Chinese Journal of Tropical Crops
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25 November 2023, Volume 44 Issue 11 Previous Issue   
Special Topic of Orchid Science
Advances in Mutagenesis Breeding Research of Orchids
LUO Weiyu, SU Qinglian, ZENG Ruizhen, YUAN Yun, WEI Qian, ZHANG Zhisheng
2023, 44 (11):  2135-2148.  doi: 10.3969/j.issn.1000-2561.2023.11.001
Abstract ( 5 )   HTML ( 3 )   PDF (1041KB) ( 4 )  

Mutagenesis breeding is a fast and effective breeding method, which has special important significance for the improvement of ornamental plant varieties. Orchids have important ornamental, medicinal, edible and/or cultural value, and broad market prospects. Studies have shown that mutagenesis causes the character changes including plant type, leaf character, flower number, flower size, flower type, flower color, flowering time, ornamental period, insect resistance, disease resistance, stress resistance in orchids, and to date, at least 930 mutants and 16 new orchid cultivars have been generated. This paper reviews the achievements of mutagenesis breeding research in orchids, clarifies current situation and factors affecting the effect of orchid mutation breeding, summaries mutation mechanism of orchids, and finds the methods to further improve the efficiency and effect of orchid mutation breeding. It would provide references for creating new varieties of orchids by better utilizing mutation breeding technology and further clarifying the mutation mechanism of orchids.

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Research Progress on Influencing Factors and Regulatory Methods of Phalaenopsis Spiking
HE Yaping, XIAO Wenfang, CHEN Heming, LYU Fubing, YAN Shijuan, AI Ye, LI Zuo
2023, 44 (11):  2149-2156.  doi: 10.3969/j.issn.1000-2561.2023.11.002
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Phalaenopsis has a rich variety of flower colors, a long-lasting flowering period, a unique flower type, and high ornamental value. It is considered the most popular and commercially valuable orchid in the market. The axillary buds at the base of each leaf of Phalaenopsis orchid can form pedicels, but most of the axillary buds are in a dormant state during the flowering period. Studying the germination law of latent axillary buds can help improving the proportion of multiple flower stalks in Phalaenopsis, and has good application prospects. The main environmental factors (temperature, plant hormones, light environment, nutrient accumulation, etc) affecting the germination of Phalaenopsis bud and the regulatory methods to promote flower bud germination will be reviewed, in order to provide a reference for the application of regulating more flower bud germination in Phalaenopsis industry in the future.

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Research Advance in Mycorrhiza of Paphiopedilum
LI Yixuan, FANG Lin, CHEN Hong, LI Lin, WU Kunlin, ZENG Songjun
2023, 44 (11):  2157-2166.  doi: 10.3969/j.issn.1000-2561.2023.11.003
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In the Orchidaceae family, Paphiopedilum exhibits extraordinary ornamental value and promising market prospects due to its unique appearance. However, Paphiopedilum is at risk of extinction worldwide due to slow growth rate, high environmental demands, and continuous collection by humans. Artificial seedlings can serve as a potential solution for ex situ conservation, reintroduction and commercial production. Unfortunately, artificial propagation is challenged due to low seed germination rate, slow growth rate, and limited resistance to ecological environments. Mycorrhizal symbiosis between plants and fungi has played a crucial role in the natural development of Paphiopedilum, particularly endophytic fungi, which significantly contribute to seed germination and plant growth. This article presents an analysis of features of endophytic fungi of Paphiopedilum, methods of endophytic fungi separation, types and functions of research, and the application prospects, while also outlining future research direction for the conservation of wild Paphiopedilum, seed germination and commercialization. This study aims to provide a comprehensive reference for future research on Paphiopedilum conservation, seed germination and commercialization.

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Metabolomics Analysis of Anthocyanin in Different Flower Colors of Phalaenopsis-Type Dendrobium
WU Meiqing, LIAO Yi, LU Shunjiao, YIN Hantai, YU Wengang, LI Chonghui
2023, 44 (11):  2167-2178.  doi: 10.3969/j.issn.1000-2561.2023.11.004
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In order to further investigate the anthocyanin biosynthetic pathway in Phalaenopsis-type Dendrobium (Den-Phals) varieties with different flower color, this study used widely targeted metabolomics technology to analyze the anthocyanin composition and content of five Den-Phals varieties with blue-purple, light peach-red, red, purple-red and deep purple-red floral color, respectively. The results showed that a total of 38 metabolites were identified from the tested Den-Phals, most of which were glycosides and acylated derivatives of cyanidin (Cy), peonidin (Pn), pelargonidin (Pg), delphinidin (Dp) and petunidin (Pt). There were significant differences in the composition and content of anthocyanin in five Den-Phals varieties. Metabolite difference analysis showed that Cy and Dp glycosides in purple flowers were significantly higher than those in light peach red flowers. The contents of three Dp-type glycosides in blue-purple flowers were significantly higher than those in other colored varieties. Only one Cy-type glycoside was screened out, and the content of which in light pink flowers was high. The content of Cy-type glycosides was the highest in most samples. With the increase of cyanidin and delphinidin and the derivatives, the color of flowers deepened and turned to red-purple. It was speculated that Cy-type glycosides made Den-Phals tend to purple while Dp glycosides gave Den-Phals a blue-purple tone. Based on the above results, it is speculated that there are acylation synthesis pathways of the above five anthocyanins in Den-Phals, which can provide a basis for flower color formation mechanism and flower color improvement of Den-Phals.

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Differential Molecular Mechanism of Dendrobium Flower Pattern Using mRNAs/microRNAs Regulatory Network
ZANG Rui, CHEN Yu, ZHAO Mei, HAO Yuheng, AO Die, LI Kaili, FANG Shidi, ZHAO Qiming, HE Fengmei
2023, 44 (11):  2179-2187.  doi: 10.3969/j.issn.1000-2561.2023.11.005
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The molecular mechanism of the formation of flower diversity is still unclear. In this study, the transcriptome and microRNA sequencing of Dendrobium officinale and D. loddigesii flower organs were performed, and the miRNA-mRNA network and target protein interaction network were constructed by cytoscape and compared at the molecular level. The results of transcriptome studies showed that mRNAs related to flower development had MADS-box AP3 gene, hormone genes, etc. The miRNA-mRNA network analysis showed that the differentially expressed miRNAs and mRNAs were involved in the differential formation of floral pattern, and miR5179 and its target genes AP3, miR160 and their target genes ARFs, miR164 and its target genes NAC021, miR159 and the target genes GAM1, bdi-miR159 and the target genes CKX9, miR167b-3p and the target genes ANT may be involved in floral pattern formation, and the above miRNA was upregulated in D. officinale. The protein interaction pathway showed that two kinds of dendrobium had the key gene of AP3, D. loddigesii lacked four genes: CUL1, TIR1, TIR1A and RBS; there weret wo differentially expressed ARFs genes in the auxin response interaction pathway of two Dendrobium, and D. loddigesii lacked one ARFI gene; D. loddigesii flora specifically possessed the protein interaction pathway of auxin signal transduction target genes constituted by the NAC021 gene. Differential expression of miRNAs/mRNAs in D. officinale and D. loddigesii and protein interaction of target genes may be the main mechanisms leading to the difference between the two Dendrobium flower types.

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Selection of Suitable RT-qPCR Reference Genes for Floral Scent Biosynthesis in Phalaenopsis I-Hsin Venus
ZHANG Yangting, ZHANG Yanping, HUANG Jingyan, WANG Wenjun, TONG Yan, ZHAO Kai, ZHOU Yuzhen
2023, 44 (11):  2188-2195.  doi: 10.3969/j.issn.1000-2561.2023.11.006
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Phalaenopsis I-Hsin Venus is an excellent variety of fragrant flowers, with elegant flower types, long flowering period and rich floral fragrance, which has high garden ornamental value. ACT1, ACT2, ACT3, GAPDH, EF1α, TUA, TUB and Ubi, were selected as the candidate internal reference genes based on the transcriptome data of different flower development stages in Phal. I-Hsin Venus to select the appropriate reference genes for RT-qPCR analysis of the correlated genes in the biosynthesis pathway of the floral scent in Phalaenopsis I-Hsin Venus. The expression of the candidate internal reference genes in inflorescences at different stages was detected by RT-qPCR. The candidate internal parameter genes were analyzed by combining three internal parameter gene stability analysis software: geNorm, NormFinder and BestKeeper. The results of comprehensive analysis showed that ACT1 was the most stable and could be used as the best internal reference gene for the expression analysis of Phal. I-Hsin Venus.

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Phalaenopsis arunachalensis, a Little Known Species of Phalaenopsis in China
DONG Xu, XU Shisong, YANG Hubiao, WANG Qinglong, YUAN Langxing, LIU Zhen, WANG Zhunian, YANG Guangsui, YIN Junmei, WANG Jiabao, HUANG Mingzhong
2023, 44 (11):  2196-2198.  doi: 10.3969/j.issn.1000-2561.2023.11.007
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Phalaenopsis arunachalensis K. Gogoi & Rinya is reported as a little known species from Medog, Tibet Autonomous Region, China. The detailed morphological descriptions and photos are also provided. This species is similar to P. honghenensis F. Y. Liu, P. wilsonii Rolfe and P. taenialis (Lindl.) Christenson & Pradhan. However, this species has a minutely spurred that bends down, the free part of lateral lobes which are less than half length of the mid-lobe, and dagger-like of lateral lobes ends, which can distinguish from other species.

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Diversity and Distribution Pattern of Orchids in Gexigou National Nature Reserve, Sichuan, China
ZHENG Wei, YAN Qi, SHI Yaoqiang, WU Xiaogang, LI Gang, LIU Shengxiang, LI Bajin, ZHANG Hui, XIE Guimo, YANG Shaoping
2023, 44 (11):  2199-2207.  doi: 10.3969/j.issn.1000-2561.2023.11.008
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In order to understand the diversity and distribution characteristics of orchids in Gexigou National Nature Reserve in Sichuan province, a systematic survey was conducted using the line transect combined with sample sampling method from 2020 to 2021. A total length of 95 km including 17 lines were set up, covering various function, habitat and elevation of the reserve. 34 species of Orchidaceae in 20 genera were investigated. Among them, 10 were rare and endangered species, five were wild species under second-class national protection; and two (Liparis brunnea and L. rostrate) were new record species in Sichuan province. Most of them were mainly in terrestrial orchid of life type and absolutely dominant in north temperate zone of geographical. A total of 6703 orchids of 34 species were investigated and distributed in 290 sites. Galearis spathulata and Neottia megalochila were the most and least abundant species respectively, and Epipactis helleborine was the most widely-distributed. Among them, the height of Cypripedium guttatum showed a significant negative correlation with elevation, while there was a slightly correlation in other species. Most of Orchidaceae genera were distributed in 3100-3300 m, followed by 2900-3100 m, while the maximum number of individuals were distributed in 3900-4100 m. The number of Cephalanthera longifolia was positively correlated with slope significantly, the number of Galearis spathulata was positively correlated with elevation significantly, the number of Herminium lanceum was positively correlated with vegetation significantly, the number of whole genus of Herminium was positively correlated with elevation moderately. In general, the orchid resources in Gexigou Nature Reserve are relatively rich, and more distribute in the middle elevation. The number of some species is significantly correlated with environmental factors. Compared with the previous investigations in this reserve, the number of orchid species is significantly increased. However, three national protected species of Cypripedium with high ornamental value were not found, and the population of one species of Cypripedium decreased. Therefore, it is urgent to protect, publicize and enforce the law on orchids in the reserve.

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Genetic Diversity of Wild Paphiopedilum hirsutissimum Populations in Southwest China with SSR Markers
XU Yan, CHEN Zhiguang, XU Yufeng, GE Hong, YANG Shuhua, ZHAO Xin, KOU Yaping, YU Xiaonan, JIA Ruidong
2023, 44 (11):  2208-2218.  doi: 10.3969/j.issn.1000-2561.2023.11.009
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Paphiopedilum hirsutissimum (Lindl. ex Hook. f.) Stein is a rare and endangered plant in China with high ornamental value. In order to explore the genetic characteristics of wild P. hirsutissimum resources in Southwest China, and contribute to the protection and utilization of the wild resources, this study used SSR molecular marker to analyze the genetic diversity and population genetic structure of 190 P. hirsutissimum resources which collected from six populations in Guangxi, Yunnan and Guizhou provinces in Southwest China. In this study, the results showed that ten pairs of primers with good amplification effect were selected from 115 pairs of primers, and 50 alleles were detected by 10 pairs of SSR primers of 190 P. hirsutissimum. The average number of alleles (Na) was five and the average number of effective alleles (Ne) was 2.4835. The average Shannon information index (I) was 0.8592. The average observed heterozygosity (Ho) was 0.4518 and the average expected heterozygosity (He) was 0.4387. The average polymorphic information content (PIC) was 0.3996 and the average Nei’s expected heterozygosty (h) was 0.4370. In this six wild P. hirsutissimum populations, the number of alleles (Na) was from 2.8000 to 4.3000. The number of effective alleles (Ne) was from 1.9655 to 2.4060. The observed heterozygosity (Ho) was from 0.3891 to 0.4839. The expected heterozygosity (He) was from 0.3795 to 0.4683. The Shannon information index (I) was from 0.6584 to 0.8369, and the Nei’s expected heterozygosty (h) was from 0.3648 to 0.4382. In the six wild populations, the genetic diversity of Guangxi Yachang (GYC) and Guizhou Wanfeng Lake (QWF) populations was generally higher (h=0.4382, h=0.4276), while the genetic diversity of Guangxi Mulun (GML) population was relatively low (h=0.3648). Analysis of molecular variance (AMOVA) results showed that genetic variation mainly occurred among individuals within the population (94%), while genetic differentiation within populations was very small (6%). UPGMA cluster analysis based on genetic distance showed that the genetic distance of the six P. hirsutissimum populations was very small, there was no obvious group division and the genetic distance was not completely related to the geographical location. The results of Structure and principal coordinate analysis were consistent with those of UPGMA cluster analysis. Structure and principal coordinate analysis results showed that there was homogenization among the populations, and there was no obvious group division. In summary, the genetic diversity of Paphiopedilum hirsutissimum resources is relatively rich, which can provide a theoretical basis for the protection and utilization of wild P. hirsutissimum resources in Southwest China.

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Ploidy Identification of Cymbidium Hybrid Seedlings by Flow Cytometry
LENG Qingyun, LU Jinping, HUANG Shaohua, XU Shisong, LI Haiyan, NIU Junhai, YIN Junmei
2023, 44 (11):  2219-2226.  doi: 10.3969/j.issn.1000-2561.2023.11.010
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Ploidy identification is one of the basic tasks for germplasm resources and new variety breeding of tropical flowers. In this study, flow cytometry was used to identify the ploidy level of Cymbidium hybrid seedlings. aiming at the key links such as the type of dissociated liquid, the tissue organ of the test material, evaluation indexes such as the nuclear dissociation, the clear degree of the main peak, the easy degree of sampling, and the coefficient of variation. A technique system was established and the stomatal traits of different ploidy were analyzed. The results showed that CyStain UV Precise P was the best of the three dissociated liquid and young leaves were most suitable. Six triploid and two tetraploid were identified from the offspring of the three hybrid combinations. There were significant differences in stomatal length, width and density between different ploidy (P<0.05). The rank of stomatal length and stomatal width was tetraploid>triploid>diploid, while stomatal density was reversed. This study is valuable for the polyploid selection and use of different ploidy germplasm resources for cross breeding of Cymbidium hybrid.

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Numerical Classification of Phalaenopsis Flower Colour Based on Phenotype
WANG Shiyao, ZHANG Guo, YANG Shucai, JIANG Shuanli, WANG Ruihua, WANG Jun
2023, 44 (11):  2227-2235.  doi: 10.3969/j.issn.1000-2561.2023.11.011
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The study aimed to define the floral colour of Phalaenopsis precisely and establish a classification system of Phalaenopsis based on floral colour phenotypes. In this study, the floral colour phenotypes of 146 Phalaenopsis germplasm resources were determined using colourimeter and RHSCC, and the floral colours of each Phalaenopsis were named by cluster analysis and combined with ISCC-NBS system, and the quantitative classification was studied. The results showed that the colour of Phalaenopsis petals and sepals did not differ much, and the lip petals were darker and more colourful than petals and sepals. The classification results based on cluster analysis of L*, a* and b* of petals could not fully characterize the classification of Phalaenopsis flower colours. The ISCC-NBS system divided Phalaenopsis flower colour into yellow, brown, red, violet, pink, purple and white groups. Each group had a preferable correspondence with L*, a* and b* parameters of the CIE Lab system, which could realize the quantitative description of different Phalaenopsis flower colour and the colour group classification was reasonable. Phalaenopsis were rich and colourful, and there were significant differences between different colour groups of Phalaenopsis, but lacking blue-green colour. Overall the brightness and colouration of Phalaenopsis were negatively correlated, and could be divided into two groups, the first type containing yellow, brown, violet, pink, purple and white groups, the second type containing red colour groups.

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Rapid Multiplication and in vitro Flowering of Mengzia foliosa (King & Pantl.) W.C. Huang, Z.J. Liu & C. Hu (Orchidaceae)
SHAO Li, ZENG Xinhua, HUANG Weichang
2023, 44 (11):  2236-2243.  doi: 10.3969/j.issn.1000-2561.2023.11.012
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In this study, the effects of different concentration and ratio of growth regulators on seed germination, rapid propagation and in vitro flowering of Mengzia foliosa were studied. The results showed that the seeds had strong activity and high germination rate at 105 days after artificial self-pollination, and 1/2MS+0.6 mg/L NAA+1.0 mg/L 6-BA medium was more suitable for seed germination. Meanwhile, 2.0 mL/L 6-BA without NAA was more suitable for cluster bud proliferation; and 1/2MS+1.0 mL/L NAA+30 g/L potato homogenate medium was the best for rooting and hardening-off. In addition, 1.0 mg/L 6-BA+0.5 mg/L NAA+1.0 mg/L PP333 medium had the highest flowering rate. Our results not only contribute to the conservation and sustainable use of the species, but also provide important materials and technical support for wild population restoration and reconstruction.

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Omics & Biotechnology
Identification and Analysis of ACA/ECA Gene Family in Hevea brasiliensis
YANG Jingjing, FANG Yongjun, ZHANG Hongtao, LONG Xiangyu, QIN Yunxia, YANG Jianghua, XIAO Xiaohu
2023, 44 (11):  2244-2255.  doi: 10.3969/j.issn.1000-2561.2023.11.013
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Ca2+ is a second messenger in plant cells, and Ca2+-ATPase (ACA/ECA), as an important protein in Ca2+ transport, plays a crucial role in ensuring the balance of intracellular calcium ions and abiotic stress in plants. To investigate the biological function of Ca2+-ATPase (ACA/ECA) in Hevea brasiliensis, 45 ACA/ECA gene family members were identified from the rubber tree genome using the ACA/ECA protein sequence of the model plant Arabidopsis as a probe, including 38 ACA members and 7 ECA members. Expasy and Plant-mPLoc were used to comprehensively analyze the physicochemical properties, gene structure, chromosomal localization, phylogeny, and expression patterns of the 45 members. Physicochemical properties analysis showed that the number of amino acids encoded by each member was 86-1142, the molecular weight of amino acids ranged from 10 048.04 to 125 412.75 Da, and the protein products were mostly localized on the cytoplasmic membrane, with a few members localized on the endoplasmic reticulum, chloroplast, vacuole, and nucleus. In evolutionary, members of the ACA/ECA family were clearly clustered into ACA and ECA two branches, and rubber tree members were always in close proximity to cassava in both branches, indicating that there was a close relative between rubber tree and cassava during phylogeny. Chromosomal localization revealed that the 45 members of the ACA/ECA family were unevenly distributed across 14 chromosomes and one contig in rubber tree, in which, chromosomes 2 and 9 had the largest member distribution of 11 members. Expression analysis showed that there were obvious differences in the expression of some members in different groups, HbACA31 was expressed the most in leaves, HbACA36 was expressed the most in latex, and the expression of HbACA36 was obviously up-regulated in latex after treatment with ethenol. In addition, we also analyzed the influence of tapping on ACA/ECA gene expression, and found that HbACA36 was at a high expression level during tapping of the two rubber tree varieties, PR107 and Reyan 8-79, and significantly up-regulated during tapping of high-yield variety Reyan 8-79,which speculated that HbACA36 was involved in the regulation of rubber biosynthesis and played an important role. The results presented in this study reveal the physicochemical properties and expression patterns of ACA/CA family members for the first time in rubber tree, and would provide a foundation for further investigation of the biological functions of ACA/ECA genes in H. brasiliensis, especially in the regulation of the rubber production.

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Expression and Function Analysis of a MiNAC7 Gene in Response to Flowering and Abiotic Stress in Mango
HUANG Xing, XU Huanhuan, LI Kaijiang, HE Xinhua, XIA Liming, LU Tingting, LIANG Rongzhen, LUO Cong
2023, 44 (11):  2256-2264.  doi: 10.3969/j.issn.1000-2561.2023.11.014
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NAC transcription factors are a large gene family in plants which play important roles in regulating plant growth and development, signal transduction, stress response, etc. In the previous study, the NAC gene, named MiNAC7, was isolated by the yeast two-hybrid system. In this study, the bioinformatics, expression patterns and gene functions of genes were studied. Bioinformatics analysis showed that the MiNAC7 gene was located on chromosome 10, with 4 introns and 5 exons. The length of the coding region of the MiNAC7 gene was 1137 bp, encoding 379 amino acids, the theoretical isoelectric point was 4.88, the molecular weight of the protein was 93.41 kDa, and the amino acid sequence contained a conserved NAM domain. Phylogenetic tree analysis showed that mango MiNAC7 and pistachio PvNAC26 had the closest genetic relationship and the highest homology, and the amino acid sequence similarity was 69.64%. Promoter sequence analysis showed that the promoter region of the MiNAC7 gene contained light response elements, gibberellin response elements and auxin response elements. Expression analysis showed that the expression level of the MiNAC7 gene was high in the stems and buds of juvenile tissues, low in flowers, high in the stems of adult tissues, and low in flowers and leaves. At the same time, it was found that the MiNAC7 gene maintained a high expression level in the leaves at the vegetative growth stage and a low expression level in the leaves at the flowering transformation stage and flower development stage. Overexpression of the MiNAC7 gene led to a late-flowering phenotype in transgenic Arabidopsis. The overexpression of the MiNAC7 gene in Arabidopsis significantly reduced the expression level of the floral-promoting genes AtFT and AtAP1, while the expression level of the late-flowering gene AtFLC was significantly increased. Stress treatment showed that Arabidopsis with excessive expression of the MiNAC7 gene improved its resistance to drought and salt and improved its resistance to GA3 but was more sensitive to ABA. This study shows that the mango MiNAC7 gene not only affects flowering but also participates in the response to abiotic stress, which would lay a foundation for further research on the gene regulatory network of the MiNAC7 gene involved in regulating mango flowering and stress response.

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Establishment and Application of a Method for Genome Size Estimation of Piper L. Using Flow Cytometry
WU Baoduo, HU Lisong, FENG Jianxiong, FAN Rui, GUO Fen, JI Xunzhi, HAO Chaoyun, YAN Lin
2023, 44 (11):  2265-2272.  doi: 10.3969/j.issn.1000-2561.2023.11.015
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Six nuclear extracts and three leaves of different development stage were screened to establish the flow cytometry (FCM) method for determining the genomic size of Piper L. and Cucumis sativus L. was used as the DNA reference standard. Result showed that the modified nuclear extract and inverted leaf were suitable for the flow cytometry of Piper L. The genome size of cultivated varieties, including Kuching, Aman and EMAS, ranged from 667.94 to 719.32 Mb, and Reyin1, microphylla variety from Cambodia, Panniyur-1 and 73F5 ranged from 759.69 to 785.38 Mb. Therefore, the genome size was significantly different among cultivated varieties. Furthermore, it is speculated that the chromosome numbers of P. wallichii and P. hancei, with genome size larger than 900 Mb, should be larger than 52 with a multiple of 13, and the number of chromosomes of germplasm, with genome size ranged from 600 to 800 Mb, may be 52. The hybrid not only had similar phenotypes to cultivated varieties and P. flaviflorum, but also had an average genome size of cultivated varieties and P. flaviflorum. Therefore, it is speculated that the hybrid may be the hybrid offspring of cultivated varieties and P. flaviflorum. This study would provide a method for the determination of the genome size of Piper L. germplasm, and provide a technical basis for the study of Piper L. germplasm evaluation and distant hybridization.

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Selection and Verification of the Reference Genes of Euphorbia maculata Based on Transcriptome Sequencing
XU Jianfeng, GUI Mingming, ZHANG Yongkang, YAO Zizhao, HUANG Shenghe, RAO Zechang
2023, 44 (11):  2273-2280.  doi: 10.3969/j.issn.1000-2561.2023.11.016
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The flavonoid quercetin is one of the main active ingredients in Chinese medicinal herb, Euphorbia maculata. At present, there are few reports on quercetin biosynthesis gene, and real-time quantitative PCR (RT-qPCR) is one of the common methods in gene research. But it is a prerequisite for relative quantitative RT-qPCR to have suitable reference genes. In order to obtain a suitable reference gene, this experiment was based on the results of transcriptome sequencing, EmSE, EmUBC, EmGDI1, EmLSM12, EmGBP, EmSYP22, EmTRI1, EmRSZ21, EmRPL7 and EmUBQ obtained by our research group in the early stage were selected as candidate reference genes. RT-qPCR was used to detect the expression of the candidate reference genes in roots, stems, leaves and fruits of E. maculata during vegetative and reproduction stages. The expression stability of the candidate genes was analyzed using geNorm, NormFinder and BestKeeper software. The results of different software were integrated and ranked by the geometric mean method. The ranking was in the order EmGDI1>EmUBQ>EmSE>EmUBC>EmLSM12=EmGBP>EmTRI1>EmSYP22>EmRSZ21=EmRPL7. EmC4H, a key enzyme gene in quercetin biosynthesis, was analyzed using EmGDI1 as the reference gene. The difference of EmC4H expression in different tissues at different stages was consistent with the trend of transcriptome sequencing. The reference gene EmGDI1 developed in this study could be used as a suitable reference gene for tissue-specific expression studies of different stages of E. maculata.

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Germplasm Resources, Genetics & Breeding
Selection and Analysis of Disease-Resistant Germplasms in Hybrid F1 Generations of Rubber Tree
LI Boxun, HUANG Guixiu, HE Ligang, CAI Jimiao, FENG Yanli, YU Wencai, LIU Zhongliang
2023, 44 (11):  2281-2291.  doi: 10.3969/j.issn.1000-2561.2023.11.017
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Corynespora leaf fall disease is one of the most severe leaf diseases in major rubber planting countries worldwide, which can cause serious yield and economic losses. The selection, creation, and utilization of resistant germplasms are the most effective prevention and control strategies for this disease. This study evaluated the disease resistance of 821 F1 populations from three hybrid combinations Yunyan 277-5×IAN 873, RRIC103×Reyan 8-79, Yunyan 277-5×Reken 525 and identified the level of disease resistance. Based on the evaluation results of disease resistance, 32 candidate F1 generation individual plants were selected from two hybrid combinations that conform to normal distribution. Using three divergent clusters of Corynespora cassiicola and two evaluation methods to identify the resistant candidate F1 clone seedlings, five resistant germplasms were ultimately obtained by measuring the activity of defense enzymes and analyzing the expression characteristics of disease-related genes in five new resistant germplasms. Further investigation confirmed that the interaction relationship between five new resistant germplasms and multi host C. cassiicola during the infection process. This study provides excellent germplasm materials and a theoretical support for the early selection, cultivation, and utilization of resistant germplasms for the disease resistance of Corynespora leaf fall disease.

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Genetic Diversity Analysis and Molecular ID Construction of Mango Germplasm Based on SSR Fluorescence Markers
TANG Yujuan, LUO Shixing, HUANG Guodi, SONG Enliang, LI Riwang, ZHAO Ying, ZHANG Yu, MO Yonglong, TANG Yingying
2023, 44 (11):  2292-2304.  doi: 10.3969/j.issn.1000-2561.2023.11.018
Abstract ( 4 )   HTML ( 1 )   PDF (1559KB) ( 3 )  

Abundant germplasm resources are the basis for mango variety breeding and industrial development. For better protection and utilization of mango germplasm resources, the TP-M13-SSR marker developed by our team previously were used to analyze the genetic diversity and construct molecular ID of 145 mango germplasms containing local cultivars, bred varieties and wild relative species, which stored in the nursery of Guangxi Innovation Base of mango germplasm resources conservation. The results showed that the average number of observed alleles for the 12 primer pairs was 3.2838, the average observed heterozygosity (Ho) was 0.5858, the average expected heterozygosity (He) was 0.6725, the average Shannon index (I) was 1.3383, and the average Nei’s gene diversity index (Na) was 0.6702. The polymorphism information content (PIC) of the 12 primer pairs ranged from 0.5036 to 0.7827, with an average value of 0.6396. All the primers were highly polymorphic sites. The result suggested that TP-M13-SSR primer could provide data support for genetic diversity analysis of mango. The genetic similarity coefficient of 145 materrials varied from 0.5676 to 1.000, with an average of 0.7417. The genetic similarity coefficient between Irwin and Indian No. 1 was 1.000. The minimum genetic similarity coefficient was 0.5676, between M. persiciformis 20-2 and Dadouxiang mango, M. persiciformis 20-2 and Shuoshuai mango, Jinhuang mango and Guire 10-1 mango. All the 145 mango germplasms were divided into two groups when the genetic similarity coefficient was 0.7060. Group I contained 108 mango species and 20 M. persiciformis species, accounting for 88.90% of the total number of germplasms. Group Ⅱ contained 17 specimens, all of which were M. persiciformis species. Group I could be further divided into five subgroups when the genetic similarity coefficient was 0.7330, among which subgroups I-1 and I-3 were the most abundant, accounting for 91.92% of all mango germplasms. The results of UPGMA clustering analysis showed that M. persiciformis were not clustered strictly according to the species relationship, and the overall clustering result of mango germplasms was basically consistent with its geographical origin. All the 145 materials were amplified by 12 pairs of SSR fluorescent primers to obtain the fingerprint map, and the molecular ID was obtained by the assignment of numbers and letters combination. Each pair of primers could distinguish 12.4 germplasms on average, and the identification rate was significantly higher than that of previous studies, indicating that TP-M13-SSR had more advantageous than denaturing polyacrylamide gel electrophoresis in mango germplasm identification. This study would provide scientific basis for the collection and utilization of germplasm resources and variety breeding of mango. It is also proposed for molecular identification of bred varieties, which is of great significance to the development of mango industry for providing methods for molecular identification and intellectual property of bred varieties.

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Somatic Cmbryogenesis of Piper nigrum L. Through Micropylar Tissues of Episperm
DING Yawen, DAI Jinfu, CEN Yi, FAN Rui, WU Baoduo, HAO Chaoyun, HU Lisong
2023, 44 (11):  2305-2311.  doi: 10.3969/j.issn.1000-2561.2023.11.019
Abstract ( 3 )   HTML ( 0 )   PDF (7944KB) ( 3 )  

Black pepper (Piper nigrum L.) is a world-famous spice crop, known as ‘the king of spice’. The establishment of tissue culture and regeneration technology of black pepper has important application value in improving the efficiency of seedling breeding and industrial development. Here, we investigated the effects of explant sterilization, callus induction, and embryo differentiation in somatic cell regeneration systematically in black pepper, and established the tissue culture technology using the elite cultivar ‘Reyin-1’ as the material. The results showed that episperm micropylar tissues could be employed as the explants. The ripe fruits were first sterilized with 75% alcohol for 45 s, and then peeled seeds were sterilized for 45 s in 75% alcohol, followed by 6-10 min of soaking in 0.1% mercuric chloride. The episperm was removed from the plantlet after two weeks culture in darkness, which was used as the explant for somatic embryogenesis. The callus was induced from episperm micropylar tissues within two-month subculture. The optimal culture medium for callus induction and embryogenic callus differentiation was MS+1.5% sucrose+0.80 mg/L 2,4-D+ 1.200 mg/L KT, and the callus induction rate was 88.14 %, and differentiation rate of embryogenic callus was about 58.18%. The embryo callus were transplanted on somatic embryo induction medium. Somatic embryos were induced on the medium MS+1.5% sucrose+0.40 mg/L 2,4-D+0.600 mg/L KT, with a induction rate of 13.33%. After two months of rooted culture, tissue culture plantlets were obtained, and the optimal medium for rooting culture was 1/2MS+1.5% sucrose+0.25% activated carbon. In conclusion, the study provided a combine method of alcohol and mercuric chloride for explants sterilization, identified that micropylar tissues of the episperm was the best explants for somatic embryogenesis in black pepper. The medium and culture conditions of callus induction, embryo differentiation, rooting culture during somatic embryogenesis were further explored in black pepper. The results would lay a foundation for high-efficiency tissue culture seedling and transgenic breeding in black pepper.

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Analysis of Leaf Character of Thirteen Germplasm Resources of Camellia sect. Chrysantha
ZHANG Hongmeng, GAO Yuan, YANG Ziyun, CHEN Longqing, WU Tian
2023, 44 (11):  2312-2321.  doi: 10.3969/j.issn.1000-2561.2023.11.020
Abstract ( 8 )   HTML ( 2 )   PDF (1393KB) ( 3 )  

Eight germplasm resources of Camellia sect. Chrysantha from Daweishan Mountain of Yunnan province were studied to provide scientific basis and research materials for the exploitation and utilization of Camellia plants. In this study, the leaf length and width of 13 cultivars of Camellia sect. Chrysantha were evaluated by direct observation of leaf phenotype, using five known germplasm resources as the control and mature leaves as the test materials, the content of six mineral elements (Cu, Zn, Fe, Mn, Ca, Mg) was determined by the wet ashing method, the contents of four functional components (tea polyphenols, total polysaccharides, total saponins and total flavonoids) were determined by the UV photometer method, the relationship between them and germplasm resources of Camellia sect. Chrysantha was analyzed by the cluster analysis. The results showed that the leaf shape of 13 Camellia sect. Chrysantha could be divided into two types: elliptic and oblong, and there were great differences in leaf area, leaf color and leaf serration. The highest and lowest content of Cu, Zn, Fe, Mn, Ca and Mg in leaves was 2.1, 1.9, 4.3, 6.6, 1.4 and 1.8 times, respectively. The content of total saponins, tea polyphenols, polysaccharides and flavonoids was 10.9, 5.8, 5.0 and 2.0 times, respectively. The correlation analysis showed that tea polyphenols had a significant negative correlation with leaf length/leaf width, a significant positive correlation with leaf color and leaf quality, and a significant negative correlation with leaf length/leaf width and zinc, there were significant positive correlations between leaf color and manganese, villi and copper. The results of cluster analysis showed that the 13 germplasm resources could be divided into 5 groups when the distance coefficient was 4.5, group I (including ‘Honghe 1’ Camellia sect. Chrysantha, JHC-2, JHC-3, JHC-4) and Group IV (including ordinary Camellia sect. Chrysantha, JHC-8, Camellia pubipetala Wan et Huang) contained relatively high contents of the constituents, JHC-2 and JHC-8 may have higher medicinal and health-care functions, but the mineral element contents and contents were generally higher, so the later development and utilization value was higher. The results of this study would be useful for further studies on the accumulation mechanism of important functional components in Camellia sect. Chrysantha, it would also provide theoretical basis for breeding new varieties of Camellia and further development and utilization of Camellia group plants in the future.

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Plant Cultivation, Physiology & Biochemistry
Effects of Reduced Fertilization on Main Agronomic Traits, Yield and Fertilizer Utilization Rate of Sugarcane under Mulched Drip Fertigation System
PENG Lishun, CAO Zhengying, CAI Wenwei, GAN Yimei, YANG Benpeng
2023, 44 (11):  2322-2329.  doi: 10.3969/j.issn.1000-2561.2023.11.021
Abstract ( 2 )   HTML ( 1 )   PDF (981KB) ( 2 )  

The present study revealed the effects of chemical fertilizer reduction patterns on growth, cane yield and fertilizer utilization rate of sugarcane, in order to provide a theoretical basis for the application of mulched drip fertigation and find out the best pattern to reduce fertilizer in sugarcane. Seven different treatments were conducted: no fertilization (CK0), conventional fertilization (CK1), mulched drip irrigation + conventional fertilization (CK2), mulched drip fertigation T100 (fertilizer amount equated with CK1) and T80, T70, T60 (20%, 30% and 40% reduction of fertilizer amount of T100). Main agronomic characters, cane yield, sucrose content, sugar yield, economic benefit and nutrient utilization rate of sugarcane were measured in the seven different treatments. The results showed that compared with CK1, the millable stalk number in CK2 increased significantly, the tillering rate, plant height, millable stalk number and stalk formation rate in T100 were all improved significantly, the effective stalk number and stalk formation rate in T80 and T70 were improved significantly. Compared with CK1, the cane yield in CK2, T100, T80 and T70 increased significantly by 13.64%, 32.20%, 27.00% and 20.18%, respectively. In the different drip fertigation treatments, there was no significant differences for cane yield between T100 and T80, but the cane yield for T70 and T60 decreased significantly in contrast to T100. There was no significant difference in sugar content among the different drip fertigation treatments. The change trend of sugar yield and cane yield among the treatments was consistent. Sugar yield in T100 and T80 was higher than that in other treatments, and there was no significant difference between that in T100 and T80. Compared with CK1, the net benefit in T100 and T80 increased significantly by 4534.4 yuan/hm2 and 3953.8 yuan/hm2, respectively. The net benefit in T70 and T60 decreased significantly compared with T100, and the net benefit in T60 was even 2350 yuan/hm2 lower than that in CK1. The fertilizer utilization rate of all drip fertigation treatments was significantly higher than that of CK1, and the average utilization rates of nitrogen, phosphorus and potassium fertilizers in T80 were the highest in all the treatments, reached 48.36%, 27.70% and 68.95%, respectively, which was 28.42, 17.95 and 30.71 percentage points higher than that of CK1, respectively. Overall, in the middle fertility of laterite soil, the application of mulched drip fertigation pattern for T80 could obtain ideal cane yield and benefit.

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Source-sink Exchange During the Growth and Development of Pleione formosana
HAN Ru, YOU Le, JIANG Mingtao, WANG Dekai, CHEN Ni, ZHAI Junwen, WU Shasha
2023, 44 (11):  2330-2342.  doi: 10.3969/j.issn.1000-2561.2023.11.022
Abstract ( 3 )   HTML ( 1 )   PDF (59578KB) ( 2 )  

The specific transport routes of photosynthetic compounds affect the resource allocation of nutrients at different growth stages. The most important is to affect the quality of flowering and fruit, and promote the artificial cultivation and production of ornamental plants. In this study, Pleione formosana, an endangered species, was used as the material, carboxylfluorescein (CFDA) tracer and laser scanning confocal microscopy were used to study the assimilate transport directions in four different growth and development periods, namely, dormancy period, blossom period, vigorous growth period and half dry period to understand the function of source and reservoir of different organs in different periods. During the dormancy period, assimilates were introduced from mother pseudobulb to bulb plate and finally transported to bud for the growth and development of new bud. In the blossom period, the assimilates were transported irreversibly from the mother bulb to the flower organs to ensure the quantity and quality of flowering. In the vigorous growth period, the assimilates mainly supplied the growth of the leaf and new bulbils at the apex of the mother pseudobulb. In the half dry period, the two daughter pseudobulbs competed with each other for nutrients from the mother pseudobulb, while some assimilates were still used to satisfy the growth and development of apex bulbils. The results showed that pseudobulb, as a nutrient storage organ, played an important role in assimilate distribution and generation regeneration succession in four different periods. Its special function would provide reference for artificial cultivation and commercial production in the future.

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Effects of Intermittent Flooding and Ammonia Nitrogen Stress on Morphology and Physiology of Excoecaria cochinchinensis, Nerium oleander, Fagraea ceilanica
LAN Pingyu, RONG Hang, YAO Zuofang, YANG Gairen, LIU Lian, HE Tieguang
2023, 44 (11):  2343-2353.  doi: 10.3969/j.issn.1000-2561.2023.11.023
Abstract ( 3 )   HTML ( 1 )   PDF (1136KB) ( 2 )  

In order to deepen the understanding of the tolerance of Excoecaria cochinchinensis, Nerium oleander and Fagraea ceilanica to intermittent flooding and high concentration of ammonia nitrogen, an intermittent flooding cultivation experiment was conducted with six aqueous solutions of ammonia nitrogen concentration (0, 0.5, 7.5, 14.5, 21.5, 28.5 mg/L). After 90 days of continuous test, the morphological and physiological indexes of the three plants were determined, aimed to provide theoretical references for the application of the three plants in rain garden, subsurface flow wetland, intermittent flooded green space and other environments. The results showed that: (1) Compared with the soil culture control, under intermittent flooding, all three plants showed adaptive changes in enhancing oxygen delivery to the roots, such as enlarged stem base lenticels and adventitious root formation, and significantly promoted the height growth of N. oleander. However, the growth rate of E. cochinchinensis and F. ceilanica was slowed down due to the influence of flooding; (2) Under the interaction of intermittent flooding and ammonia nitrogen, the plant height of N. oleander increased with the increase of ammonia nitrogen concentration, while the plant height growth of E. cochinchinensis and F. ceilanica was significantly inhibited after the ammonia nitrogen reaching 14.5 mg/L, and when the ammonia nitrogen was 21.5 mg/L, the leaves of E. cochinchinensis appeared symptoms such as reduced leaf area and green loss; (3) Intermittent flooding had no significant effect on the activities of antioxidant enzymes and the contents of chlorophyll, malondialdehyde (MDA), free proline (Pro) and soluble protein (SP) of the three plants. On the 60th days, the activities of antioxidant enzymes, free proline and soluble protein of the three plants fluctuated to some extent, but on the 90th day, each index had little difference compared with soil culture; (4) Under the interaction of intermittent flooding and ammonia nitrogen, on the 90th day, N. oleander could recover to the normal level even when the ammonia nitrogen was 28.5 mg/L. After the ammonia nitrogen was 14.5 mg/L, E. cochinchinensis and F. ceilanica tended to produce more antioxidant enzymes, MDA and free proline, less soluble protein, with obvious physiological disorders. It could be seen that N. oleander showed active morphological adaptation and strong resistance in the aqueous solution with ammonia nitrogen concentration of 0-28.5 mg/L, and could grow in a certain length of time and intermittent flooding environment. Although E. cochinchinensis and F. ceilanica also showed strong adaptability, the tolerance to ammonia nitrogen was weaker than that of N. oleander, and they only grew well in the range of 0-14.5 mg/L ammonia nitrogen concentration aqueous solution. Therefore, the three plants could be used in the intermittent flooded green space, and N. oleander has strong adaptability to high concentration ammonia nitrogen water environment.

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