In Vitro Regeneration from Leaf Explants of Pineapple (Ananas comosus L, Merr)

Multiple shoots were regenerated from leaf explants obtained from in vitro grown shoot cultures of pineapple. Each leaf was horizontally cut into three pieces (～ 0.5 cm, basal, middle and tip) and cultured onto MS basal medium supplemented with 2% sucrose and various growth regulators.The explant containing the basal part of the leaf gave rise to tiny protuberances which grew into shoots.The highest number of shoots were obtained on MS basal medium supplemented with 2,4-D (0.90 µM) and 2iP (0.98 pM).These shoots were subcultured ontowhite’s basal medium supplemented with 1% sucrose, NAA (0.54 µM) and IBA (1.97 µM). Plantlets produced in vitro were transferred to paper cups containing autoclaved soil or Soilrite, hardened in the greenhouse and established in soil.The protocol provides an easy propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Ultrastructure of the Water-absorbing Trichomes of Pineapple (Ananas comosus, Bromeliaceae)

An ultrastructural study of dry and wet trichomes from the upperleaf surface of pineapple revealed that shield cells were normallydead and stalk cells were often living. In stalk cells of drytrichomes, polysaccharide material was present within vacuolesand a layered material was present between the plasma membraneand cell wall. In stalk cells of trichomes wet for 12 h vacuoleswere enlarged and appeared to contain little material. Layeredmaterial between the plasma membrane and cell wall was alsoabsent. In both wet and dry trichomes, numerous mitochondriawere present and plasmodesmata were observed connecting stalk,foot, and adjacent epidermal and mesophyll cells. It is suggestedthat the large numbers of mitochondria, which indicate highmetabolism, and the numerous plasmodesmata connecting the cells,provide indirect evidence for the function of the trichomesin uptake of dissolved nutrients. Ananas comosus (L.) Merr., pineapple, trichomes, ultrastructure, Bromeliaceae     

菠萝种质目标起始密码子(SCoT)遗传多样性分析

利用SCo T分子标记技术对来自9个国家或地区的46份菠萝种质进行了遗传多样性分析,并对SCo T标记在菠萝研究中的效率做了探讨。结果表明,SCo T标记在菠萝种质中具有丰富的多态性,引物多态性条带百分比在75%~100%之间,平均为94.61%;引物的有效等位基因数(Ne)、Nei's基因多样性指数(H)、Shannon's信息指数(I)和多态性信息含量(PIC)平均值分别为1.45、0.27、0.41和0.28,表明SCo T标记具有较高的多态性检测效率。基于SCo T标记计算获得的遗传相似系数对菠萝种质做聚类分析,46份菠萝种质可被划分为5个类群,其中,第Ⅰ类群所包含的菠萝种质数量最多,占菠萝种质总数的84.78%。主成分分析获得了与聚类分析不尽一致的结论,但两者反映的种质亲缘关系基本一致。本研究结果将为我国菠萝种质的鉴定、保存和科学利用提供一定的理论依据。     

Polymorphic microsatellite and cryptic simple repeat sequence markers in pineapples (Ananas comosus var. comosus)

A rapid method for isolating microsatellite loci in pineapples, based on the 5′‐anchored polymerase chain reaction technique, revealed 137 microsatellite loci (consisting of 62 dinucleotide, 24 trinucleotide, 49 tetranucleotide and 2 hexanucleotide repeats) and 16 cryptically simple repeat sequences. We report on the characterization of 19 polymorphic microsatellite loci and one cryptic simple repeat loci in pineapples. The number of alleles per locus ranged from two to four while the observed heterozygosity ranged from 0.1705 to 1. These markers are useful as tools for detecting levels of genetic variation in pineapple varieties for germplasm management and crossbreeding purposes.     

Routes of Ethephon Uptake in Pineapple (Ananas comosus) and Reasons for Failure of Flower Induction

Ethylene-releasing agents such as ethephon (2-chloroethylphosphonic acid) are used widely to induce flowering in pineapples (Ananas comosus (L.) Merrill). However, ethephon treatment is less reliable in summer, particularly if plants are treated on abnormally hot days. [¹⁴C]ethephon was used to follow uptake and translocation in leaf tissues. Up to 30% of the ethephon entered the leaf within 4 h, and up to 60% by 24 h. Uptake was dramatically modified by temperature, relative humidity, solution pH, and the surface on which solution droplets were placed. Entry occurred across the leaf cuticle and probably also by way of stomatal pores, and label was recovered at all depths within the leaf. ¹⁴C label entered more rapidly through the abaxial epidermis than through the adaxial epidermis. Low-volume spray applications to whole plants resulted in rapidly drying droplets mainly on the adaxial, distal epidermis and were rather ineffective at inducing flowering, possibly because little ethephon or ethylene reaches the shoot apex. High-volume sprays may facilitate ethephon entry because solution accumulates in leaf axils and hence remains in prolonged contact with abaxial epidermis of leaf bases close to the shoot apex. When poured into the center of the plant, 20% of a normal commercial ethephon dose induced full flowering even under adverse temperatures. It is suggested that high-volume evening spraying and avoidance of hot days may reduce the incidence of flowering failure. Received March 20, 1998; accepted September 6, 1999     

菠萝花发育相关基因AcMADS1的克隆与组织表达特性分析

MADS-box转录因子在植物的发育过程特别是控制花器官的诱导与发育中起关键作用。利用同源克隆结合RACE技术, 从菠萝(Ananas comosus)花中分离出1个新的菠萝MADS-box基因, 命名为AcMADS1(GenBank登录号为KC257408)。AcMADS1基因的编码区为726 bp, 编码241个氨基酸, 蛋白质分子量为27.50 kDa, 等电点为9.26。序列比对和系统进化树分析表明, AcMADS1具有保守的MADS-box及半保守的K区, 属于AGL6亚家族MADS-box蛋白。生物信息学分析表明, AcMADS1是亲水碱性蛋白, 二级结构主要以α-螺旋、无规则卷曲和折叠延伸链为蛋白质骨架, 三级结构中蛋白核心结构符合转录因子与DNA结合的常见功能域MADS-box, 而且作为转录因子定位于细胞核中。组织特异性表达分析表明, AcMADS1基因在菠萝果肉以及花器官的雌蕊、花瓣和萼片中均有表达, 但在雄蕊以及营养器官的根、茎和叶中几乎不表达; 且在花器官早期发育过程中大量表达, 后期呈下降趋势。因此推测这个基因可能在菠萝花器官发育和开花诱导过程中起重要作用。     

16S Ribosomal DNA Characterization of Nitrogen-Fixing Bacteria Isolated from Banana (Musa spp.) and Pineapple (Ananas comosus (L.) Merril)

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.     

Carbon Dioxide Exchange and Acidity Levels in Detached Pineapple, Ananas comosus (L.), Merr., Leaves during the Day at Various Temperatures, Oxygen and Carbon Dioxide Concentrations

The effects of temperature, O₂, and CO₂ on titratable acid content and on CO₂ exchange were measured in detached pineapple (Ananas comosus) leaves during the daily 15-hour light period. Comparative measurements were made in air and in CO₂-free air. Increasing the leaf temperature from 20 to 35 C decreased the total CO₂ uptake in air and slightly increased the total CO₂ released into CO₂-free air. Between 25 and 35 C, the activation energy for daily acid loss was near 12 kcal mol⁻¹, but at lower temperatures the activation energy was much greater.     

16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril)

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.     

Earthworm Communities in the Pineapple (Ananus comosus) and Mixed Fruit Plantations of West Tripura, India

Present studies on the community characteristics of earthworms revealed the occurrence of 11 species of earthworms in the pineapple (Ananus comosus) and 14 species in the mixed fruit plantations of west Tripura (India). While 9 species of earthworms namely Drawida assamensis, Drawida papillifer papillifer, Drawida nepalensis, Kanchuria sp., Metaphire houlleti, Eutyphoeus gigas, Eutyphoeus scutarius, Eutyphoeus comillahnus and Pontoscolex corethrurus are of common occurrence to both the pineapple and the mixed fruit plantations, two and five earthworm species namely Kanchuria sumerianus, Eutyphoeus sp. and Metaphire posthuma, Perionyx excavatus, Lampito mauritii, Amynthus alexandri, Eutyphoeus gammiei are restricted to the pineapple and the mixed fruit plantations respectively. Earthworms were found mostly within 15 cm depth of soils having temperature 25–25.8 °C, moisture 18.8–22.4 %, water holding capacity 26–31.7 % and organic matter content 2.4–4.0 %. Mean earthworm densities (158 ind. m^?2) was significantly higher (p < 0.01, t = 9.67) and biomass (36.67 g m^?2) significantly lower (p < 0.01, t = ?5.98) in the pineapple plantation than the mixed fruit plantation (density 93 ind. m^?2, biomass 56 g m^?2). High density value of earthworms in pineapple plantation is linked with dominance of D. assamensis and high biomass value in mixed fruit plantation was due to the higher relative abundance of larger species like E. gigas, E. scutarius, E. comillahnus and E. gammiei. Compared to the mixed fruit plantation, significantly (p < 0.05) higher index of dominance, lower index of diversity, species richness index and species evenness were recorded in the pineapple plantation.     

Development of Pseudocercosporella herpotrichoides (Fron) Deighton var. herpotrichoides and var. acuformis on Wheat Plants Measured by ELISA

Using the ELISA method, the development of Pseudocercosporella herpotrtchoides var. herpotrichoides and var. acuformis in a susceptible cultivar of winter wheat was compared under controlled and held conditions. In the greenhouse, var. acufornis grew less vigorously, was slower in penetrating the coleoptile and the successive leaf sheaths and in colonizing the stem tissue than var. herpotrichoides. In the field, these differences were confirmed on the last leaf sheaths and the stem. At ripening stage, however, identical ELISA values were measured for both varieties. Moreover, a significant variation was observed between the individual isolates of each variety. Comparison of the effect of both varieties of P. herpotrichoides on 20 wheat cultivars characterized by different resistance levels showed significant interactions. The cultivars carrying the Pch-1 gene always remained the less diseased genotypes. In general, var. acuformis developed less antigen in the cultivars than var. herpotrichoides. It is concluded, that in tests for resistance to P. herpotrichoides mixtures of many highly pathogenic isolates of both fungus varieties should be used. Less complex mixtures or single isolates may result in wrong estimates of resistance.     

Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro

Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20–80 fold more than in anther but similar to that of non-embryogenic callus, which was 20–50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity.     

Flowering and Growth Response of Peanut Plants (Arachis hypogaea L. var. Starr) at Two Levels of Relative Humidity

Peanut plants (Arachis hypogaea L. var. Starr) grown under two different relative humidities were used in all experiments. All plants were germinated and grown to flowering in the greenhouse. At this time, one group was moved to a growth room under 95% relative humidity. After 50 days the humidity of the growth room was lowered to 50%. The second group was moved into a growth room at 50% relative humidity and after 50 days the humidity was raised to 95%.     

菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。