A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

Micropropagation of Pistachio from mature trees

Multiple shoots were induced from nodal segments of mature trees of Pistacia vera L. on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BA). Maximum shoot production was obtained from shoot tips taken from in vitro proliferated shoots when cultured on solidified MS medium containing 8.8 μM BA. The multiplication rate was 20 microshoots per explant on the 30th day. Rooting of microshoots was achieved in MS medium supplemented with indole butyric acid (IBA). Rooted plantlets reassumed independent growth after a short period of acclimatisation. Stable regenerated plants were established in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Germination stimulant from root exudates of Vigna unguiculata

Root exudate of Vigna unguiculata was extracted from a soil system consisting of charcoal and vermiculite. Germination stimulating activity for Striga gesnerioides was found in extracts of the soil system, and an active compound was isolated. The chemical structure of the active ingredient was determined to be (+)-4-O-acetylorobanchol, based on analysis of the spectral data of 1-D and 2-D NMR together with nuclear Overhauser effect (NOE) experiments. Application of the active compound to the seeds of S. gesnerioides at a concentration of 0.35 × 10⁻⁹ mol/disk led to 69% germination. The germination observed with application of GR-24, a positive control, at 0.57 × 10⁻¹⁰ mol/disk was 80%.     

Micropropagation of Taxus mairei from mature trees

Micropropagation of mature Taxus mariei was achieved using bud explants derived from approximately 1,000-year-old field-grown trees. Bud explants derived from 1-year-old stecklings raised from rooted cuttings of these trees were included in this study for comparison. Steckling-derived explants collected in different seasons showed high survival rates and good growth performance in vitro, whereas the survival of mature tree-derived explants was highly season dependent, with satisfactory survival in early spring only. During successive subculturing, tissue browning was observed in cultures of both origins, but the problem was circumvented by supplementing the basal medium with 1 g l^-1 activated charcoal and 100 mg l^-1 silver nitrate. The steckling-derived cultures performed better than mature tree-derived cultures in terms of shoot multiplication and rooting ability. New shoots of both origins exhibited plagiotropic growth, but steckling-derived shoots had a smaller plagiotropic angle (near vertical) than mature tree-derived shoots. In both cases, the plagiotropic angles decreased after these subcultures were maintained in shoot multiplication conditions for half a year. Orthotropic growth was restored in 25% and 10% of the steckling- and mature tree-derived cultures, respectively.     

Differentiation in explants from mature leguminous trees

Stem and petiole explants, obtained from mature trees, ofAlbizzia lebbeck,Cassia fistula andC.siamea callused and differentiated shoot-buds and later shoots on B5 medium supplemented with either 0.5 mg/l IAA + 1 mg/l BAP or BM + 2 mg/l NAA + 0.5 mg/l BAP. The stem explants were more responsive than the petiole explants. InA.lebbeck, the IAA substituted medium favoured differentiation from both types of explants. However, inC.fistula, the type of explants rather than the medium composition had an overriding influence on shoot differentiation since those from petiole hardly responded in either medium. It has been possible to obtain plantlets from bothA.lebbeck andC.fistula under conditions conducive to rooting. Plantlets ofA.lebbeck have also been successfully transferred to the field.     

大蒜根系分泌物的化感作用

以苍山白蒜和蔡家坡紫蒜为材料,采用水培方法收集根系分泌物,研究了2个大蒜品种的根系分泌物对莴苣种子发芽和幼苗生长及对黄瓜枯萎病菌、西瓜枯萎病菌的化感效应.结果表明:2个大蒜品种的根系分泌物对莴苣种子发芽和幼苗生长均表现为低浓度(0.1、0.2 g·mL-1)促进、高浓度(0.4、0.6 g·mL-1)抑制,高浓度时蔡家坡紫蒜的抑制作用大于苍山白蒜;对黄瓜枯萎病菌和西瓜枯萎病菌的菌丝生长及孢子萌发均表现为抑制作用,随着根系分泌物浓度的提高,抑制作用增强,其中黄瓜枯萎病菌较敏感,且蔡家坡紫蒜的抑制作用大于苍山白蒜.     

Micropropagation ofDalbergia sissoo from nodal explants of mature trees

A method for micropropagation ofDalbergia sissoo has been developed. Single node segments obtained from coppice shoots of a mature tree (20 – 25 year old) produced 3–4 shoots per explant on Murashige and Skoog (MS) medium containing 4.4 x 10⁻⁶ M benzylaminopurine (BAP) and 4.4 × 10⁻⁷ M of Β-naphthoxy acetic acid (NOA) (shoot multiplication medium) within 4 weeks. Thein vitro regenerated shoots were 3 – 4 cm in length and provided 2 to 3 culturable nodal segments which on shoot multiplication medium again produced 3–4 shoots. Following this procedure 18–24 shoots were produced from single nodal segment within 60 d. 80 % of the shoots directly produced five roots when they were firstly treated with MS medium supplemented with 10⁻⁵ M indole-3-butyric acid (IBA) and subsequently transferred to half strength liquid MS medium containing 1 % activated charcoal followed by half strength liquid MS free hormones, vitamins and activated charcoal. Thein vitro raised plants were hardened for survival after transplantation to soil by exposing them to various humidity conditions, gradually from higher to low, with nearly 100 % transplant success. Acknowledgement: Authors are grateful to CSIR and DST, New Delhi for financial assistance.     

转基因植物根系分泌物对土壤微生态的影响

随着转基因植物商品化进程的加快,对其进行生态风险性评价日益引起学者的重视。诸如转基因逃逸到其它亲缘物种中、产生超级杂草和病毒、昆虫产生耐受性及生物多样性遭受破坏等问题已在部分转基因作物中显现。本文综述了转基因植物中根系分泌物对土壤微生态的影响。     

Nitrogen in shoot litter,root litter and root exudates from nitrogen-fixingAlnus incana

Summary A pot experiment withAlnus incana (L.) Moench growing in sand was set up to compare the amounts of nitrogen released from plants shoot litter with that released below ground as root litter and/or root exudation. No nitrogen fixation by free-living microorganisms was found in the sand and the increased nitrogen content of the plant + soil system was therefore due to nitrogen fixation byFrankia in the alder root-nodules. Most of the nitrogen released from the plants was in the nitrogen-rich leaf and other shoot litter. Only small amounts of nitrogen were found in the drainage water from the pots and were recorded as increased nitrogen content of the sand.     

Decomposition of root exudates in soil

The conversion of synthetic root exudates, i.e. of a mixture of amino acids, organic acids and sugars, added to soil in a single dose or continuously, was studied. After the addition of a single dose, the root exudates were gradually mineralized and after 76 hours, 85% of carbon had been released in the form of carbon dioxide. The extent and rate of mineralization was not influenced by the simultaneous addition of ammonium phosphate. The continuous addition of substrate formed a model artificial rhizosphere. In the steady state, 93% of the carbon in the added substrate was mineralized to carbon dioxide. The conversion of organic acids, sugars and amino acids and the mineralization of nitrogen was studied simultaneously by chromatography. In soil continuously enriched with root exudates, phenomena similar to the rhizosphere effect in nature were observed both in the numbers of microorganisms and in the relative incidence of the nutritional groups of bacteria.     

Regulation and function of root exudates

Root-secreted chemicals mediate multi-partite interactions in the rhizosphere, where plant roots continually respond to and alter their immediate environment. Increasing evidence suggests that root exudates initiate and modulate dialogue between roots and soil microbes. For example, root exudates serve as signals that initiate symbiosis with rhizobia and mycorrhizal fungi. In addition, root exudates maintain and support a highly specific diversity of microbes in the rhizosphere of a given particular plant species, thus suggesting a close evolutionary link. In this review, we focus mainly on compiling the information available on the regulation and mechanisms of root exudation processes, and provide some ideas related to the evolutionary role of root exudates in shaping soil microbial communities.     

植物根系分泌物的生态效应

根系分泌物是在一定的生长条件下 ,活的且未被扰动的根系释放到根际环境中的有机物质的总称 ,在植物主动适应和抵御不良环境中具有重要作用 :(1)通过化感作用影响根际微生物和周围其它植物的生长 ,并进一步改善植物的生态环境 (生物因素 ) ;(2 )通过对土壤中矿质元素的溶解、螯合作用、迁移和活化等作用 ,不仅在营养缺乏的情况下提高矿质营养元素的有效性 ,而且在面临重金属胁迫时能降低根际中金属污染物的活性 ,减少植物对金属的吸收。根系分泌物在植物与环境的相互作用中起着传递信息的作用     

B-Vitamins in root exudates of cotton

Summary Cotton field soils fromFusarium wilt-free zone (KPT) and a wilt-sick zone (PLD), and root exudates of diploid and amphidiploid strains of cotton grown in these two soils were analyzed for B-vitamins by microbiological assay technique employing X-ray mutants ofNeurospora crassa andN. sitophila.PLD soil was found to contain choline, pyridoxine,p-aminobenzoic acid, traces of biotin, and inositol. KPT soil was found to contain pyridoxine, choline, thiamine,p-aminobenzoic acid, and traces of biotin and inositol.Thiamine, biotin, pyridoxine, andp-aminobenzoic acid were found in higher amounts in the exudates of diploid cotton strains, especially K 2 and 6186.9 in KPT soil. In addition to the above, choline was also found in the exudates of diploid strains grown in PLD soil.Amphidiploid strains showed comparatively lower amounts of these vitamins in the root exudates with a few exceptions.In the inoculated series, inositol was absent in most cases and the contents of other vitamins were reduced to varying extents compared to the respective healthy controls.Part of Doctoral thesis, University of Madras.     

根系分泌物研究方法(综述)

根系分泌物是植物与土壤进行物质、能量与信息交流的重要载体物质.研究根系分泌物的种类、数量对了解植物与土壤及根际微生物相互作用机理具有重要指导意义,根系分泌物研究方法的选择对研究结果有重要的影响.本文着重讨论根系分泌物收集、分离纯化及鉴定的常用方法,并列举特定根系分泌物的研究方法,旨在为根系分泌物的研究提供借鉴和参考.     

Micropropagation of Tamarix gallica from nodal explants of mature trees

Tamarix gallica L. was micropropagated from four-to six-node explants taken from mature trees. Shoot proliferation was induced on Linsmaier and Skoog medium containing 30 g l^-1 sucrose, 7 g l^-1 agar, 200 mg l^-1 reduced glutathione (basal medium) and supplemented with 3.3 M benzyladenine. Adding 0.5 or 1.0 M indole-3-butyric acid (IBA) to the basal medium increased lateral shoot formation and ease of rooting. Microcuttings repeatedly subcultured on 1.0 M IBA produced well-developed roots, a high number of axillary shoots and could be acclimatized in the greenhouse.     

Study of soybean and lentil root exudates

Summary Some polyphenolic compounds have been identified in root exudates of two species of legumes (both cultivated in sterile conditions): for lentil, three desoxy-5 flavones (4′,7-dihydroxy, 3′,4′,7-trihydroxy and 4′,7-dihydroxy, 3′-methoxyflavones) and for soybean: two isoflavonoids, coumestrol and daidzein (or 4′,7-dihydroxyisoflavone). Identified compounds are polyphenolic aglycones whereas roots contain the corresponding glycosides, showing that a hydrolysis takes place at the time of exudation. Compounds are observed in plantlet exudates as soon as the first day after planting. Their daily quantitative variations parallel those of total phenolic compounds. Exudated coumestrol and 4′,7-dihydroxyflavone amounts are estimated at about 20% of root content in these molecules.     

Comparison of maize root mucilages isolated from root exudates and root surface extracts by complementary cytological and biochemical investigations

Summary A strategy to obtain fractions enriched in mucilages secreted by root caps or produced by the rhizodermis of axenicallygrown maize seedlings is proposed. It involves a two-step procedure allowing the successive collection of root exudates and surface extracts from the same set of intact, sterile maize plants. Cytological controls were performed at each phase of collection. Whereas root cap mucilage is easily collected in water after one day's extraction, under conditions favouring secretory activity, rhizodermal mucilage remains tightly adherent to the root surface. It can be better extracted using neutral saline buffer assisted by gentle shaking at low temperature. Acidic saline buffer is unsuitable as it induces cell lysis and release of cell wall components.Biochemical analyses confirm that fractions enriched in root cap mucilage contain very high levels of fucose and galactose, high levels of arabinose, xylose and glucose and trace amounts of mannose. Fractions enriched in rhizodermal mucilage contain large amounts of glucose, moderate amounts of arabinose, xylose, mannose and galactose and trace levels of fucose. Isoelectric focusing and SDS-PAGE indicate that there are numerous similarities in the protein composition of materials enriched in root cap mucilages from root exudates or aqueous root surface extracts. However, specific protein bands that could be characteristic of rhizodermal mucilage are obtained using neutral saline buffer extracts. According to these biochemical data, the two-step procedure used in the present study appears to be useful for further biochemical characterization of both types of mucilages.Abbreviations BSA bovine serum albumin - BSTFA N,O-bis (trimethylsilyl)-trifluoroacetamide - DTT dithiothreitol - i. d. internal diameter - MW molecular weight - PATAg periodic acid-thiosemicarbazide-silver proteinate - PVPP polyvinylpolypyrrolidone - RE root exudates - RSE root surface extracts - TMCS trimethylchlorosilane - TMS trimethylsilyl     

Enhancement of embryogenic culture initiation from tissues of mature sweetgum trees

 Male inflorescences, female inflorescences, and leaves collected from dormant buds of three sweetgum (Liquidambar styraciflua) trees were tested for induction of somatic embryogenesis following treatment with thidiazuron, naphthaleneacetic acid (NAA) or different combinations of the two. Explants were placed into culture either within a few days after collection or following 2 months of storage at –15  °C. Although embryogenic cultures were obtained from all three trees, embryogenesis induction was strongly affected by genotype (source tree), with 100% of the staminate inflorescence explants from one tree producing embryogenic cultures in one experiment. Embryogenesis induction was also influenced by explant type, with staminate inflorescences up to five times more likely to produce an embryogenic culture than female inflorescences. No embryogenic cultures were obtained from leaf explants. While treatment with plant growth regulators was not required for embryogenesis induction from inflorescence explants, culture on medium with NAA alone resulted in the highest production of repetitively embryogenic cultures and cultures producing proembryogenic masses. Dormant buds stored for 2 months at –15  °C were still able to produce embryogenic cultures, although frozen storage decreased this ability by over one-half for staminate inflorescences. Received: 20 January 1999 / Revision received: 18 April 1999 / Accepted: 29 April 1999     

7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed

Root exudates from Acroptilon repens (Russian knapweed) were found to be phytotoxic and the phytotoxin in the exudate was identified as 7,8-benzoflavone (alpha-naphthoflavone), (1), not previously known as a natural product. In tests on growing seedlings both 1 and its isomer 5,6-benzoflavone (2) were phytotoxic. Flavone, a structural analog of 1 and a known granular leaf and stem exudate of other plant species, was also phytotoxic and more potent than 1 or 2.