Effect of ABA,arginine and sucrose on protein content of date palm somatic embryos

Abscisic acid (ABA), arginine and sucrose were evaluated for their effects on the morphology, germination rates and protein content of date palm somatic embryos (SE). Different concentrations of these supplements in the culture medium were used. The comparative study of SE length and thickness between treated and untreated SE revealed no differences, except for ABA (20 μM), which increased thickness. A decrease of water content (WC) in favor of an increase in dry weight (DW) was observed in all treated SE, especially with sucrose (90 g l⁻¹) and ABA (20 μM). Only ABA (20 and 4 μM) caused a proliferation rate of the cultures higher than those in the control. Although all the tested compounds increased protein content, ABA (20 μM) was more effective in protein enrichment than arginine and sucrose treatments. The SDS-PAGE protein profiles showed a significant difference between treated and untreated SE. A protein band of 22 kDa, identified as glutelin in a previous work, was accumulated after treatment with 20 μM ABA or 3 mM arginine. These findings may contribute to further understanding of the mechanisms involved in the accumulation of specific storage proteins in several plants.     

Varying plant density and harvest time to optimize cowpea leaf yield and nutrient content

Plant density and harvest time were manipulated to optimize vegetative (foliar) productivity of cowpea [Vigna unguiculata (L.) Walp.] canopies for future dietary use in controlled ecological life-support systems as vegetables or salad greens. Productivity was measured as total shoot and edible dry weights (DW), edible yield rate [(EYR) grams DW per square meter per day], shoot harvest index [(SHI) grams DW per edible gram DW total shoot], and yield-efficiency rate [(YER) grams DW edible per square meter per day per grams DW nonedible]. Cowpeas were grown in a greenhouse for leaf-only harvest at 14, 28, 42, 56, 84, or 99 plants/m2 and were harvested 20, 30, 40, or 50 days after planting (DAP). Shoot and edible dry weights increased as plant density and time to harvest increased. A maximum of 1189 g shoot DW/m2 and 594 g edible DW/m2 were achieved at an estimated plant density of 85 plants/m2 and harvest 50 DAP. EYR also increased as plant density and time to harvest increased. An EYR of 11 g m-2 day-1 was predicted to occur at 86 plants/m2 and harvest 50 DAP. SHI and YER were not affected by plant density. However, the highest values of SHI (64%) and YER (1.3 g m-2 day-1 g-1) were attained when cowpeas were harvested 20 DAP. The average fat and ash contents [dry-weight basis (dwb)] of harvested leaves remained constant regardless of harvest time. Average protein content increased from 25% DW at 30 DAP to 45% DW at 50 DAP. Carbohydrate content declined from 50% DW at 30 DAP to 45% DW at 50 DAP. Total dietary fiber content (dwb) of the leaves increased from 19% to 26% as time to harvest increased from 20 to 50 days.     

Physical and cellular structure changes of Rastali banana (Musa AAB) during growth and development

A study on the physical and physiological characteristics of Rastali bananas (Musa AAB) was carried out throughout the 12 weeks after emergence of the first hand to determine the optimum growth stage of Rastali bananas. The fruit length and diameter followed a trend similar to that observed in fruit fresh weight throughout fruit development, and three physiological stages (S1–S3) of sigmoid growth were identified. The growth rate was slow during S1 (1st to 4th week), rapid during S2 (5th to 10th week) and remained constant during S3 (11th and 12th week). Peel cells underwent periclinal growth, which was accompanied by an increase in fruit peel and pulp thickness as the fruit developed. Moreover, the pulp colour became more vivid and yellow as the fruit developed. The peel moisture content decreased while pulp moisture content increased during fruit growth and development. The pulp firmness increased from the first until seventh week, and it decreased from the seventh week until twelfth week of the experiment. Carbon dioxide (CO₂) production was the highest at S1. However, CO₂ production declined until the 10th week and remained constant at a low level of 50 mL kg⁻¹ h⁻¹ during S3. Ethylene (C₂H₄) could not be detected throughout fruit growth and development. Thus, Rastali bananas showed physiological maturity at week 11 and week 12 after emergence of the first hand with constant fruit growth.     

Flowering,fruit set and development of the fruit and seed of sweet pepper (iCapsicum annuum L.) cultivated under conditions of high ambient temperature

Sweet pepper (Capsicum annuum L.) cvs E-84066 and Yolo Wonder were grown under ambient conditions in plastic covered greenhouses. Anthesis commenced five and six weeks after planting and rose to a peak of 39 (E-84066) and 19 (Yolo Wonder) flowers per week respectively after which there was a rapid fall. Temperatures throughout anthesis were high (>38°C (maximum) and >16°C (minimum)) and this is likely to have contributed to the magnitude of the flux. Fruit set was highest in the early stages of the crop, but subsequently declined as the majority of flowers aborted. Overall, fruit weight was correlated with seed weight and seed number, and declined throughout the life of the crop. By contrast, seed size and the percentage germination remained constant. The latter is of particular importance for seed crops where seed quality is of primary concern.     

百合类体细胞胚和体细胞胚的形态学与组织学研究

 以东方百合‘Siberia’的无菌苗叶柄为试材,诱导产生初始分生结节组织。其后对分生结节组织进行分化再生采用体式显微镜观察和石蜡切片分析的方法对诱导得到的类体细胞胚和体细胞胚再生结构进行形态学和组织学观察。百合类体细胞胚结构呈球形至心形结构或芽状,组织内部主要由薄壁细胞组成,与母体组织存在着明显的维管组织联系,具有多个维管组织中心,其外形与体细胞胚极为相似。百合体细胞胚发源于分生结节表面新生的胚性愈伤组织,经历球形、椭圆形和子叶形胚阶段发育形成完整的植株,在整个发育过程中与母体愈伤组织在整个发育过程中无维管组织联系,且它的根芽两极同时发生,相互联系。研究结果表明,两种再生结构虽然具有极相似的外观形态,但是在发育方式和内部组织结构上截然不同。     

Synchronization of anthesis and enhancement of vegetative growth in coffee (Coffea arabica L.) following water stress during floral initiation

SUMMARY

The possibility of using water stress during floral initiation and development, to synchronize flowering in potted coffee trees of cvs Catuai Rojo and Mundo Novo was investigated. Moderate and severe cyclic and constant water stress had little effect on vegetative growth during floral initiation. However, upon rewatering, shoot growth was significantly greater in plants where leaf water potential [¨,] had declined to -2.5 MPa compared with plants where ¨, was maintained above -0.5 MPa. The period of floral initiation was not influenced by water stress and occurred only under short days (<12 h). In contrast, a y, of -2.5 MPa significantly reduced the number of inflorescences compared with plants maintained at a ¨| of -0.5 or -1.5 MPa. This reduction was associated with leaf drop in stressed plants. Therefore, regular irrigation during the period of floral initiation is recommended. Water stress (¨, of -1.5 or -2.5 MPa compared with -0.5 MPa) accelerated floral development with no deleterious effects on floral differentiation. Once flower buds are fully differentiated they enter dormancy and reach anthesis only if trees are stressed and rewa-tered. Flower buds remain dormant if trees are watered regularly or a constant water stress provided. A constant period of water stress in the late stages of floral development after floral initiation is complete provides a means of increasing the proportion of fully differentiated dormant flower buds (mature buds). This could represent a practical method to achieve synchronized flowering in field conditions where there is irrigation and a reliable dry season in the late stages of floral development.     

草莓果实发育过程中糖代谢相关基因的表达分析

 为了分析草莓（Fragaria × ananassa Duch.）果实发育过程中可溶性糖积累与相关基因表达量的关系,以‘杜克拉’草莓7个发育时期的果实为试材,利用实时荧光定量RT-PCR的方法,分析果实发育中蔗糖转运蛋白及糖代谢关键酶（酸性转化酶、蔗糖合成酶和蔗糖磷酸合成酶）4个基因的表达量以及可溶性糖含量的变化趋势。结果表明,蔗糖、葡萄糖和果糖含量随着果实发育都呈逐渐增加的趋势,尤其蔗糖积累与果实成熟的关系较密切。随着果实发育,蔗糖磷酸合成酶基因表达量呈逐渐增加的趋势,尤其在果实发育的后期增加较快;蔗糖合成酶和酸性转化酶基因表达量除了白熟期和转色期外,呈逐渐降低的趋势;蔗糖转运蛋白基因表达量呈先升后降,降后又升的变化趋势,尤其在白熟期前急剧增加。结合可溶性糖和基因变化趋势分析表明,蔗糖转运蛋白和蔗糖磷酸合成酶在草莓果实发育过程中发挥着重要的作用。     

仙客来体细胞胚发生和发育过程中淀粉粒的变化

 以仙客来（Cyclamen persicum Mill.）开花植株的新生叶片为外植体，诱导筛选胚性愈伤组织，并使其进一步发育成体细胞胚。组织切片观察结果表明：胚性愈伤组织由胚性和非胚性细胞组成，胚性细胞多以胚性细胞团的形式存在，胚性细胞团起源于诱导的胚性决定细胞。体细胞胚起源于单个胚性细胞，经多细胞原胚、球形胚、心形胚和鱼雷胚等时期发育成完整植株。在体细胞胚的发生发育过程中，淀粉粒出现4次积累高峰，分别为胚性细胞、球形胚、早期鱼雷胚和成熟胚发育成完整的小植株时期，淀粉代谢与体细胞胚发生、发育及小植株的形态建成密切相关。     

Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l⁻¹ kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.     

Anti-oxidant enzyme responses during in vitro embryogenesis in Catharanthus roseus

Summary

The present study was carried out to analyse the activities of several anti-oxidant enzymes at various stages of somatic embryogenesis in Catharanthus roseus. The hypothesis was that anti-oxidant enzymes accumulated as part of a cellular defence mechanism in response to stress. We therefore measured superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities in various in vitro-grown tissues such as embryogenic and non-embryogenic calli, and in embryos at various stages. SOD activity increased gradually from the early embryogenic stage to heart-shaped stage embryos, but declined in the later stages (i.e., torpedo-shaped and cotyledonary embryos). In contrast, APX activity was high in non-embryogenic callus and decreased rapidly during the stage of embryo initiation. This pattern was the same for CAT. The maximum CAT activity was observed in non-embryogenic callus, then it declined almost linearly at the embryonic and post-embryonic developmental stages. The effect of exogenous hydrogen peroxide (H₂O₂) on in vitro embryogenesis was also evaluated. Lower H₂O₂ levels (0.025 mM) promoted embryo formation, whereas higher levels (0.10 mM) inhibited embryogenesis in C. roseus. Finally, higher soluble protein, free amino acid, and proline contents were found in embryogenic calli compared to non-embryogenic calli.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.     

Somatic embryogenesis and plant regeneration in Psidium guajava L. cv. Banarasi local

A protocol for plant regeneration by somatic embryogenesis was developed in guava cv. Banarasi local by using immature zygotic embryo explants. Best induction of somatic embryogenesis was achieved from 10-week-old zygotic embryos on MS medium supplemented with 2,4-d (4.52 μM) and 5% sucrose. Maximum number of somatic embryos was produced when zygotic embryo explants were transferred to growth regulator free full strength MS basal medium after 8 days treatment with 2,4-d. Full strength MS basal medium containing 5% sucrose was most favorable for maturation of somatic embryos. Highest frequency of conversion and normal plantlet production were recorded from elongated torpedo stages of somatic embryos on half strength MS medium containing 3% sucrose. Over 90% of rooted shoots survived acclimatization.     

扁桃杧(Mangifera persiciformis)体胚发生及再生体系建立

以扁桃杧(Mangifera persiciformis Wu&Ming)未成熟珠心组织为外植体,建立体胚发生及再生体系,同时对幼苗进行茎尖染色体计数和胚根组织形态学观察。结果表明,在改良的B5基本培养基+2,4-D1.0mg·L-1+Gln400mg·L-1+6%蔗糖上培养4～5周后可诱导胚性愈伤组织分化。继代培养基与成熟培养基交替培养能有效降低胚性愈伤组织的褐化并保持旺盛的分化能力。培养3～4个月后,大部分体胚均能发育成熟,26.03%的体胚畸形。体胚在改良B5培养基+Gln400mg·L-1+4%蔗糖上的萌发率较低,仅为8.39%。次级体胚以直接体胚发生方式于萌发体胚的下胚轴产生。幼苗的生根不理想,生长极为缓慢;其茎尖染色体数目为2n=2x=40;胚根形态学上端内部维管组织解体,愈伤化,结构松散。     

龙眼TIR1和ABP1基因的克隆及其在体胚发生过程中的表达分析

AIM: To investigate the expression and the role of histone deacetylase 8 (HDAC8) in cardiac hypertrophy and the effects of valproic acid sodium (VPA, a histone deacetylase inhibitor) on the expression of HDAC8 and cardiac hypertrophy in two-kidney two-clip (2K2C) renovascular hypertensive rats. METHODS: Male SD rats were randomly divided into sham operation group, 2K2C group, high dose VPA (400 mg穔g^-1穌^-1) treatment group, low dose VPA (200 mg穔g^-1穌^-1) treatment group and candesartan (10 mg穔g^-1穌^-1) treatment group.Four weeks after surgery,the rats were intraperitoneally injected with VPA for 4 weeks. Sham operation and 2K2C rats were given vehicle for 4 weeks. All animals were sacrificed 8 weeks after surgery. The ratio of left ventricular weight to body weight (LVW/BW) was calculated and pathological changes of the myocardium were observed with HE staining. The mRNA expression of atrial natriuretic factor (ANF) and HDAC8 was examined by RT-PCR. The protein level of HDAC8 was also measured by Western blotting analysis. RESULTS: Compared with the control rats, the mRNA and protein expression of HDAC8 significantly increased in the myocardium in 2K2C rats while the mRNA and protein expression of HDAC8 was significantly decreased by VPA treatment in 2K2C rats. The mass index (as measured by LVW/BW) and cardiomyocyte cross areas were markedly increased and myocardial fibers were disordered in 2K2C rats, but these parameters were markedly reversed after treated with VPA for 4 weeks, indicating that VPA attenuated cardiac hypertrophy. Moreover, VPA also decreased the mRNA expression of ANF. CONCLUSION: HDAC8 may play an important role in cardiac hypertrophy in 2K2C renovascular hypertensive rats. VPA inhibits the expression of HDAC8 and prevents the development of cardiac hypertrophy.     

河套蜜瓜子叶诱导形成体细胞胚的研究

 以河套蜜瓜成熟种子子叶为外植体, 研究体细胞胚途径再生培养的主要影响因子, 并对体胚发生过程进行了观察。结果表明; 体细胞胚发生对培养基糖源浓度要求较高, MS附加60 g/L的糖源效果最好; 最佳植物生长调节剂组合为2,4-D 2.0 mg/L + 6-BA 1.0 mg/L; 1日苗龄子叶为最佳外植体源; 在诱导培养基上经暗培养1周、光培养1周, 再转入胚发育培养基, 有利于成熟胚的产生; 体细胞胚可产生在愈伤组织内部或表面、外植体表面、不定根上及外植体维管束的束中分生组织等部位。     

Effect of miR-19a on lipid catabolism in hepatocyte LO2

AIM: To observe the effect of microRNA-19a (miR-19a) on the lipid catabolism of hepatocyte LO2, and to explore the potential mechanism. METHODS: miR-19a was over-expressed or silenced by transfection of miR-19a mimics or miR-19a inhibitor into LO2 cells, then the mRNA level of miR-19a was detected by real-time PCR. The potential target of miR-19a was found by the method of bioinformatics through internet website. The effect of miR-19a on the 3' UTR of peroxisome proliferator-activated receptor α (PPARα) was measured by dual luciferase reporter assay, and the protein level of PPARα and its 2 major downstream rate-limiting enzymes involved in lipid catabolism, acyl-coenzyme a dehydrogenase (ACADM) and carnitine palmitoyltransferase 1A (CPT1A), were detected by Western blotting. Meanwhile, the effect of miR-19a on the generation of ketone body was measured by beta-hydroxybutyric acid (β-OHB) detection assay. RESULTS: The mRNA level of miR-19a was dramatically elevated by the transfection of miR-19a mimics, and sharply decreased by the transfection of miR-19a inhibitor (P<0.05). PPARα was found as a potential target of miR-19a, and dual luciferase reporter assay and Western blotting confirmed the regulatory effect of miR-19a on the expression of PPARα, with the protein level changes of ACADM and CPT1A. miR-19a mimics down-regulated, while miR-19a inhibitor up-regulated the concentration of β-OHB in LO2 cells (P<0.05). CONCLUSION: miR-19a regulates the lipid catabolism of hepatocytes by targeting the PPARα and its 2 downstream rate-limiting enzymes.     

Somatic embryogenesis and plant regeneration in Opuntia ficus-indica (L.) Mill. (Cactaceae)

We present here a method for the regeneration of the prickly-pear (Opuntia ficus-indica (L.) Mill.) through somatic embryogenesis (SE). Direct SE was induced by cultivating shoot apices devoid of leaf primordia on semisolid Murashige and Skoog (MS) basal medium supplemented with 4-amino 3,5,6-trichloropicolinic acid (picloram) at 4 mg l⁻¹. Somatic embryogenesis was influenced by the type, age, physiological and developmental stage of the explants, and also by light conditions, wounding, and the sucrose concentration in the induction medium. A histological analysis confirmed SE by revealing the presence of a closed vascular system in the developing embryos and the absence of a vascular connection between the somatic embryos and the explant.     

设施栽培中‘春捷’桃糖积累与相关酶活性的变化

 以设施栽培中‘春捷’桃为试材, 研究了其果实发育过程中糖积累与蔗糖代谢相关酶- 酸性转化酶(A I) 、中性转化酶(N I) 、蔗糖合成酶( SS) 及蔗糖磷酸合成酶( SPS) 活性的关系。结果表明:果实发育早期, 果实中的可溶性糖主要是还原糖; 中后期, 还原糖维持在相对较低的水平, 非还原糖大量积累。在整个发育过程中, 蔗糖持续积累, 尤其在成熟期急剧增加, 而果糖和葡萄糖含量在果实发育前期呈下降趋势, 中后期维持在一个相对较低的水平。A I、N I在果实发育前期活性最强, 之后迅速下降, 中后期维持在较低水平; SS在果实发育前期分解方向活性很强, 后期以合成方向为主, 并在果实成熟期保持较高水平; 在果实发育过程中SPS活性呈上升趋势, 但上升幅度较小。葡萄糖含量与A I、N I、SS-h活性显著正相关; 果糖含量与A I、SS-h显著正相关; 蔗糖含量与SS-s活性显著正相关, 与SPS不相关。     

荔枝组织培养中胚状体产生的类型及分析

以荔枝２０—３０ｄ龄幼胚为外植体诱导产生愈伤组织，并筛选建立了幼胚胚性愈伤组织系。胚性愈伤组织在附加２，４－Ｄ的ＭＳ培养基上诱导胚状体分化，在附加ＮＡＡ和ＩＢＡ的培养基中促进胚状体的发育和成熟。在体外胚胎发生过程中，形成大量各种类型的胚状体，其中出现不少异常胚；胚性愈伤组织继代培养过程中存在大量的染色体变异，这可能是产生异常胚的重要原因之一。