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Agrobacterium tumefaciens-Mediated Transformation of Leaf Callus in Eucommia ulmoides     Minhao Liu,Junlei Xu,Jing Ye,Zhouqi Li,Ruishen Fan,Long Li Scientia Silvae Sinicae   2020,56(2 ):79 -88. DOI:10.11707/j.1001-7488.20200209 Abstract  （527） HTML （19） PDF（pc） （2588KB）（406） Save Objective: To optimize the callus regeneration system for Eucommia ulmoides leaf explants, to assess the sensitivity of callus to Km and Cef, and to determine the optimum factor levels for Agrobacterium tumefaciens-mediated transformation, in order to allow for genetic transformation of adult plant of E. ulmoides. Method: Callus was induced from leaves of E. ulmoides adult plants. It was inoculated in different concentrations of MS medium, NAA and 6-BA to optimize culture conditions for adventitious bud induction and regeneration. In addition, different concentrations of Km and Cef were added to the culture medium to study callus sensitivity. Based on the obtained callus receptor system for leaves, an L16 (45) orthogonal array experiment was conducted to explore the effect of different conversion factors on Agrobacterium-mediated transformation efficiency. Transformation with an optimum combination of factors was used to obtain Km-resistant buds, which were assessed as transformants by GUS histochemical staining and PCR. Result: MS medium at 3/4 strength promoted the induction and growth of adventitious buds in E. ulmoides. The optimum medium for induction of adventitious buds in callus was 3/4MS + 0.27 μmol·L-1 NAA + 4.4 μmol·L-1 6-BA, with induction rate of 83%±10.0%; The optimal medium for adventitious bud rejuvenation was 3/4MS + 0.054 μmol·L-1 NAA + 4.4 μmol·L-1 6-BA, with an average growth length of (2.47 ±1.33) cm. Sensitivity testing of antibiotic and bacteriostatic agents showed that in the selected medium for genetic transformation, the optimal concentrations of Cef and Km for screening were 200 mg·L-1 and 70 mg·L-1, respectively. The orthogonal test showed that the optimum combination of factors for Agrobacterium-mediated transformation was 5 days of pre-culture, 10 min of infection time and 3 days of co-cultivation. Genetic transformation of about 200 calli was carried out using the optimum instantaneous conversion system, and three Km-resistant buds were obtained. GUS histochemical staining showed that the GUS gene was expressed in these Km-resistant buds, and PCR analysis demonstrated the presence of the NPTⅡ gene. Conclusion: For E. ulmoides callus, this study defined an adventitious bud induction medium, 3/4MS+0.27 μmol·L-1 NAA + 4.4 μmol·L-1 6-BA, and an adventitious bud rejuvenation medium:3/4MS + 0.054 μmol·L-1 NAA + 4.4 μmol·L-1 6-BA. Agrobacterium-mediated transformation of callus involved 5 days of pre-culture, 10 min of infection time and 3 days of co-cultivation, and the selection medium, 3/4MS + 0.054 μmol·L-1 NAA + 4.4 μmol·L-1 6-BA + 200 mg·L-1 Cef + 70 mg·L-1 Km. Three resistant buds were obtained using this system. PCR analysis and GUS histochemical staining indicated that T-DNA had been integrated into the genome of these Km-resistant buds. This report demonstrated that genetic transformation could be studied in a mature E. ulmoides tree, laying a foundation for studying gene functions and for objective-specific improvement of E. ulmoides. Table and Figures丨 Reference丨 Related Articles丨 Metrics

Codon Usage Bias and Its Influencing Factors in Pinus massoniana Transcriptome     Peihuang Zhu,Yu Chen,Lingzhi Zhu,Rong Li,Kongshu Ji Scientia Silvae Sinicae   2020,56(4 ):74 -81. DOI:10.11707/j.1001-7488.20200408 Abstract  （205） HTML （12） PDF（pc） （2310KB）（260） Save Objective: Heterologous expression is an important means of plant protein function verification and molecular breeding. Codons are important factors for efficient expression of heterologous genes. An analysis of codon bias of gene coding region of Pinus massoniana transcriptome was conducted in order to provide a theoretical basis for molecular breeding of P. massoniana. Method: Analysis of codon parameters and bias in P. massoniana transcriptome by using CodonW, EMBOSS and other codon analysis software. According to the neutral mapping, ENc-GC3s correlation analysis and bias analysis speculates the main reasons for codon bias. The optimal codons of P. massoniana were screened by comparing the relative synonymous codon usage(RSCU) of high and low expression gene samples, and the codon bias differences between P. massoniana and Arabidopsis thaliana, Nicotiana tabacum, Populus tremula, Escherichia coli and Saccharomyces cerevisiae were analyzed by codon usage frequency ratio. Result: The average GC content of P. massoniana transcriptome coding sequence(CDS) codon was 44.95%, and GC content in the third position of codon was 38.95%, especially preferred A/T. Statistical analysis of high and low expression gene samples indicated that the difference of the relative synonymous codon usage (RSCU) in the transcriptome of P. massoniana was small, the 27 codons such as TTA, CAA, TGT and GGT can be used as the optimal codons for P. massoniana. Neutral mapping, ENc-GC3s correlation analysis and bias analysis showed that the formation of P. massoniana codon preference may be mainly affected by mutation, and secondly affected by multiple factors such as natural selection. Codon usage frequency analysis showed that the codon bias in P. massoniana was less different from N. tabacum, A. thaliana and P. tremula. The difference with E. coli was the largest, and the difference with S. cerevisiae was smaller than E. coli. Conclusion: Among the 27 optimal codons screened, 25 codons have the third one as A/T. P. massoniana prefers the third codon as A/T. The formation of P. massoniana codon preference may be mainly affected by mutation, and secondly affected by multiple factors such as natural selection. N. tabacum is the preferred plant organisms for heterologous expression of P. massoniana and S. cerevisiae may be superior to E. coli in the microorganism. Table and Figures丨 Reference丨 Related Articles丨 Metrics

Morphological and Anatomical Observations of Floral Organs and Sterility Analysis of Fargesia yuanjiangensis     Ling Huang,Lin Deng,Caihua Chu,Hui Zhan,Shuguang Wang Scientia Silvae Sinicae   2020,56(4 ):64 -73. DOI:10.11707/j.1001-7488.20200407 Abstract  （248） HTML （8） PDF（pc） （6417KB）（102） Save Objective: The inflorescences and floral organs of Fargesia yuanjiangensis were studied by morphological and anatomical observations. The inflorescence characteristics, flowering characteristics and seed setting rate of F. yuanjiangensis were analyzed, and the sterility was tried to shift through tissue culture, in order to provide a new theoretical evidence for reproductive biology and breeding of F. yuanjiangensis. Methods: The morphological and anatomical characteristics of floral organs were described by means of anatomical observation. The development process of female and male gametophytes was studied using paraffin sections. The pollen viability was determined using TTC staining. The development of anthers under field and tissue culture conditions was compared and analyzed. Results: The inflorescence of F. yuanjiangensis is terminal or occasionally lateral with phyllomophore, and surrounded by a foliage leaf. The whole inflorescence is uniaxial branched, and the top spikelet develops earlier than the base one, which is finite inflorescence. The base floret of a single spikelet opens first, so it belongs to infinite inflorescence. Each spikelet contains 4-5 small florets and the average length of the florets is 0.9 cm. Each floret is connected by one spikelet, and the tip of the spikelet is swollen with white fluff. There are 2 pieces of glumes at the base of the spikelet. Each floret contains 2 opposite bracts, 3 lodicules, 1 pistil and 3 stamens. The lodicules are located between palea and stamen. When the florets are maturing, lodicules absorb water, yellowish-green or purple anthers are extended outer with four chambers. The immature anther wall consists of four layers:epidermis, endothecium, middle layer and tapetum from outside to inside. The anther develops to the dyad, and the tapetum begin to degenerate, the tapetum cells belong to the glandular type. The mature pollen grains are two-cell type with an average diameter of 43.92 μm. The pollen grains are dyed red by TTC with pollen viability of 54.78%, yet the germination rate is extremely low and even without germination. The pollen sterility can be summarized into four types:tapetum overdevelopment, anther wall shrinkage, pollen grains degradation, empty pollens without nuclei and cytoplasm. The ovary has 2 carpels and unilocular, superior and parietal placentation with dual-integument enclosing the anatropous ovule, and their stigmas have 2 extremely feathery branches, with short column and long stigma, homogamy and herkogamy. Under tissue culture condition, the flower buds can normally develop into spikelets and blossom, the inner and outer lemma and palea open, and the anthers extend out of the spikelet. The fibrous layer of anther wall became thicker and more continuous, but most pollen grains still appear empty shell. Conclusion: F. yuanjiangensis is a mixed inflorescence, uniaxially branched and open wind-borne flower. It is a typical male sterility and cannot form effective pollen, which is the main reason for the low seed setting rate of F. yuanjiangensis. The spikelet of F. yuanjiangensis can continue to bloom under tissue culture and the anthers extend out of spikelets, but the pollen development is still abnormal. The tissue culture techniques cannot change the sterility phenomenon of F. yuanjiangensis, suggesting that the stamen sterility of F. yuanjiangensis may not be caused by nutrient deficiency. Table and Figures丨 Reference丨 Related Articles丨 Metrics

Changes of Endogenous Hormones during Fruit Development and Their Relationship with Embryo Abortion in Ziziphus jujuba 'Lengbaiyu'     Haiyan Ren,Yongkang Wang,Ailing Zhao,Xiaofang Xue,Guihua Gong,Xuemei Du,Dengke Li,Junjie Du Scientia Silvae Sinicae   2020,56(4 ):55 -63. DOI:10.11707/j.1001-7488.20200406 Abstract  （315） HTML （6） PDF（pc） （2638KB）（103） Save Objective: The endogenous hormones, including auxin, gibberellins, and abscisic acid, play an important role in regulating the embryonic development. The dynamics of indole-3-acetic acid (IAA), gibberellic acid (GA4), cis-abscisic acid (cABA), and trans-abscisic acid (tABA) were studied, in embryos and flesh at four important developmental stages of the same jujube (Ziziphus jujuba) variety, in order to reveal the relationship between the embryo abortion and the changes of endogenous hormones during fruit development and to provide evidence for clarifying the mechanism of jujube embryo abortion and theoretical guidance for salvage, germplasm innovation, especially cross breeding of jujube embryos. Methods: The development of fruit and immature embryos in 20-50 days of Z. jujuba 'Lengbaiyu' were observed during different development stages, including pro-embryo, small globular embryo, globular embryo and torpedo shaped embryo, and the concentration of IAA, GA4, cABA, and tABA in embryo and flesh of normal embryo and aborted embryo during the four embryonic development stages was analyzed using liquid chromatography-mass spectrometry (HPLC-ESI-MSn). Results: 1) The IAA content was significantly higher in the embryo than in the flesh. The IAA content in the embryo increased gradually for the normal embryo, and gradually decreased for the aborted embryos. The IAA content in the flesh displayed an inverted V-shape for the normal embryo, and a normal V-shape for the aborted embryo. 2) The content of GA4 was significantly lower in the embryo than in the flesh and it was at a lower level. The GA4 content was lower in the normal embryos than in the aborted embryos. The GA4 content in the flesh showed an increase earlier followed by a decrease later in both the normal and the aborted embryos. 3) cABA showed a gradual decrease in the development of flesh and embryo. The cABA content was greater in the normal embryos and lower in the aborted embryos respectively than in the flesh. 4) The content of tABA was higher in the embryo than in the flesh. The content of tABA displayed a tendency of gradual decrease in the normal embryos, and a tendency of gradual increase in the aborted embryos and even higher than that in the normal embryos at a later stage. The tABA content in the flesh for both the normal and the aborted embryos displayed an earlier decrease followed by a slow increase later. 5) The lower level of GA4, higher level of IAA in the embryos, the lower levels of tABA, cABA and the high IAA/GA4 were beneficial to the normal development of immature embryos. The decrease of IAA, the continuous increase of tABA, and the decrease of (GA4+IAA)/ABA ratio in embryos were the important factors leading to embryo abortion. Conclusion: In the early stage of jujube embryo development, higher growth hormones are required, and the increase content of tABA in the embryo at the later stages may cause embryo abortion. Table and Figures丨 Reference丨 Related Articles丨 Metrics

Comparative Analysis of Chloroplast Genome Characteristics between Paeonia jishanensis and Other Five Species of Paeonia     Xiaojun Zhou,Kai Zhang,Zhengfeng Peng,Shanshan Sun,Huiyuan Ya,Yanzhao Zhang,Yanwei Cheng Scientia Silvae Sinicae   2020,56(4 ):82 -88. DOI:10.11707/j.1001-7488.20200409 Abstract  （277） HTML （7） PDF（pc） （1921KB）（270） Save Objective: China's wild peony has been regarded as precious germplasm resources at home and abroad. Wild Paeonia jishanensis is considered as one of the most important ancestral species of cultivated tree peonies. The analysis of chloroplast genome (cp DNA) characteristics of Paeonia, including P. jishanensis, has important theoretical and practical values for elucidating the phylogeny of Paeonia and developing improved varieties. Method: Chloroplast data of P. jishanensis were obtained by high-throughput sequencing. The cp DNA data of P. ostii, P. ludlowii, P. delavayi, P. veitchii and P. obovata were downloaded from NCBI database. Comparative analysis of the six peony cp DNA was performed using Geneious 8.0 and EMBOSS 6.4.0. Result: The cp DNA sequence of P. jishanensis was 152 628 bp with 112 genes, 25 988 codons encoding 78 proteins. There are 19 genes (including 4 rRNA, 7 tRNA, 8 protein coding genes) repeat in the IR (Inverse Repeat) region. A total of 143 SSR loci were searched, the mononucleotide repeat are the most common (116, accounting for 81.12%), and there was no hexanucleotide repeat motif. Although the chloroplast genome of Paeonia is conservative, there are still some changes in the boundary of IR and LSC(large single-copy) between different species. The rpl2 gene of P. ostii extends 718 bp to the LSC region, while that of other species are completely located in the IR region. Conclusion: Paeonia cp DNA genome size and gene content are highly conserved. The cp DNA of P. obovata and P. delavayi was the largest (152 698 bp), and that of P. ostii was the smallest (152 153 bp). The codons of cp DNA protein coding genes and the base composition of SSR loci in P. jishanensis prefer to use A/T. Among 143 SSR loci, 134 loci were A/T loci. The distribution of cp DNA SSRs in P. jishanensis has heterogeneity, 14 SSR loci were located in IR region, 103 in LSC region and 26 in SSC region. Analysis showed that boundary change of LSC/IRb was the main reason for the expansion or contraction of IR region in Paeonia. The results provide a basis for the studies of phylogeny and origin of cultivation, and for the development of molecular markers and the breeding of elite cultivars of Paeonia plants. Table and Figures丨 Reference丨 Related Articles丨 Metrics

Cloning and Salt Tolerance Analysis of Transcription Factor HSFA4 from Betula platyphylla     Zhongyuan Liu,Zheng Liu,Ying Xu,Shanshan Liu,Zhilan Tian,Qingjun Xie,Caiqiu Gao Scientia Silvae Sinicae   2020,56(5 ):69 -79. DOI:10.11707/j.1001-7488.20200508 Abstract  （381） HTML （19） PDF（pc） （5330KB）（218） Save Objective: Heat shock transcription factor (HSF) plays an important regulatory role in plant response to abiotic stress. The aim of this study was to identify the stress resistance function of BpHSFA4 in birch(Betula platyphylla) in order to lay a theoretical foundation for using the gene in tree breeding through genetic engineering. Methods: The BpHSFA4 cDNA sequence was obtained by searching with birch transcriptome libraries. Further, the BpHSFA4 gene was cloned by RT-PCR. And the BpHSFA4 cDNA sequence was analyzed using bioinformatics tools. Quantitative real-time RT-PCR was used to monitor the expression patterns of BpHSFA4 genes in the leaves, stems and roots of birch under different stress treatments. The vector of overexpression pROKⅡ-BpHSFA4 and silencing expression vector of pFGC5941-BpHSFA4 were constructed and transient transformed into birch. The empty pROKⅡ was also transient infected into birch as a control. The MDA content, H2O2, SOD activity, POD activity, electrolyte leakage and diaminobenzidine(DAB), nitrotetrazolium blue chloride (NBT) and Evans blue staining under NaCl stress were measured and compared between transgenic (OE, SE) and control (CK) plants. Results: The results showed that the cDNA length of BpHSFA4 was 1 170 bp. The protein encoded by this BpHSFA4 gene was 389 amino acids. The relative molecular weight of BpHSFA4 protein was 44.15 kDa, with isoelectric point 5.14. The typical HSF domain was found in the BpHSFA4 sequence.The results of qRT-PCR showed that the expressions of BpHSFA4 gene were different under abiotic stresses and hormone treatments compared to the control. Furthermore, the expressions were significantly different at least at one stress point time for each treatment. In addition, the expression patterns of BpHSFA4 gene were more significantly changed under NaCl stress. To determine whether BpHSFA4 displayed a successful overexpression(OE) or a suppressed expression (SE), the transcript levels of BpHSFA4 in the three types of transformed birch plants were measured by qRT-PCR. The results showed that the expression of BpHSFA4 was significantly higher in OE plants and clearly lower in SE plants, especially 12 h after transformation, indicating that these transient expression plants were suitable for further study. Under NaCl stress, The NBT and DAB staining and H2O2 content also showed that overexpression of BpHSFA4 markedly reduced ${\rm{O}}_2^{\bar \cdot }$ and H2O2 accumulation, while suppressed expression of BpHSFA4 led to elevated accumulation of ${\rm{O}}_2^{\bar \cdot }$ and H2O2. The SOD and POD activities of transgenic birch plants showed that OE had the highest SOD and POD activities, and that SE plants had the lowest SOD and POD activities. The Evans blue staining and MDA content and electrolyte leakage showed that cell death greatly decreased in the OE plants, but greatly increased in the SE plants. Conclusion: BpHSFA4 gene can respond to abiotic stresses and hormone treatments, and stronger under salt stress, it indicates that the gene may be involved in the response to salt stress in birch trees. All results indicated that overexpression of BpHSFA4 improved the salt stress tolerance by increasing the ability to scavenge reactive oxygen species (ROS), the activities of SOD and POD to reduce the accumulation of H2O2 and ${\rm{O}}_2^{\bar \cdot }$, thereby reducing cell damage or cell death and enhancing birch resistance. BpHSFA4 was proved to be a candidate gene responding to stresses. Table and Figures丨 Reference丨 Related Articles丨 Metrics

Characterization and Phylogenetic Analysis of Ostrya multinervis Chloroplast Genome     Ming Jiang,Shisheng Ke,Junfeng Wang Scientia Silvae Sinicae   2020,56(5 ):60 -68. DOI:10.11707/j.1001-7488.20200507 Abstract  （270） HTML （10） PDF（pc） （3561KB）（163） Save Objective: Ostrya multinervis is a species endemic to China, and it is listed as a rare and endangered wild plant, and a species with extremely small population in Zhejiang Province. The wild O. multinervis trees are found to be very rare in Zhejiang Province with very few young-aged plants and a declining population. During the evolution process, the chloroplast genomes are relatively conserved and thus provide useful information for genetic relationship analysis and phylogenetic studies. In the present study, based on the assembly of chloroplast genome, sequence characteristics were determined to confirm its structure and gene organization. By phylogenetic analysis, relationships and taxonomic ranks with its relative were identified. Methods: Genomic DNA of O. multinervis was extracted by using CTAB method, and a library was constructed for high-throughput sequencing with HiSeq X Ten with the paired-end strategy. The chloroplast genome was assembled by using NovoPlasty, and PCR was applied to isolate the border sequences following sequencing verification. SSR loci were detected by MISA and a phylogenetic tree was generated using PhyML 3.0. Results: A total of 19 869 412 clean reads were obtained by getting rid of both adaptors and low quality sequences. The result indicated that the complete O. multinervis chloroplast genome was 159 583 bp in length containing a typical quadripartite structure, and the lengths of large single copy, small single copy, and inverted repeat were 88 514 bp, 18 953 bp, and 26 058 bp, respectively. The O. multinervis chloroplast genome contains 131 genes, with 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Among them, ycf1 and ycf15 were pseudogenes, and their sequences were shorter than normal ones. A total of 58 SSR loci were predicted from O. multinervis plastome, and 53 of which were mononucleotide repeats. Multiple sequence comparison showed the highest similarity between O. multinervis and O. rehderiana (99.1%) and the lowest was between O. multinervis and O. japonica (98.7%). The best fit model for constructing a phylogenetic tree was GTR+G+I, and the Akaike information criterion and Bayesian information criterion were 556 643.629 64 and 556 954.642 07, respectively. The phylogenetic analysis indicated that 12 plants could be divided into two groups, and Alnus japonica and A. rubra from the tribe Betuleae were grouped into one clade, while 10 plants from tribe Coryleae were grouped into another clade. O. multinervis and the other three Ostrya species clustered in the same clade with 100% of bootstrap support. Conclusion: Based on the high-throughput sequencing, the chloroplast genome of O. multinervis was assembled, and its structure and gene organizations were confirmed. Higher similarities in chloroplast genome sequences were found between O. multinervis and other three Ostrya species, and they shared the same clade. The assembly, sequence comparison and phylogenetic analysis provide a basis for genetic structure and population diversity studies in the future. Table and Figures丨 Reference丨 Related Articles丨 Metrics