 μM KT. The average frequency for all nine genotypes was 74.2%. Embryogenic calluses of the nine genotypes of elite trees were subcultured on RJW basal medium containing 8.06 μM NAA, 1.11 μM BA, and 1.16 μM kinetin. The calluses of three lines, 3^#, 9^#, and 2^#, were actively proliferated but others were not. Somatic embryogenesis was induced from the embryogenic callus in genotypes of 3^#, 9^#, and 2^# on RJW medium with ABA and 60 g l⁻¹ sucrose. Cotyledonary somatic embryos were subjected to a drying process. The drying of embryos by uncapping the culture bottle for 5 days on a clean bench resulted in a high frequency of germination of somatic embryos (87% in RJW medium). However, plantlet conversion from germinated embryos was greatly reduced and the optimal medium for plant conversion was 1/2 WPM or 1/2 BMI medium. In conclusion, we have, for the first time, established a plant regeneration system via somatic embryogenesis in the Korean spruce, which can be applied for rapid micropropagation of elite trees.     

The effectiveness of various nitrogen sources in white spruce [Picea glauca (Moench) Voss] somatic embryogenesis

The effects of glutamine-based dipeptides, glutamine and casein hydrolysate, as well as the deletion of organic nitrogen, were investigated during white spruce [Picea glauca (Moench) Voss] somatic embryogenesis. There were no differences in the fresh weight increase of the tissue masses grown on initiation medium with different combinations of organic nitrogen. This was also the case for subsequent growth on kinetin medium, except that glutamine alone produced a significantly lower fresh weight increase than the other organic nitrogen combinations. Without organic (i.e. with only inorganic) nitrogen in the medium, the fresh weight increase was significantly less than with organic nitrogen on both initiation and kinetin medium. No differences were found between the dry/fresh weight ratios obtained with the various nitrogen treatments. The number of mature embryos produced per gram fresh weight when cultured in the absence of organic nitrogen was significantly higher than that obtained in its presence. There were no differences in the total number of mature embryos produced in cultures grown with various organic nitrogen combinations or without organic nitrogen. There were large clone differences with respect to the number of mature somatic embryos per gram tissue and the total number of somatic embryos produced. Hence, nitrogen type influences culture growth rate but not the number of mature somatic embryos produced. The latter was clone dependent.Abbreviations BA 6-benzylaminopurine - CH casein hydrolysate     

Rhizobial Nod factors stimulate somatic embryo development in Picea abies

 Nod factors are lipochitooligosaccharides (LCOs) secreted by rhizobia. Nod factors trigger the nodulation programme in a compatible host. A bioassay was set up to test how crude (NGR234) and purified (NodS) Nod factors influence cell division and somatic embryogenesis in a conifer, Norway spruce (Picea abies). The Nod factors promoted cell division in the absence of auxin and cytokinin. More detailed studies showed that NodS stimulates development of proembryogenic masses from small cell aggregates and further embryo development. However, stimulation was only observed in low-density cell cultures. Our data suggest that rhizobial Nod factors substitute for conditioning factors in embryogenic cultures of Norway spruce. Received: 20 January 1999 / Revision received: 26 March 1999 / Accepted: 27 April 1999     

青杄中sPPa1的cDNA序列克隆及其生物信息学分析

以青杄(Picea wilsonii)均一化cDNA文库为模板,通过RACE方法克隆得到青杄PPa1基因cDNA全长,对该cDNA序列、核苷酸序列的相似性、理化性质、疏水性、二级结构、三级结构及是否跨膜进行了分析预测;进行了多序列比对并构建了系统树,同时对PPa1在青杄各组织中的表达量进行了检测.结果表明:青杄PPa1基因共由216个氨基酸组成,分子量为24.55 kD,理论PI为5.83,属可溶性蛋白;二级结构主要由α-螺旋、不规则卷曲和β-折叠构成;PPa1在青杄花粉中表达量最高.研究为进一步研究青杄PPa1的功能奠定了基础.     

Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]

 Trees were regenerated from six white spruce embryogenic clones after cryopreservation for 3 and 4 years, respectively. Genetic stability was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprints. Somaclonal variation was detected in some in vitro embryogenic cultures 2 and 12 months after they were re-established following cryopreservation but not in the corresponding regenerated trees. These results suggest that trees regenerated from cryopreserved cultures in subsequent years are primarily genetically stable in the genomic regions tested and that variation observed due to the in vitro culture process infrequently affects trees regenerated from normally maturing and germinating somatic embryos. However, trees regenerated from somatic embryos that matured or germinated abnormally in in vitro culture exhibited altered RAPD fragment patterns. Received: 20 July 1998 / Revision received: 15 October 1998 / Accepted: 14 December 1998     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

红皮云杉胚性愈伤组织诱导技术研究

以红皮云杉未成熟胚为外植体进行胚性愈伤组织诱导实验,利用L₁₆(4²×2)混合水平正交设计研究基础培养基、光照条件、未成熟胚采集时期对胚性愈伤组织诱导的影响,以此为基础对不同的培养温度梯度进行了筛选。结果表明：改良RJW基本培养基为最适宜的基础培养基,光照条件以暗培养为宜,未成熟胚的最适宜的采集时间7月20日,适宜培养温度为22℃。当未成熟胚在添加1.0 mg·L^-1 BA,5.0 mg·L^-1 NAA,20 g·L^-1蔗糖,450 mg·L^-1 L-谷氨酰胺、750 mg·L^-1水解酪蛋白的改良RJW培养基,22℃下暗培养时,胚性愈伤组织诱导率最高,达到81.3%。