A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

Age-Dependent Variations of Volatile Emissions and Inhibitory Activity Toward <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> in Tomato Leaves Treated with Chitosan Oligosaccharide

We investigated variations in the level and composition of volatiles emitted by tomato leaves at different ages. Our focus also included their antifungal properties and responses to chitosan oligosaccharide. Based on leaf position, the release of volatiles decreased over time. Young leaves produced high levels of C6-aldehyde, which is mainly composed of hexenal, while the volatiles emitted by more mature leaves largely comprised terpenes, particularly β-phellandrene and caryophyllane. In young upper leaves, the main components (up to 86% of the total) were hexenal, β-phellandrene, and caryophyllane. Their levels decreased steadily over time, from 386.3 μg g⁻¹ fresh weight (FW) in young leaves to 113.2 μg g⁻¹ FW in old tissues. Volatiles emitted from young leaves exhibited the best antifungal activity against spore germination and hyphal growth by Botrytis cinerea and Fusarium oxysporum. Leaves became more susceptible to oligosaccharide treatment with increasing age. When young tissues were exposed to chitosan, we found declines in both the quantity of volatiles and their ability to inhibit fungal growth. Compared with the control, the amount of volatiles from young tissues was 88.4% lower after such treatment. In contrast, contents of volatiles from old and adult leaves were dramatically increased by chitosan oligosaccharide. Likewise, their inhibitory effect was significantly enhanced. Therefore, our results suggest that these volatiles are responsible for antifungal activity and may play a role in age-related resistance by tomato.     

Regulation of source: sink relationship,fruit set,fruit growth and fruit quality in European plum (<Emphasis Type="Italic">Prunus domestica</Emphasis> L.)—using thinning for crop load management

Demand for large fruit of uniform size is increasing in the market; thinning is a means to achieve consistently large fruit and to overcome possible alternate (biennial) bearing for the small-fruited European plum (Prunus domestica L.). However, chemical thinning agents for stone fruits are scarce and/or often ineffective. Hence, the objective of this work was to study possibilities of enhancing fruit growth and to improve fruit quality, viz size using plum as a model crop. Nine-year-old ‘Ortenauer’ plum trees, trained to spindles, with maximum flowering intensity (score value 9) near Bonn, Germany were mechanically, chemically or hand-thinned. Un-thinned plum trees in the same rows served as control. Trees were either mechanically thinned at full bloom on 20 April 2009 with a rotor speed of either 300, 400 or 500 rpm, and half of those trees additionally treated with ATS (15 L/ha) and an ethylene releasing compound 35 days after full bloom or manually thinned. The objective of 1/3 flower removal was successfully achieved even with the slowest rotor speed of 300 rpm. The number of fruit per branch was significantly reduced from 152 to 67–76, equivalent to a (source: sink) leaf: fruit ratio of 5:1. Mechanical thinning significantly enlarged fruit mass from 28 g in the un-thinned control to 30–32 g with rotor speeds of 400 or 500 rpm. Additional chemical thinning with ATS and an ethylene-releasing compound resulted in no further increase in fruit mass. Inner fruit quality (sugar) of the plums appeared unaffected by either mechanical or chemical thinning, except for fruit firmness: Plums thinned with an ethylene releasing compound were softer and ripened earlier than respective control fruit. The most efficient method of flower removal and fruit mass enlargement was mechanical blossom thinning with 400 rpm, which may provide a suitable replacement for chemical and/or manual thinning. Alternatively, the mechanical thinning can be combined with either of those options. Mechanical blossom thinning was as effective to overcome or avoid alternate/biennial bearing as the ethylene releasing compound in the previous year. The results are discussed with respect to stone fruit being more difficult to thin than pome fruit, because the former do not exhibit leaves at the time of blossom thinning. Stone fruits develop within a shorter time and a larger number of (smaller) leaves (source) are required for the same fruit growth and final fruit size (sink). A lower threshold of fruit removal has to be exceeded before the remaining stone fruit grow faster and final fruit mass and sugar (and possibly fruit firmness) increase, while acidity remains unaffected by fruit set. An upper saturation threshold is reached fairly quickly without further effects.     

Quantitative analysis of carbon balance in the reproductive organs and leaves of <Emphasis Type="Italic">Cinnamomum camphora</Emphasis> (L.) Presl

We investigated seasonal changes in dry mass and CO₂ exchange rate in fruit and leaves of the evergreen tree Cinnamomum camphora with the aim of quantitatively determining the translocation balance between the two organs. The fruit dry mass growth peaked in both August and October: the first increase was due to fruit pulp development and the second to seed development. Fruit respiration also increased with the rapid increase in fruit dry mass. Therefore, the carbohydrates required for fruit development showed two peaks during the reproductive period. Fruit photosynthesis was relatively high in early August, when fruit potentially re-fixed 75% of respired CO₂, indicating that fruit photosynthesis contributed 15–35% of the carbon requirement for fruit respiration. Current-year leaves completed their growth in June when fruit growth began. Current-year leaves translocated carbohydrates at a rate of approximately 10–25 mg dry weight (dw) leaf⁻¹ day⁻¹ into other organs throughout the entire fruit growth period. This rate of translocation from current-year leaves was much higher than the amount of carbohydrate required for reproduction (ca. 3 mg dw fruit⁻¹ day⁻¹). Given the carbon balance between fruit and current-year leaves, carbohydrates for reproduction were produced within the current-year fruit-bearing shoots. C. camphora would be adaptive for steadily supplying enough amount of carbohydrate to the fruits, as there was little competition for carbohydrates between the two organs. As assimilates by leaves are used for processes such as reproduction and the formation of new shoots, photosynthesis by reproductive organs is considered to be important to compensate for reproductive cost.     

Ploidy stability of somatic embryogenesis-derived <Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast. plants as assessed by flow cytometry

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.     

Enhanced production and secretion of rutin and GABA in immobilized cells of mulberry tree (<Emphasis Type="Italic">Morus bombycis</Emphasis> K.)

Cell immobilization has been proposed as a useful technique for mass production and efficient purification of secondary metabolites. In this study, we compared the bio-productivity of ligand-free and Ca-alginate-immobilized mulberry cells for rutin and γ-amino butyric acid (GABA). In the leaves of Subong mulberry plants (M. bombycis K.) grown in a greenhouse, GABA accumulated as the leaves aged; a more than a 20-fold increase of GABA was observed in leaves undergoing senescence than in younger leaves. In contrast, more rutin was detected in mature leaves than in young leaves and those undergoing senescence. The production of total proteins in ligand-free leaf callus cells dramatically increased until 6 days after incubation in liquid suspension media (from 6.5 mg/g callus at day 0–14.5 mg/g callus), and by day 15 dropped to levels similar to those seen in the 0-day control. In contrast, immobilized cells showed a slight increase and then an insignificant decrease in protein content during the 15-day incubation period. Interestingly, immobilized mulberry cells more efficiently produced and secreted rutin and GABA into the suspension media than ligand-free cells. KN, a cytokinin, enhanced this production while 2,4-dichlorophenoxyacetic acid(2,4-D), an auxin, alleviated the effect of KN. As a result, incubation of the immobilized Subong cells in a full-strength Murashige and Skoog (MS) liquid medium containing 1 mg/l of 2,4-D and 0.1 mg/l KN, among the hormone combinations in the medium we tested, produced the highest amounts of rutin (8.2 μg/g callus cells) and GABA (305 μg/g callus cells) and secreted the largest amounts into the suspension media.     

Rapid extraction of genomic DNA from leaves and bracts of dove tree (Davidia involucrata)

Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction. A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and the restriction enzyme digestion needed for Southern blotting.     

Magnesium deficiency of kiwifruit (Actinidia deliciosa)

Magnesium deficiency was associated with large yield reductions in a five-year-old commercial kiwifruit (Actinidia deliciosa) orchard. The effect on yield resulted primarily from a reduction in fruit numbers, there being no difference in mean fruit weight between fruit harvested from affected and unaffected vines. Magnesium deficiency had no deleterious effect on postharvest storage characteristics of fruit stored at 0.5–1°C for 18 weeks; fruit from deficient vines were firmer but had slightly lower soluble solids than fruit from control vines. Although deficiency symptoms were first observed on the basal leaves of the non-fruiting shoots mid season, indications of the impending deficiency could be established very early in the season using foliar analysis. Magnesium concentrations in youngest fully expanded leaves (YFEL) on the affected vines were less than 2.0 g kg⁻¹ DM four weeks after budbreak and remained below this value for the rest of the season; concentrations in YFEL on unaffected vines did not decrease below this value and gradually increased after fruitset to 4.5 g kg⁻¹ DM at harvest. To avert potential production losses, it is suggested that soluble magnesium fertilizers (containing at least 200 kg ha⁻¹ Mg) should be broadcast early in the season if foliar magnesium concentrations less than 2.0 gkg⁻¹ DM are measured four–six weeks after budbreak.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

A simple protocol for isolating genomic DNA from chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> tratt) for RFLP and PCR analyses

Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols, polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analyses.     

An improved method for isolating high-quality DNA from<Emphasis Type="Italic">Vitis vinifera</Emphasis> nuclei

We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A_260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.     

DNA isolation protocol for seaweeds

We report a DNA isolation protocol for red seaweeds. Recovering DNA of high quality and quantity is a prerequisite for ensuring suitable applications, such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and sequencing. Isolation of DNA from seaweeds has proven difficult because of coprecipitation of polysaccharides. Our process minimizes this contamination, which is mostly due to the highly hydrocolloidal content of algal cell walls. This protocol, using 2 steps, is based on a preliminary enzymatic digestion of cell wall with specific enzymes (Novozymes) followed by centrifugation, allowing isolation of DNA on the pellet. This provides a higher yield of DNA, in the range of 40 μg (Palmaria palmata) and 18 μg (Gracilaria verrucosa) from 50 mg of fresh frozen pellet.     

Phenology of figs in Budongo Forest Uganda and its importance for the chimpanzee diet

This paper reports on the phenological patterns of figs in Budongo Forest, Uganda, and how it relates to chimpanzee food availability in different seasons. In addition, we analysed the dung of chimpanzees to understand the composition of fruits in their diet. The aim of our study was to assess Ficus phenology and how it affects chimpanzee diet. Fifteen species of figs were monitored for fruit (syconium) and leaf phenology between June 2000 and 2001. Ficus fruit production varied significantly between and within species, and also with tree trunk and crown diameters. Fig fruit production was asynchronous and individual fig trees produced crops from one to five times in a year. In addition to fruits, chimpanzees fed on young leaves of some Ficus species. Shedding of old Ficus leaves coincided with the dry season, followed by appearance of young leaves. The dry season in Budongo is a period of general fruit scarcity. The combination of fig fruits and young leaves make up the most important food in the diet of chimpanzees. From the chimpanzee dung, more than 78% of seeds comprised fig ‘seeds’ (nutlets) and the rest of the diaspores were from other tree species. Our findings suggest that chimpanzees disperse large number of diaspores in their dung, thereby serving as important agents of natural forest regeneration.     

Extraction of High Quality of RNA and Construction of a Suppression Subtractive Hybridization (SSH) Library from Chestnut Rose (Rosa roxburghii Tratt)

Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and β-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 μg RNA g⁻¹ fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes. Qiang Xu and Xiaopeng Wen - Contribute to this work equally Revisions requested 3 November 2005; Revisions received 18 January 2006     

The effect of phosphorus deficiency on nutrient uptake,nitrogen fixation and photosynthetic rate in mashbean,mungbean and soybean

Phosphorous (P) fertilization is the major mineral nutrient yield determinant among legume crops. However, legume crops vary widely in the ability to take up and use P during deficiency. The aim here was to compare P uptake and translocation, biological nitrogen fixing ability and photosynthetic rate among mashbean (Vigna aconitifolia cv. ‘Mash-88’), mungbean (Vigna radiata cv. ‘Moong-6601’) and soybean (Glycine max L. cv. ‘Tamahomare’) during deficiency in hydroponics. Two treatments, the withdrawal of P from the solution (P-deprivation) and continued P at 160 μM (P sufficient) were effected at the pod initiation stage. Plants were grown for 20 days. Short-term labeling with ³²P showed the uptake and distribution of P into plant parts. Withdrawal of P from the solution reduced biomass, photosynthetic activity, and nitrogen fixing ability in mungbean, and mashbean more than in soybean. P deprivation decreased P accumulation more than N accumulation. The decrease was more severe in mungbean and mashbean than soybean. More P was translocated and distributed into leaves in soybean than in mungbean and mashbean. Leaf P amount was more correlated to leaf area than to photosynthetic rate per unit leaf area among all three legume species. The results indicate that selection for increased efficiency of P utilization and leaf area may be used to improve leguminous crops.     

A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (<Emphasis Type="Italic">dahi</Emphasis>)

A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was ~50 μg/ml log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications. The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction (PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid, cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus.     

Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components

A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.     

Ethanol production from hydrolysed agricultural wastes using mixed culture of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis. Acid hydrolysis of fruit and vegetable residues gave 49–84 g reducing sugars l⁻¹ and 29–32 g ethanol l⁻¹ was then obtained. Alkaline hydrolysis did not give significant amount of reducing sugars. Enzymatic hydrolysis of fruit and vegetable residues yielded 36–123 g reducing sugars l⁻¹ and 11–54 g ethanol l⁻¹.     

Growth Rate and Water Relations of Citrus Leaf Flushes

Growth rates of seasonal leaf flushes of ‘Valencia’orange [Citrus sinensis (L.) Osbeck] were measured and waterrelations characteristics of young (new) and over-wintered (old)citrus leaves were compared. New flush leaves had lower specificleaf weights and lower midday leaf water potentials than comparablyexposed old leaves. Spring and summer flush new leaves had higherosmotic potentials than old leaves. These differences becamenon-significant as the new leaves matured. During summer conditions,water-stressed new leaves reached zero turgor and stomatal conductancealso began to decrease in them at higher leaf water potentialsthan in old leaves. Old leaves were capable of maintaining openstomata at lower leaf water potentials. Opened flowers and newflush leaves lost more water, on a dry weight basis, than flowerbuds, fruit or mature leaves. The results illustrate differencesin leaf water potential and stomatal conductance which can beattributed to the maintenance of leaf turgor by decreases inleaf osmotic potentials as leaves mature. These changes in citrusleaf water relations are especially important since water stressresulting from high water loss rates of new tissues could reduceflowering and fruit set. Citrus sinensis (L.) Osbeck, orange, Citrus paradisi Macf., grapefruit, growth rate, leaf water relations, osmotic potential, water potential, stomatal conductance     

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis> Bge. var. <Emphasis Type="Italic">major</Emphasis> N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.