Impact of nitrogen supply on growth,steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni

This work investigated the agronomic, physiological and biochemical response of Stevia rebaudiana Bertoni grown under different nitrogen (N) rates. A pot trial in open air conditions was set up in 2012 with the aim to evaluate the effect of four N rates on the biometric and productive characteristics, steviol glycoside (SG) content as well as on leaf gas exchanges, chlorophyll fluorescence, photosynthetic pigments, Rubisco activity and N use efficiency. N deficiency caused a decrease in leaf N content, chlorophylls and photosynthetic CO₂ assimilation, resulting in a lower dry matter accumulation as well as in reduced SG production. The application of 150 kg N ha^? 1 seems to be the most effective treatment to improve rebaudioside A (Reb A) content, Reb A/stevioside ratio, photosynthetic CO₂ assimilation, stomatal conductance, N use efficiency, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) and PSII efficiency. The results demonstrate that by using an appropriate N rate it is possible to modulate the SG biosynthesis, with a significant increase in the Reb A content and in the ratio between Reb A and stevioside. This finding is of great relevance in order to obtain a raw material designed to meet consumer needs and bio-industry requirements for high-quality, Reb A content, and safe and environmentally friendly products.     

Production,maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

A method is described for producing and maintaining Stevia rebaudiana suspensions and regeneration of plants from calli derived from cell suspensions. Suspension cultures composed of isolated cells (ca. 10%) and cellular aggregates (5–100 cells) were obtained in 20–30 days by using friable callus as the initial inoculum in liquid medi with BA (0.5 mg/l)+2,4-D (1.0 mg/l), and periodic filtering (100–500 m sieves) with 6–7 days interval between subcultures. Cultures derived from actively growing calli are mainly diploid (2n=22) whereas those derived from senescent calli showed a wide variation in chromosome number (55–200). Stock cell suspensions which had been maintained for 3 years were plated on basal LS agar medium with BA (0.5 mg/l)+2,4D (0.5 mg/l) to form callus. Calli originating from predominantly 2n cell suspensions when transferred to medium with K (2.0 mg/l)+NAA (0.02 mg/l) were able to form buds. Shoot elongation and further rooting of isolated shoots was better on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological characters and chromosome number was found amongst regenerated plants.Abbreviations BA Benzylaminopurine - 2,4-D 2,4 - Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - LS Linsmaier & Skoog     

Main sugar composition of floral nectar in three species groups of Scrophularia (Scrophulariaceae) with different principal pollinators

In some angiosperm groups, a parallelism between nectar traits and pollination syndromes has been demonstrated, whereas in others there is not such relationship and it has been explained as due to phylogenetic constraints. However, nectar trait information remains scarce for many plant groups. This paper focuses on three groups of Scrophularia species, with different flower sizes and principal pollinators, to find out whether nectar sugar composition is determined by pollinator type or reflects taxonomic affinities. Since the species we examined have protogynous flowers, and gender bias in nectar sugar composition has been noted in few plant groups, we also investigated whether sexual phase influenced Scrophularia nectar composition. The sugar composition was found to be similar in all species, having high‐sucrose nectar, except for the Macaronesian Scrophularia calliantha, which was the only species with balanced nectar; this last kind of nectar could be associated with the high interaction rates observed between S. calliantha and passerine birds. The nectar sugar composition (high in sucrose) was unrelated to the principal pollinator group, and could instead be considered a conservative taxonomic trait. No gender bias was observed between functionally female and male flowers for nectar volume or concentration. However, sexual phase significantly affected sucrose percentage in the largest‐flowered species, where the female phase flowers had higher sucrose percentages than the male phase flowers.     

甜菊醇糖苷生物合成关键基因的导入和鉴定分析

甜叶菊（Stevia rebaudiana Bertoni）生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点,被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1,构建植物基因过量表达载体,以单独或组合的形式将这些基因导入到甜叶菊中,获得转基因植株。与野生型对照植株相比,单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高,总糖苷、莱包迪苷A含量及占比没有明显变化;单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低,而甜菊醇双糖苷含量显著增加;单独导入SrUGT76G1的转基因植株中,总糖苷含量显著提高,莱包迪苷A含量达到10%以上,比对照提高了2倍,而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似,其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达,培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。     

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni)

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l⁻¹). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l⁻¹) and gibberellic acid (GA₃; 0.5 mg l⁻¹). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%).     

The impact of different seed priming agents and priming durations on stand establishment and biochemical attributes of Stevia rebaudiana Bertoni

Stevia (Stevia rebaudiana Bertoni) is an alternative sugar crop currently gaining importance in several geographic regions of the world. Generally, crop is propagated by seeds; however, low seed germination hinders the adaptability of the crop in different cropping systems. Seed priming with different compounds improves germination of several arable crops under stressful and benign environmental conditions. This study evaluated the role of different seed priming agents and priming durations on stand establishment, allometric and yield-related traits, and steviol glycosides contents of Stevia in two different experiments. The first experiment consisted of five different seed priming agents (i.e., hydropriming with distilled water, ascorbic acid, potassium chloride, benzyl amine purine and unprimed seeds) and five priming durations (0, 8, 16, 24 and 32 h). Seed priming with potassium chloride (KCl) and benzyl amine purine (BAP) for 32 h improved stand establishment and biochemical attributes of Stevia seeds. Therefore, these two seed priming agents along with unprimed seeds were included in the second experiment to quantify their impact on allometric and yield-related traits and steviol glycosides contents. Seed priming with both KCl and BAP resulted in better allometric traits (plant height, number of leaves per plant, leaf area and chlorophyll index) compared with unprimed seeds. Similarly, seed priming with KCl resulted in higher fresh and dry biomass production of stem and leaves. Nonetheless, higher stevioside and rebaudioside A contents were recorded for the seeds primed with KCl, whereas unprimed seeds resulted in the lowest values. It is concluded that Stevia seeds must be primed with KCl for 32 h before sowing to get higher seed germination, leaf yield and steviol glycosides contents. Nonetheless, the role of KCl priming in improving abiotic stress tolerance of Stevia must be explored in the future studies.     

The use of esterase polymorphism for analysis of the genetic diversity and structure of stevia (Stevia rebaudiana Bert. Bertoni) populations

Native polyacrylamide gel electrophoresis was used in the current study to identify polymorphisms in α- and β-esterase loci in the leaves of Stevia rebaudiana Bert. Bertoni. The esterases produced from the Est-2 and Est-4 loci were observed as strongly stained bands, and four alleles were detected at each of these loci from the 428 analysed S. rebaudiana plants. The observed and expected mean heterozygosity were higher in populations maintained by genetically breeding plants for higher glycoside production than in plant populations propagated by seeds or maintained by vegetative propagation. The esterase analysis employed in this study showed that the artificial selection of plant samples with specific uniform characteristics, such increased height and precocious flowering, may lead to the fixation of alleles and α/β-esterase phenotype patterns in S. rebaudiana populations. The uniform α/β-esterase phenotype may be applied in the monitoring of genetic stability in selected populations for specific traits of interest.     

Insect pollinators of anise plants (Pimpinella anisum L.) and the important role of honey bees (apis mellifera L.) on their yield productivity

The present investigation aimed to identify the insect pollinators and the efficiency of honeybees in pollination of anise plant during flowering periods of 2008 and 2009 seasons at Assiut Governorate, Egypt. Obtained results indicated that the total insect counts attained 11 species, belonging to four orders; Hemiptera (one species), Coleoptera (two species), Diptera (five species) and Hymenoptera (three species). Honeybees were the most abundant of hymenopterous insects during anise flowering and the daily peak activities were detected between 12.00 noon and 2.00 p.m. for the two seasons. Regarding the effect of prevailing air temperature and relative humidity percentage (RH%) on the occurrence of insect pollinators, it is clear that the effects of the two factors were moderate in the two seasons. Also, the results showed a significant effect of open pollination on the seed yield/feddan (1024.12 kg) followed by honeybees pollination (781.55 kg), while insect exclusion recorded the lowest value (300.24 kg).     

Study on the bioactivity of steviol and isosteviol in stevia (Stevia rebaudiana Bertoni)

Steviol and isosteviol are diterpene metabolites that are produced from a shared biosynthesis pathway with gibberellins in stevia leaves. These compounds share a chemically similar structure with gibberellins. An experiment was conducted to find the bioactivity properties of steviol and isosteviol in stevia. During vegetative growth, external solutions of steviol and isosteviol (1.6 × 10^?5 and 1 × 10^?5 M, respectively) were sprayed on stevia shoots and then the stevia growth and metabolites were assessed. The results showed that the stevia leaf growth was reduced by external application of steviol. The leaf growth was not affected by external isosteviol while the stevia height and stem dry weight significantly increased. The antioxidant capacity and steviol glycosides content were increased by external steviol. Our results implicated that the isosteviol bioactivity is similar to that of gibberellin hormone. On the other hand, steviol induces the antioxidant system and stimulates the steviol glycosides biosynthesis in stevia leaves.     

Effect of two plant growth retardants on steviol glycosides content and antioxidant capacity in Stevia (Stevia rebaudiana Bertoni)

Two greenhouse experiments were conducted to study the effect of two plant growth retardants, Chlorocholine chloride (CCC) and Paclobutrazol (PBZ), on growth, Steviol glycosides (SVglys) content and antioxidant capacity in Stevia (Stevia rebaudiana Bertoni). Five concentrations of CCC (0, 250, 500, 750 and 1,000 ppm) and PBZ (0, 6, 12, 18 and 24 ppm) with three replications were applied to Stevia plants as treatments based on completely randomized design. CCC was sprayed on Stevia shoots, but PBZ was applied as a drench. The obtained results showed that CCC reduced plant height but improved leaf and stem dry weight, especially with 750 ppm concentration. Total SVgly content and consequently SVglys yield were significantly reduced by CCC application, and 1,000 ppm of CCC concentration was more effective than other treatments. PBZ had no effect on Stevia height while it significantly enhanced stem and dry weight at 12 ppm. Moreover, PBZ remarkably increased total SVglys contents, SVglys yield, and Rebaudioside A/Stevioside ratio. Total antioxidant capacity significantly varied with CCC and PBZ and the highest activity was obtained with 1,000 and 12 ppm of CCC and PBZ, respectively. The results of these experiments indicated that, although CCC and PBZ are plant growth retardants and act as anti-gibberellins, only CCC reduced plant height and SVglys production in Stevia. On the contrary, PBZ at 12 ppm concentration, improved Stevia growth, SVglys production, and antioxidant capacity.     

Spectral lights trigger biomass accumulation and production of antioxidant secondary metabolites in adventitious root cultures of Stevia rebaudiana (Bert.)

Stevia rebaudiana (S. rebaudiana) is the most important therapeutic plant species and has been accepted as such worldwide. It has a tendency to accumulate steviol glycosides, which are 300 times sweeter than marketable sugar. Recently, diabetic patients commonly use this plant as a sugar substitute for sweet taste. In the present study, the effects of different spectral lights were investigated on biomass accumulation and production of secondary metabolites in adventitious root cultures of S. rebaudiana. For callus development, leaf explants were excised from seed-derived plantlets and inoculated on a Murashige and Skoog (MS) medium containing the combination of 2,4-dichlorophenoxy acetic acid (2, 4-D, 2.0 mg/l) and 6-benzyladenine (BA, 2.0 mg/l), while 0.5 mg/l naphthalene acetic acid (NAA) was used for adventitious root culture. Adventitious root cultures were exposed to different spectral lights (blue, green, violet, red and yellow) for a 30-day period. White light was used as control. The growth kinetics was studied for 30 days with 3-day intervals. In this study, the violet light showed the maximum accumulation of fresh biomass (2.495 g/flask) as compared to control (1.63 g/flask), while red light showed growth inhibition (1.025 g/flask) as compared to control. The blue light enhanced the highest accumulation of phenolic content (TPC; 6.56 mg GAE/g DW), total phenolic production (TPP; 101 mg/flask) as compared to control (5.44 mg GAE/g DW; 82.2 mg GAE/g DW), and exhibited a strong correlation with dry biomass. Blue light also improved the accumulation of total flavonoid content (TFC; 4.33 mg RE/g DW) and total flavonoid production (TFP; 65 mg/flask) as compared to control. The violet light showed the highest DPPH inhibition (79.72%), while the lowest antioxidant activity was observed for control roots (73.81%). Hence, we concluded that the application of spectral lights is an auspicious strategy for the enhancement of the required antioxidant secondary metabolites in adventitious root cultures of S. rebaudiana and of other medicinal plants.     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

Consequences of southern cassowary (Casuarius casuarius,L.) gut passage and deposition pattern on the germination of rainforest seeds

In Australia's tropical rainforests the endangered southern cassowary, Casuarius casuarius, L., is the largest native frugivore and many plant species, because of the size of their fruits or seeds, are thought to be largely dependent on cassowaries for their dispersal. In this study we asked whether the contribution of cassowaries to plant recruitment extends beyond removing seeds from the vicinity of the parent. To do this we conducted germination trials involving 17 rainforest plant species to test whether cassowary consumption and seed deposition pattern alter germination probability or time to germination. Twenty‐four per cent of species showed changed germination probabilities, with one species showing no germination without cassowary consumption, and 35% showed changed time to germination. However these differences did not translate into any significant effects when considered across all species. We examined gut scarification, fruit pulp removal (de‐inhibition) and deposition in faecal material as mechanisms for changing germination success; each contributed to the changed success of individual species. There was a negative effect of seed clumping on five species. We conclude that cassowary consumption can modify germination performance in a minority of rainforest plants and that the effect is generally positive. Although the effect on large seeded species was small it is most likely to be important as the cassowary is the only animal in Australia able to internally process large numbers of these seeds.     

Digestion time and intactness of seeds ingested by Sturnira lilium (E. Geoffroy, 1810) (Mammalia,Chiroptera)

Bats are responsible for many ecological services, such as seed dispersal of several plant species, contributing to the processes of succession and forest regeneration. A factor that can interfere with this process is the animal digestion, which can affect germination, altering the patterns of seedling distribution. The effects of seed passage through bats’ guts varies with the species, leading to some discrepancies in the literature. In this study, we tested the digestion time of one Phyllostomidae bat species, Sturnira lilium, in two Neotropical plants: Solanum paniculatum and Ficus organensis, and the effects on seed germination. The experiment was conducted in captivity and the germination tests were made in laboratory conditions. The results suggested that most seeds ingested by S. lilium are dispersed within 40 min for both species and the digestion seems not to significantly affect the germination of F. organensis, despite the slight acceleration of germination. In S. paniculatum, germination occurred only in the control (39%), whereas in the treatments, all the seeds remained dormant during the 25 experimental days. In this case, the digestion of S. lilium possibly contributes to the formation of seed banks, randomizing the temporal distribution of seedlings.     

野生人参种子的超低温保存

人参（ＰａｎａｘｇｉｎｓｅｎｇＣ．Ａ．Ｍｅｙ．）是珍贵的药用植物，由于长期过度采挖，资源枯竭，现存于我国东北地区的野生人参已处于濒临绝灭的边缘，被列为国家一级重点保护植物［１，２］。所以在加强其就地保护的同时，人参种质资源的迁地保存也是非常必要的。按...     

Germination of Prosopis juliflora (Sw.) D.C. seeds at different osmotic potentials and temperatures

The effects of osmolytes, osmotic potential (Ψ_s), temperature, and their interactions on the germinability, germination rate, and other germination parameters of the invasive shrub Prosopis juliflora, which grows in the semiarid environmental conditions of the Caatinga in northeast Brazil, were evaluated. To study the effects of polyethylene glycol (PEG) and NaCl stress and temperature on germination, two separate experiments were carried out at the Plant Ecophysiology Laboratory of the Federal University of Pernambuco in 2011. The overall germinability decreased significantly with increases in both PEG (one‐way anova , F_4,75 = 111.21, P ≤ 0.001) and NaCl (one‐way anova , F_4,75 = 12.82, P ≤ 0.001); however, the effects were more accentuated with PEG than NaCl. The PEG‐treated seeds maintained their germinability, even when they were subjected to a Ψ_s = ?1 MPa after being rinsed and allowed to germinate on deionized water. In contrast, NaCl‐treated seeds usually lost their ability to germinate; this fact was possibly linked to the accumulation of Na⁺ and Cl^? in the cells, which may have contributed to a loss of membrane function that led to the death of the embryos. Although numerous studies describing seed germination in the presence of osmolytes have been conducted, studies that show the interactions between osmolytes, osmotic potentials, and temperature are scarce. The present study is the first to describe these interactions for P. juliflora seeds.     

Flower and nectar attributes of pepper (Capsicum annuum L.) plants in relation to their attractiveness to honeybees (Apis mellifera L.)

Flower morphology, nectary structure, nectar traits and rates of honeybee foraging on pepper plants were studied. The nectary appears as swellings on the basal part of the ovary. The nectariferous cells are smaller and denser than the neighbouring parenchyma. Stomata are present in the nectary epidermis, but do not appear on the other parts of the ovary epidermis. Seven pepper breeding lines were grown near a bee yard in Rehovot. Five to six fold differences in nectar volume were found between the extreme genotypes. Nectar volumes were higher during noon and afternoon hours, as compared with morning hours. High correlation coefficients between nectar volume and sugar concentration were found. These were significant for the four high nectar yielding genotypes, ranging between r = 0.65 to r = 0.94. Male-fertile flowers produced more nectar and higher sugar concentration than sterile ones. Skewed distribution was observed in nectar volume of F₂ populations, but relatively low heritability values were calculated. Pepper nectar contains fructose and glucose only. The former occupies 52–82 % of the total sugar content. Pepper genotypes varied in frequency of honeybee visits and significant correlation between sugar quantity and number of honeybee visits per flower was evident. Fertile pepper flowers are not very attractive to honeybees and male-sterile flowers are even less so. The considerable variation in nectar characteristics can be exploited to increase attractiveness to honeybees, thus facilitating bee pollination in commercial production of F₁hybrid seeds and improve fruit quality.     

环境因子对黄顶菊种子萌发的影响

黄顶菊是一种入侵性极强的外来杂草,其种子对环境的适应是其成功入侵的前提.通过室内条件下黄顶菊种子的物理特性、环境因子对其种子萌发及幼苗生长影响等方面的研究,可为探讨黄顶菊快速蔓延的原因与揭示黄顶菊成功入侵机制提供理论依据.结果表明,黄顶菊种子极多而轻与黄顶菊大面积扩散密切相关,温度、光照和种子埋藏深度是决定黄顶菊种子萌发的关键因素.黄顶菊种子萌发的温度范围为5～40℃,25℃最适于种子的萌发,25～35℃条件下有利于幼苗的生长;黄顶菊种子是光敏感型种子,在土壤表面的萌发率最高(74.44%),大于3cm的深层土壤中则不能萌发;pH 对黄顶菊种子的萌发影响不明显,但其幼苗在酸性条件下生长较好;黄顶菊种子的萌发能忍受一定的盐胁迫和干旱胁迫,但随着胁迫程度的加重,根长和苗长逐渐降低,50%的土壤含水量对黄顶菊种子萌发和幼苗生长比较适宜.     

低能碳、氮离子对甜叶菊萌发率、生长量及过氧化物酶的影响

以经过碳、氮（75×10