Table of Content

15 September 2016, Volume 36 Issue 5

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Notes on the genus Elatostema(Urticaceae) of China

WANG Wen-Tsai

2016, 36(5):  641-646.  doi:10.7525/j.issn.1673-5102.2016.05.001

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(1)Two species of the genus Elatostema(Urticaceae), E.villosistipulum and E.longgangense, are described as new from Guangxi Autonomous Rejion, and the diagnostic differences between them and their allies are given respectively; (2)E.lineolatum var. integrifolium is newly recorded to the flora of China; (3)The descriptions of pistillate capitula, pistillate flower and achene of E.brevipedunculatum and E.lineolatum var. integrifolium are given for the first time; (4)The time of occurrence of the ribbed achenes peculiar to the genus Elatostema is briefly discussed.

Two New Species of Urticaceae from Xizang

WANG Wen-Tsai

2016, 36(5):  647-649.  doi:10.7525/j.issn.1673-5102.2016.05.002

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Two species of the family Urticaceae, Pilea yadongensis and Elatostema dingjieense, are described as new from Xizang Autonomous Region. The diagnostic differences between them and their allies are given respectively.

Beccarinda baolianis,A New Species of Gesneriaceae from Fujian Province

LIN Qin-Wen

2016, 36(5):  650-652.  doi:10.7525/j.issn.1673-5102.2016.05.003

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A new species of the genus Beccarinda(Gesneriaceae), B.baolianis, is described from Fujian Province, China. The new species is distinguished from other species of this genus by its green and 5 middle-lobed calyx, and 4 free stamens.

Reduction on Littledalea tibetica Based on the Generalized Morphological Characteristics

ZHOU Yong-Hui, LIU Yu-Ping, LÜ Ting, ZHANG Xiao-Yu, CHEN Zhi, SU Xu

2016, 36(5):  653-659.  doi:10.7525/j.issn.1673-5102.2016.05.004

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We analyzed the differences among nearest related Littledalea tibetica, L.racemosa and L.przevalskyi in the genus Littledalea by morphological observation and anatomical analysis of leaf epidermises. There was very slight disparity between L.tibetica and L.racemosa in external morphology, the classification of the two species is very difficult, and the differences between L.tibetica and L.przevalskyi are distinct, the two taxa are two different species. Anatomically, the most features of L.tibetica are consistent with or similar to that of L.racemosa, while for L.przevalskyi, the discontinuity of characters between them is obviously present. Therefore, L.tibetica and L.racemosa should belong to the same species, and L.tibetica can be classified to synonymy of L.racemosa.

Anatomical Characteristics of Vegetative Organs of Two Alpine Crabapples and Their Ecological Adaptation

XU Yang, CHEN Xiao-Hong, LI Hui-Min, DING Yin-Peng, KANG Xi-Kun, LI Qian

2016, 36(5):  660-668.  doi:10.7525/j.issn.1673-5102.2016.05.005

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The experiment was conducted to study the anatomical characteristics of alpine wild crabapples and their ecological adaptation, the vegetative organ structures of Malus toringoides and Malus transitoria from 3 000 m altitude in western Sichuan Plateau with paraffin sectioning and NaOCl. The two crabapples had similar anatomical structures. The possible reason might be their closed relationships of origins and geographic distribution between them. Both of the two plants had thick cuticle and epidermis cells. There was not stomata on the upper epidermis, while a dense stomata distribution was observed on the lower epidermis. The collateral vascular bundle was rich in palisade tissues on the lower epidermis. The longitudinal ridges structure with palisade and sponge tissues existed in the petiole. The phelloderm cells were crassexinous. The secondary xylem was rather rich in the stem. There were some wood parenchyma cells with lignified cell wall in the vicinity of the vessels. Around the pith there were crassexinous cells, called perimedullary zone. The secondary structure existed in the thin roots of both of the two crabapples, in which there was a large proportion of xylem and no pith. The vessels were dense, in the form of solitary pore. Therefore, the two alpine crabapples adapted well to the dry environment of western Sichuan Plateau.

Leaf Anatomical Structure of Populus euphratica in Tarim River Basin

HUNG Wen-Juan, JIAO Pei-Pei, HUANG Jin-Hua, ZHANG Dan

2016, 36(5):  669-675.  doi:10.7525/j.issn.1673-5102.2016.05.006

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From the regional scale, three natural ranges of Populus euphratica in Alar, Luntai and Yuli County with different climate and soil conditions were chosen from the upper, middle and lower reaches of Tarim River Basin, respectively. In each site, 8 plots with the same size of 50 m×50 m were chosen and within each plot, leaves from 3 adult trees of P.euphratica were selected randomly. Routine paraffin sectioning method was used to observe and compare the leaf anatomical characters of P.euphratica, and explore their adaptive strategies and anatomical features to adapt to different environmental conditions. The results showed:(1)The basic leaf anatomical characters from 3 distribution areas were consistent with each other, and showed:a. The upper and lower epidermis were both mutiple epidermis and consisted of double layer of cells; b. Palisade tissue of mesophyll was differentiated close to upper and lower epidermis, and the leaf belonged to isolateral leaf. The mucous cells which relatively deep stained were also visible in the mesophyll tissue. c. The "bundle sheath" consisting of thick-walled cells was visible outside the vein vascular bundle. (2)From the whole point of the Tarim River Basin, there were vary degrees of variations among the leaf anatomical structure indexes. The range of variation coefficient was from 0.029 to 1.786, and the thickness variation coefficient of upper epidermal cells was smallest, while the blade thickness was largest. (3)The quantitative values of leaf anatomical structure index varied in different areas:leaf thickness, palisade tissue thickness, cuticle layer thickness, lower epidermis thickness and the grid/sea showed Yuli County >Luntai County >Alar County, which indicated that P.euphratica stored water and reduced water loss to adapt to the relatively dry environment through increasing the thickness of leaf, cuticle layer and epidermal cells. (4)There was significant correlation between the structure indexes and altitude, latitude and longitude. The thickness of upper epidermal and upper cuticle layer were positively correlated with longitude significantly(P<0.05)and highly significantly(P<0.01). The leaf thickness, mesophyll thickness, palisade tissue thickness and grid/sea were negatively correlated with longitude highly significantly(P<0.01). The thickness of upper epidermal, upper cuticle layer and grid/sea were negatively correlated with altitude significantly(P<0.05)or highly significantly(P<0.01).

Megasporogenesis, Microsporogensis,and Male and Female Gametophyte Development of Ribes mandshuricum

ZHENG Bao-Jiang, ZHANG Yi-Fan, WANG Mei-Juan, WANG Hai-Zhu, XU Qi-Jiang

2016, 36(5):  676-682.  doi:10.7525/j.issn.1673-5102.2016.05.007

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We used normal paraffin sectioning method to study the megasporogenesis and microsprrogenesis and the development of male and female gametophytes during development process of Ribes mandshuricum(Maxim.) Kom. The flower of R.mandshuricum has five stamens with four locules in each. During meiosis of the mother cell, the division of the cytoplasm can be attributed to the simultaneous type. The tetrad of microspores resulted from the meiosis are simultaneously bilateral and tetrahedral. The mature pollen grains are two-celled. The development of the anther wall belongs to an elementary type. The anther wall can be divided into five layers from outside to inside, including epidermis, endothecium, two middle layers and tapetum. Tapetum belongs to glandular type. The development of embryo sac belongs to the type of polygonum. R.mandshuricum is with one ovary, the ovules in which are anatropous, numerous, crassinucellate, and double integuments. The megasporocyte divides into four linear megaspore in the form of meiosis. The megaspore in chalazal end has a functional ability. There are some abnormal phenomena in the development of the anther, leading to the pollen degeneration.

Effect of Water Table and Neighbor on Three Mosses in Hani Peatland in Changbai Mountains

GU Xiao-Nan, BU Zhao-Jun, GE Jia-Li, LIU Sha-Sha, JIANG Tao, LU Meng, GUO Zhen-Yu, DING Ji-Zhao

2016, 36(5):  683-691.  doi:10.7525/j.issn.1673-5102.2016.05.008

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The dominance of non-Sphagnum mosses such as Polytrichum strictum has increased in some peatlands of NE China while the mechanism is not clear. We performed a one-year experiment in Hani Peatland with three mosses, Sphagnum palustre, S.magellanicum and P.strictum in two typical habitats with different water table depth. We tried to explore the effects of water table and neighbor on morphological traits including height increment, biomass production and side-shoot production and biochemical traits including carbon and nitrogen content and soluble sugar content. Two Sphagnum mosses both responded to water table drawdown with the increasing of side-shoot production and nitrogen content but decreasing height increment. S.magellanicum also responded with the decreasing of soluble sugar content. Except for the inhibitive effect on biomass production and the facilitation effect on side-shoot production, no other traits in P.strictum were affected by water table drawdown. In mixed culture, two Sphagnum species mutually promoted their height increment and biomass production. Side-shoot production in S.palustre and carbon content in S.magellanicum were negatively affected by neighbor P.strictum, while neither neighbor had any effects on P.strictum. Although P.strictum did not have a direct competition effect on two Sphagnum mosses, and by virtue of its good drought tolerance, it could still get the competitive advantage and realize population expansion in peatlands in the background of climate warming and water table drawdown resulted from disturbance by human beings.

Leaf Wax Components and Surface Structure of Tillandsia Species

ZHENG Gui-Ling, ZHANG Wei, LI Peng

2016, 36(5):  692-696.  doi:10.7525/j.issn.1673-5102.2016.05.009

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Tillandsia is a kind of special plant which absorbs water and nutrients directly from the air through leaves. In order to investigate the function of the leave, five Tillandsia species were chose to analyze their leave surface structures with scanning electron microscope(SEM) and wax components with gas chromatography-mass spectrometry technology(GC-MS). The white, waxy, sunflower-shaped trichomes covered the leaf surface in all species. The foliar trichomes were made up of disc cells, ring cells and wing cells. The main wax components contained low-polarity constituents, including hydrocarbons, alcohols, ketones and esters, which may reduce the water loss effectively. In addition, the total contents of wax components among five Tillandsia species were different with the order of Tillandsia ionantha >Tillandsia brachycaulos >Tillandsia velutina >Tillandsia medusa >Tillandsia streptophylla. By the correlation analysis, the total contents of wax components were positively correlated with the total leave area of foliar trichomes, indicating that those wax components were distributed mainly on the foliar trichomes.

Barrier Zone Formation in Agarwood Formation of Aquilaria sinensis

LIU Pei-Wei, YANG Yun, ZHANG Yu-Xiu, YANG Yong, LIU Yang-Yang

2016, 36(5):  697-704.  doi:10.7525/j.issn.1673-5102.2016.05.010

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With the sliding section, histochemical staining, paraffin section, scanning electricity microscope and spectrophotometry, we studied the anatomical structure and histochemcial characteristics of barrier zone. The barrier zone was arisen from interxylary phloem. Barrier zone in different thickness located between agarwood zone and transition zone. The barrier zone was formed of abnormal parenchyma and xylem. Barrier zone contained a small amount of starch and more total sugar. Our results might be used to eliminate and delay the barrier zone, and improve whole-tree agarwood-inducing technique.

Leaf Dvelopment and Its Ecological Adaptability of Cercidiphyllum japonicum

MA Yong-Hong

2016, 36(5):  705-711.  doi:10.7525/j.issn.1673-5102.2016.05.011

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Using the paraffin-section method, we studied the morphological variations and its ecological adaptability of Cercidiphyllum japonicum, based on the developmental regularities and difference between the immature leaf and mature leaf in anatomical structure. Leaf development were existed in three stages of leaf margin curl inward, expand and mature stage. The first and second pair immature leaves base have a well developed membranous sheath, with the most developed sheath on the first pair immature leaves and more developed sheath on the second, but not found on the others. The leaf of C.japonicum is typical bifacial leaf with developed spongy parenchyma, outer convex stoma apparatus in the upper and lower epidermis, developed cuticle, vein, mechanical tissue, a amphicribral vascular bundle in min-petiole and bundle sheaths, and the C.japonicum is with strong ecological adaptability to the drought and humid conditions. There are more ergastic substance stored in vegetative organs, which can offer the main nutrition for organ during in breeding season. The first pair immature leaves with faster growth rates also satisfy little nutritional requirements for other organ development.

Species Diversity of Ground Bryophyte Communities in Junger Loess Hill-Cully Region in Inner Mongolia

HONG Xia, TIAN Gui-Quan, WU Ri-Gumala

2016, 36(5):  712-720.  doi:10.7525/j.issn.1673-5102.2016.05.012

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Based on the quadrat survey of ground bryophytes community under nine kinds of vegetation in Junger loess Hill-Cully, we studied the species diversity and its relationship with the environmental factors. (1)We found 8 families, 18 genera, and 35 species bryophytes, among which Pottiaceae and Bryaceae were dominant families, and Bryum argenteum, Didymodon vinealis, Brachythecium salebrosum, Bryum bicolour, Hypnum cupressiforme var. lacunosum, etc were dominant species; (2)Among the Alpha diversity index, Patrick index was 4-21 species, Shannon-Wiener index and Simpson diversity index were 1.078 0-2.153 3 and 0.588 9-0.826 3, respectively, and the order in natural vegetation was Juniperus rigida forest >Artemisia giraldii shrub >Prunu davidiana forest >Caragana intermedia shrub >Stipa bungeana steppe >Thymus serpyllum steppe. The three indexes of artificial forest of Populus simonii forest and Platycladus orientalis forest were close to those of the Caragana intermedia shrub, and Pinus tabulaeformis forest was close to those of the Juniperus rigida forest; (3)The main ecological factors affecting the diversity of bryophytes were soil water content, air humidity and light intensity; (4)Bata diversity index was 0.240 0-0.857 1, the species similarity between Stipa bungeana steppe and Artificial Populus simonii forest was the highest, and was the lowest between Juniperus rigida forest and Thymus serpyllum community. The bryophyte community in Juniperus rigida forest, the key area for bryophyte diversity conservation in Junger loess Hill-Cully, had the richest species diversity.

Overexpression of DmDREBa Gene Significantly Enhances Low Temperature Tolerance in Transgenic Tobacco

YANG Yan-Fang, WU Jian, ZHU Kai, LIU Li-Qing, CHEN Fa-Di, YU De-Yue

2016, 36(5):  721-729.  doi:10.7525/j.issn.1673-5102.2016.05.013

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Many environmental factors such as drought, salt stress, hormones and low/high temperature influence the growth and development of plants. It has been found that DREB transcription factors play important roles in plants against different abiotic stresses. In this study, DmDREBa(GenBank accession No.EF490996), a DREB-like gene isolated from chrysanthemum was overexpressed in tobacco plants by mediation of Agrobacterium tumefaciens under the control of the constitutive promoter CaMV 35S, and 43 transgenic tobacco plants were obtained by PCR method. 9 transgenic tobacco plants were randomly selected to perform RT-PCR investigation and 7 plants indicated RNA transcripts accumulation. The southern blot result showed that 1-3 copies of DmDREBa were randomly inserted. The tolerance of transgenic tobacco plants was increased comparing with the wild type tobacco plants under low temperature condition. The water loss of transgenic tobacco was less than wild type plants after natural transpiration. Moreover, the density of the stomata in the epidermis was observed. The result showed that the numbers of stomata in upper or lower epidermis of transgenic plants were reduced comparing to wild type tobacco. The result of t test demonstrated that the differences between the upper and lower epidermis of transgenic plants and wild type were significant. Furthermore, the content of malondialdehyde(MDA) in transgenic plants was lower than that in wild type tobacco plants at lower temperature condition, which indicated the tolerance ability of transgenic plants was increased. All these results indicated that DmDREBa improved tolerance ability of trangenic tobacco plants to the lower temperature stress. This research will provide a very useful reference for functional analysis of DREB genes in chrysanthemum in the future.

Bud Inducing from 16-year Elite Trees Explants of Three Acacia Species

WANG Hong, HUANG Lie-Jian, HU Feng

2016, 36(5):  730-738.  doi:10.7525/j.issn.1673-5102.2016.05.014

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The explants from 16-year elite trees of Acacia mangium, A.auriculiformis and A.mangium×A.auriculiformis were used to establish the efficient bud inducing technique, by studying the sterilization methods, position, gathered time and type of explants, the effects of 6-BA, sucrose, and basal medium. A.mangium was disinfectant sensitive, treatment with mercuric chloride 9 min and ethanol 15 s was the optimal, but longer time were need for A.auriculiformis and A.mangium×A.auriculiformis, treatment with mercuric chloride 18 min, ethanol 15 s and mercuric chloride 15 min, and ethanol 30 s, respectively. These three Acacia species shared common optimal position, gathered season and type:3-5^th node of shoot as explants were optimal position. It was the optimal season to gather explants in August; Type 2 explant germinated from shoots of cutting orchard (established by cutting the shoots of mature elite) was the optimal type; The optimal bud inducing medium were modify MS+6-BA 0.5 mg·L^-1+sucrose 30 g·L^-1 for A.mangium, MS+6-BA 1.0 mg·L^-1+sucrose 40 g·L^-1 for A.auriculiformis, MS+ sucrose 20 g·L^-1 for A.mangium×A.auriculiformis, with the highest germination rate of 93.33%, 92.00% and 97.33%, respectively.

Rooting Impact Factors of Callicarpa nudiflora Softwood Cuttings

LIU Shi-Chao, ZHOU Zai-Zhi, ZHANG Jin-Hao, LIANG Kun-Nan, MA Hua-Ming, HUANG Gui-Hua

2016, 36(5):  739-746.  doi:10.7525/j.issn.1673-5102.2016.05.015

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With two-year softwood cuttings, five experiments were conducted to study the effects of substrate, cutting portion, style of keeping leaves, cutting season, different types and contents of exogenous hormones on rooting of Callicarpa nudiflora cuttings. Rooting rate, hyponastic rate, rooting number and the maximum root length were measured, and a subordinate function value was used to comprehensively evaluate the rooting quality. The optimum substrate among the eight treatments was peat soil:vermiculite:river sand=2:1:1 in volume ratio, and the rooting rate and average rooting number were 74% and 5.4, respectively. The upper and middle portion of cutting were more easily for rooting than the bottom portion, but the middle portion was ranked the best. Cuttings with leaves significantly produced a higher rooting rate with comparison of leaves off. Cuttings with a pair of half leaf got the maximum rooting rate of 64%. Autumn(mainly in September) was the optimal season for cutting and its rooting number and maximum root length were significantly higher than that of spring(April) and summer(July). The cutting effect of IBA treatments within four concentration levels were all superior to that of IAA and NAA treatments. Cuttings treated with IBA(1 000,1 500 mg·L^-1) showed better rooting quality significantly, while cuttings treated with high concentration levels of NAA(2 000,2 500 mg·L^-1) were obviously not conducive to root.

Characteristics of SSR and SNP in Gentiana veitchiorum in Qinghai-Tibetan Plateau,by High-throughput Sequencing

TIAN Zun-Zhe, GAO Qing-Bo, CHEN Shi-Long, ZHANG Fa-Qi

2016, 36(5):  747-752.  doi:10.7525/j.issn.1673-5102.2016.05.016

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We used the next generation sequencing(NGS) to capture the SSR and SNP marker in Gentiana veitchiorum, sampled in Zeku, Qinghai Province. A total of 8588 simple sequence repeats were generated through lumina HiSeq^TM 2500. The dinucletide repeats were the highest(54.7%), followed by the trinucleotide repeats(41.3%). A proportion of tetranucletide, pentanucleotide and hexanucleotide repeats was less, which is 4% in total. In dinucleotide repeats, AT/TA repeats were the dominated ones, 9.85% and 9.5%, respectively. The length of nucleotide repeat and number of repeat were negative correlation, while there was a positive correlation between the total length of SSR sequences and the length of nucleotide repeat. A total of 253 789 and 249 417 SNPs were indenfied in GP-F and GP-L of G.veitchiorum. The proportion of the SNPs located in the noncoding region were 51.29% and 51.96%, respectively. The proportion of synonymous transition(48.63% and 47.96%) in coding region was significantly higher than that of nonsynonymous transition(0.08% and 0.08%), which might be caused by relatively conservative domains of functional genes.

Cloning and Expression Analysis of CjCor1 Gene cDNA from Camellia japonica(Nai Dong)

YANG Kai, SUN Ying-Kun, WANG Kui-Ling, LIU Qing-Hua, GAO Han-Dong

2016, 36(5):  753-759.  doi:10.7525/j.issn.1673-5102.2016.05.017

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COR genes play important roles in plant cold acclimation process. In order to study the Cor gene function of Camellia japonica to severe cold, adopting homologous cloning method, a 617 bp full-length cDNA sequence was cloned from leaves of C.japonica(Nai Dong) under the natural low temperature condition, named CjCor1. The total length sequence is complete, and contains an opening reading frame of 600 bp. Amino acids sequence alignment shows that CjCor1 gene encodes a protein of 199 amino acids, containing specific conservative domain structure of WCOR413 in cold acclimatization protein superfamily, and highly conservative property in species belonged to same genus. By real-time PCR analysis, comparing with southern Camellia species and common varieties, CjCor1 genes had higher expression in C.japonica(Nai Dong) under the condition of low temperature(below 0℃), and also had higher expression in higher cold resistance varieties.

Influence of Endogenous IAA and GA₃ on in vitro Lily Seedling Growth

YANG Liu, WANG Le, XIE Zhong-Kui, GUO Zhi-Hong, ZHANG Yu-Bao

2016, 36(5):  760-767.  doi:10.7525/j.issn.1673-5102.2016.05.018

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The in vitro seedlings of Lilium oriental ‘Sorbonne’ were used to study the response of plants to exogenous IAA and its inhibitor (2,3,5-Triiodobenzoic acid, TIBA), including the growth of stems, leaves and roots, and the dynamic change of endogenous hormones was analyzed by HPLC. The data showed that 10 mg·L^-1 IAA have the best significant role on growth of stems, leaves and roots simultaneously while 40 mg·L^-1 IAA and TIBA treatments inhibited growth significantly. By the hormone analysis, the endogenous hormones was influenced by exogenous IAA and TIBA application, the effect of IAA on growth was achieved probably by endogenous hormones regulation. Analysis based on the growth and the change of endogenous hormones revealed that higher level of endogenous IAA and lower level of GA₃ were disadvantageous for growth, and endogenous IAA and GA₃ played optimal role in their optimum concentrations. Endogenous Zeatin perhaps did not take part in the regulation of seedling growth. These results would be helpful in the rational use of exogenous hormones in lily planting and useful to improve economic benefit of growers.

Leaf N and P Stoichiometry of Leymus chinensis in Relation to Soil Properties in Saline-alkali Degraded Grassland

ZHAO Long, WANG Hua, LIANG Zheng-Wei, ZHANG Zhong-Hua, TANG Zhong-Hua, NIE Si-Ming, ZHAO Dong-Mei

2016, 36(5):  768-774.  doi:10.7525/j.issn.1673-5102.2016.05.019

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N and P stoichiometry of constructive species of a climax community has an important influence on the stability and productivity of community. We chose Leymus chinensis, the constructive species of Songnen grassland community to study the effects of grassland degradation on N and P stoichiometry of L.chinensis under saline-alkali stress. There were lower leaf P content(1.32 g·kg^-1), medium leaf N content(18.90 g·kg^-1) and slightly higher N/P(15.83) in degraded L.chinensis grassland compared with the L.chinensis grassland in the north of China. Leaf N and N/P were significantly positively correlated with soil total nitrogen and soil alkaline hydrolysis nitrogen. Leaf P was significantly negatively correlated with soil total nitrogen and soil alkaline hydrolysis nitrogen, but other factors were not significantly correlated with N, P and N/P. Both the significant correlations between leaf P content and soil N content and departure from leaf P content and soil P content common displayed that there might be a limitation of P in Songnen Plain at the saline alkali degradation of L.chinensis grassland.

Characterization of Microsatellites in Genome of Pinus squamata, a Critically Endangered Species in the World

RUAN Zhen-Yuan, WANG Bing-Yi, OUYANG Zhi-Qin, LIAO Li-Bin, SU Teng-Wei, QIAO Lu

2016, 36(5):  775-781.  doi:10.7525/j.issn.1673-5102.2016.05.020

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Pinus squamata is a critically endangered species in the world, therefore, developing simple sequence repeat(SSR) primers will facilitate the species genetic research and conservation. The complete genome sequence of P.squamaia was determined through Illumina genome analyzer, and 2 651 microsatellites were discovered by MISA software. Single nucleotide repeat number occupied the highest proportion of these microsatellites which may indicate a long evolution history of P.squamata. Among different repeat types, A/T contents was significantly higher than G/C contents. In terms of distinct length repeat units, the length of dinucleotide repeat microsatellite varies with the highest degree. The length of SSRs with different repeat type was negatively correlated with frequency of occurrences. The available microsatellite sequences can satisfy P.squamaia population genetics, and reveal the species bias and corresponding potential functions.

Comparative Analysis of the Performance of Different Lysis Buffers for Measuring the Ploidy Level of Salicaceae Species

GUO Wei, HU Nan, LI Xiao-Ping, LI Shu-Xian

2016, 36(5):  782-789.  doi:10.7525/j.issn.1673-5102.2016.05.021

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Polyploid breeding is an important approach in the breeding programs of Salicaceae species. Flow cytometer was commonly used to measure the ploidy level of plants. However, lysis buffer affects the result of ploidy measurement significantly. In this study, a variety lysis buffers were tested to isolate nuclei from leaf cells of different Salicaceae species. Subsequently, the nuclei suspensions were measured by a flow cytometer to obtain the values of parameters including forward light scatters(FS), side light scatters(SS), relative fluorescence intensity of propidium iodide-stained nuclei(FL), coefficient of variation of G₀/G₁ DNA peaks(CV), and debris background factor(DF). By analyzing these parameters, SLB-3 was the optimal lysis buffer in measuring the ploidy level of Salicaceae species, as it had a higher fluorescence intensity and a low coefficient of variance. SLB-3 buffer contains 0.5 mmol·L^-1 spermine·4HCl, 30 mmol·L^-1 sodium citrate, 200 mmol·L^-1 Tris, 80 mmol·L^-1 KCl, and 0.5%(v/v) Triton X-100 with pH of 7.5. In conclusion, there was a reliable experimental protocol for measuring the ploidy level of Salicaceae species, which provided a useful tool for the polyploidy breeding program in these species.

Preliminary Studies on Hybridization among Sympatric Distributed Calligonum Species in Northern Xinjiang

KANG Xiao-Shan, Sophia, DUAN Shi-Min, PAN Bo-Rong, ZHANG Yong-Zhi, TIAN Cong

2016, 36(5):  790-794.  doi:10.7525/j.issn.1673-5102.2016.05.022

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Hybridization is one key issue in taxonomy of the genus Calligonum. To certificate whether or not hybridization are existing among sympatric distributed Calligonum species, observations of flowering phenology both in natural habitats and botanical garden, artificial pollination hybridization experiments and fluorescence microscope observation on cross-pollination of pollen tubes were carried on. Florescence among the sympatric species was overlapped. Fruit rate by artificial pollination was very low, and could not germinate. Pollen tube fluorescent experiments showed that hybrid pollen could germinate on the stigma, but the pollen tube could not stretch to the ovary. Above all, affinity of interspecific gametes was low, reproductive isolation was existing, and hybridization was absent. This study has important implications for understanding the isolation and differentiation of sympatric Calligonum species.

A Protocol for the Isolation and Purification of Protoplast from Bletilla striata Leaves

XU De-Lin, ZHANG Lin, CHU Shi-Run, WEI Xin, QIAN Gang, ZHENG Ming-Hui

2016, 36(5):  795-800.  doi:10.7525/j.issn.1673-5102.2016.05.023

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To construct a protocol of isolation and purification of protoplasts from the seedling leaves of Bletilla striata. After a series of pretreating assay, the leaves were enzymatic hydrolyzed with mixed solution of mannitol, Cellulase R-10, Pectolyase Y-23 and Macerozyme R-10 for certain hours. Then the newly released protoplasts were purified through filtration and centrifugalization. Finally, the harvest protoplastswere detected in the survival rate by means of fluorescein diacetate (FDA). The preprocessing of seedlings living in dark field for 12 h and the leaves were immersed in 0.75 mol·L^-1 mannitol for 1 h was the most effective way for protoplasts isolation. The enzymes mixture of 0.75 mol·L^-1 mannitol+1.5% Cellulase R-10+0.4% Pectolyase Y-23+0.5% Macerozyme R-10 was the optimism composition of enzyme solution. The pH of 5.8 and 4 h enzyme dissolve with the revolving speed of 120 r·min^-1 at room temperature was the best environmental condition. Under this optimal condition, abundant of 4.72×10⁶ protoplasts with survival rate of 90.4% could be harvested from each gram leaf. This study gave a necessary help for protoplasts culture, cell fusion and genetic transformation of B.striata.