Molecular assessment of diversity among endophytic diazotrophs isolated from subtropical Indian sugarcane

Twenty-two endophytic bacterial isolates from the roots of sugarcane were compared morphologically, biochemically and genetically. Gram staining, colony pigment, texture and other cultural characteristics were taken for morphological characterization. Oxidation-fermentation tests for D-glucose and D-sucrose, production of acid and hydrogen from different carbon source, oxidase activity, antibiotic and drug resistance patterns were chosen as the biochemical and physiological criteria. Twelve random decamer primers were used to analyze and compare these isolates through RAPD among themselves as well as with known standard diazotrophic strains. The isolates were compared through dendrograms constructed on the basis of similarity patterns obtained from biochemical and RAPD analysis. The estimated diversity through RAPD analysis was more evident than the diversity on the basis of morphological and biochemical characters. Within Acetobacter group, the isolates showed substantial genetic diversity for future exploitation as PGPRs and diazotrophic associative endophytes.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

有机肥对桃园土壤硝态氮分布的影响

于2004—2005年在北京市平谷区有机桃园设置不同有机肥处理：2年连续施有机肥,年均67 500 kg·hm^-2（T₁）;第一年不施肥,第二年施有机肥135 000 kg·hm^-2（T₂）;第一年不施肥,第二年施有机肥67 500 kg·hm^-2（T₃）;不施肥对照（CK）,并于2006年对0～120 cm土层土壤进行取样分析,研究施用有机肥对土壤硝态氮分布和淋失的影响.结果表明：对照土壤中硝态氮分布较均匀,T₁和T₃在0～120 cm土层硝态氮浓度变化呈单峰曲线,其中60 cm以上较高,在60～120 cm逐渐降低;而T₂土壤硝态氮浓度由浅到深逐渐增加,峰值出现在100～120 cm土层,其在60 cm以下土层的硝态氮浓度在所有处理中最高,说明过量施用有机肥易导致硝态氮的淋失.相关分析表明,土壤硝态氮的浓度和分布与多年施氮总量、最近一年施氮量和检测点与树的距离呈显著相关关系,并据此建立了有机肥施用与土壤剖面硝态氮浓度之间的相关模型.     

Complementation and Expression Analysis of SoRab1A and SoRab2A in Sugarcane Demonstrates Their Functional Diversification

Mammalian and plant Rabl and Rab2 are small GTPases that regulate vesicle trafficking in the endoplasmic reticulum （ER） to Golgi compartments. Little is known about their functional diversification or potential interaction. We cloned sugarcane （Saccharum officinarum L.） Rab1A and Rab2A genes and studied their functional differences by expression and complementation experiments. We found differential expression of the two genes during sugarcane leaf development： SoRab2A expression declined from the dividing base to the maturing tip of the growing leaves, whereas SoRab1A was constitutively expressed, suggesting that SoRab2A is required for cell division and expansion and SoRablA is required for cells at all developmental stages. We used a yeast temperature sensitive ypt1-A 136D mutant strain to further investigate these shared and unique functions. Yptl is a small GTPase that regulates vesicle transport in the same cellular location as Rabl and Rab2. Neither SoRab1A nor SoRab2A alone could restore the growth of the mutant at restrictive temperatures when SoRab1A and SoRab2A were transformed separately. However, SoRab1A transformants maintained normal morphology and viability at non-permissive temperature, and resumed growth when returned to permissive temperature, whereas SoRab2A transformants died at non-permissive temperature, suggesting that SoRablA function is required for a cell＇s viability. Mutant growth was fully restored when SoRab1A and SoRab2A were co-transformed, indicating that SoRablA and SoRab2A complement each other and they both are needed to restore the function of ypt1-A136D. These results demonstrate that SoRab1A and SoRab2A serve distinct but overlapping functions, mostly by regulating the transportation of different sets of proteins.     

利用pmi标记基因筛选培育转EaDREB2B基因甘蔗

利用已构建的植物表达载体prd29a-dreb-hyg,通过酶切连接到含有磷酸甘露糖异构酶基因（pmi）的表达质粒pZMLR14上,构建植物表达载体pDREB-PMI。利用基因枪轰击转化甘蔗愈伤,经过甘露糖筛选,共获51株抗性苗,转化再生频率为4.25%。对转基因植株进行分子检测,结果表明有8株为阳性转基因无性系。氯酚红试验表明标记磷酸甘露糖异构酶基因在转基因株系中均有表达。对转基因T₁代甘蔗植株进行分子检测,结果表明EaDREB2B基因在转基因甘蔗无性系T₁代中稳定遗传。该结果为进一步研究EaDREB2B基因在甘蔗抗旱方面的作用奠定了基础。     

Doubled sugar content in sugarcane plants modified to produce a sucrose isomer

Sucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades. Recently, genes have been cloned for bacterial isomerase enzymes that convert sucrose into sugars which are not metabolized by plants, but which are digested by humans, with health benefits over sucrose. We hypothesized that an appropriate sucrose isomerase (SI) expression pattern might simultaneously provide a valuable source of beneficial sugars and overcome the sugar yield ceiling in plants. The introduction of an SI gene tailored for vacuolar compartmentation resulted in sugarcane lines with remarkable increases in total stored sugar levels. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. The lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. This remarkable step above the former ceiling in stored sugar concentration provides a new perspective into plant source–sink relationships, and has substantial potential for enhanced food and biofuel production.     

耕作深度对蔗地土壤物理性状及甘蔗产量的影响

探讨蔗地机械化耕作深度对耕层土壤物理性状及甘蔗产量构成因子的影响,阐明深松作业的针对性土壤区位和障碍因子,可为甘蔗良好耕层的构建和土壤改良策略的制定提供科学依据.本研究设置3种深松作业深度(35、40和45 cm),以不深松为对照,对蔗地土壤物理性状(紧实度、容重、含水率、孔隙度、三相容积率)和甘蔗产量构成因素及蔗茎产量等指标进行研究.结果表明: 耕作作业深度与蔗地土壤结构特性及甘蔗产量的改善提升效应呈显著正相关.深松能够打破犁底层,显著降低土壤紧实度和容重,减小机械作业相应的贯入阻力和抗剪强度,尤其对20～30 cm土层的上述因子具有显著改善效应,对甘蔗高产意义重大.深松显著提高了30 cm以内土层的液相容积率,增大了土壤水分库容,使10～30 cm土层的水分指标显著改善.10～30 cm土层是深松对耕层土壤固相容积率改善效应最显著的区位.耕作深度的增加对甘蔗有效茎数、株高、蔗茎产量及蔗糖含量具有显著的促进效应.鉴于蔗区当前较普遍的机具装备水平,我国适宜蔗区实施机械化深松的作业深度标准应不小于40 cm.     

Biodegradation and detoxification of dyes and industrial effluents by Ganoderma weberianum B-18 immobilized in a lab-scale packed-bed bioreactor

White-rot fungi are considered to be promising biotechnological tools to complement or replace the current technologies for the treatment of effluents from textile production plants. The aim of this work was to investigate the decolorization capacity of Ganoderma weberianum B-18 in solid state fermentation with sugarcane bagasse as a substrate and ligninolytic inducer as well as to decolorize and detoxify industrial effluents by this strain in a laboratory scale packed-bed bio-reactor. The results demonstrated that G. weberianum B-18 indeed showed to possess decolorization capacity in solid state fermentation with sugarcane bagasse supplemented with synthetic dyes. Moreover, fungal biomass of G. weberianum B-18 immobilized in sugarcane bagasse in a packed-bed bioreactor was shown to efficiently decolorize and detoxify different dyes and authentic industrial effluents in semi-continuous conditions. In this decolorization process, laccase enzymes secreted by the fungus played the main role. Hence, a packed-bed reactor with G. weberianum B-18 immobilized in sugarcane bagasse seems to be a suitable system for the further development of an efficient bioprocess for large-scale treatment of dye-containing wastewaters.     

甘蔗转基因甘露糖筛选系统的建立

以甘露糖作为筛选底物,对甘蔗品种新台糖22号进行临界筛选浓度测定,获得愈伤组织的继代、分化、生根的临界筛选浓度。而后应用含有甘露糖筛选标记基因pmi及绿色荧光蛋白报告基因GFP的植物表达载体,通过农杆菌介导法对新台糖22号的愈伤组织进行遗传转化。用所测定的临界筛选浓度先后进行继代、分化、生根筛选培养,获得抗性植株。对获得的抗性植株分别进行pmi基因和GFP基因的PCR检测,以及GFP显微镜荧光检测,结果证实已成功建立了高效的甘蔗转基因甘露糖筛选系统。     

Effects of biocides and rotation breaks on soil organisms associated with the poor early growth of sugarcane in continuous monoculture

Glasshouse and field experiments were conducted to determine the effects of biocides and rotation breaks on deleterious soil organisms associated with the poor early growth and subsequent yield decline of sugarcane grown in continuous monoculture. Fumigation of a soil that had been under sugarcane monoculture with minimal breaks for more than 30 years markedly improved the health and growth of the sugarcane sett and shoot root systems, increased the growth of the primary shoot and stimulated more and larger secondary shoots. It also reduced populations of culturable fungi in the rhizosphere of the sett roots and reduced colonization of the sett and shoot roots by lesion nematode (Pratylenchus zeae). Exposure of the developing sett root system for 14 days to mono-cultured sugarcane soil was sufficient to significantly retard subsequent plant growth. In field experiments, fungicide and nematicide (mancozeb + aldicarb), when applied together to land under sugarcane monoculture, was as effective as fumigation in improving early sugarcane growth and increasing sugarcane yields. Rotation breaks (alternate crops, sown pasture, bare fallow) that were in place for 54 months, increased sugarcane establishment and increased sugarcane yields to levels similar to that obtained following fumigation of land under sugarcane monoculture. Fumigation of land that had been under the rotation breaks gave plant growth responses that were in addition to that achieved by the breaks alone. A mancozeb + aldicarb treatment was as effective as fumigation in increasing sugarcane yields after a bare fallow break but accounted for only a portion of the fumigation response following the crop and pasture breaks. Improved plant nutrition may be a factor in the fumigation response following the crop and pasture breaks. Plant growth responses to fumigation and the manocozeb + aldicarb treatments that were manifested in final sugarcane yields (after one years growth) were evident as plant growth responses (sett root, shoot root and primary shoot dry weight) measured 54 days after planting. The experimental results support the concept that when sugarcane is grown as a monoculture, deleterious fungi and nematodes retard plant establishment and early plant growth and that this leads to reduced sugarcane yields.