Yang-Rui Li

Trehalose Synthase Gene Transfer Mediated by Agrobacterium tumefaciens Enhances Resistance to Osmotic Stress in Sugarcane

Trehalose synthase gene (TSase) fromGrifola frondosa was transferred into sugarcane (Saccharum officinarum L.) usingAgrobacterium-mediated method to improve sugarcane drought-tolerance. The results indicated that embryogenic callus of sugarcane was sensitive toA. tumefaciens EHA105 strain in the transformation system employed. The high frequency of PPT-resistant plants were obtained from transformated with 3 weeks callus after incubation, which reached 4.5% on average. The transgenic plants was confirmed by PCR and southern blot analysis. Some transgenic plants showed multiple phenotypic alterations and some plants demonstrated improvement tolerance to osmotic stress.

Effects of Vinasse and Press Mud Application on the Biological Properties of Soils and Productivity of Sugarcane

The present study has been conducted to investigate the effects of vinasse and press mud (PM) (sugar industry by-products) applications on sugarcane soil microbial population, its enzyme activities and biomass carbon and nitrogen in order to work out its potential to be used as bio-friendly fertilizer. The treatments were compared with traditional chemical fertilizer (CF) applications. Vinasse and PM amended soils were found to have slightly lower pH and higher total N and C content, however, the difference was insignificant with CF amended soils. Both the treatments increased sugarcane yield compared to chemical fertilizer, but this increase was not significant in PM amended soils. The populations of fungi, bacteria and actinomycetes increased in vinasse and PM amended soils. This increase in former one was significant in PM amended soils only, and that of the latter in vinasse amended soil. The biomass C and N contents were also higher in both the treatments, however, only the former one had significant difference with CF. Amongst the soil enzymes, the activities of cellulase, phosphatase and aminopeptidase were significantly higher in PM treatment while these were at par in vinasse and CF treatments. These results showed a potential possibility of substituting chemical fertilizers with vinasse and press mud which besides improving soil health and enhancing sugarcane productivity, can also solve the problem of their disposal in free environment.

Transformation of sugarcane with ACC oxidase antisense gene

The present study is an attempt to validate the function of sugarcane ACO by construction of plant expression vectors for sugarcane ACO sense and anti-sense genes and their effective transformation. Plant expression vectors for sugarcane ACO anti-sense gene was constructed and then transformed into sugarcane variety ROC22 mediated by Agrobacterium tumefaciens. The transgenic shoots were screened under the selective pressure of 40 mg/l G418 and 300 mg/l cefalexin and 19 resistant plants were obtained, two of which were further proved to be positive through detecting NPTII by PCR. The transformation rate was found 10.5%. The positive plants were found to grow slowly and were more dwarf compared to non-transgenic ones. The present work reveals the efficient genetic transformation system and development of transgenic sugarcane plant with ACO antisense gene. This work will help in understanding the ethylene and growth relationship and will also help breeders in breeding programs tailored to develop new varieties using molecular approaches.

Physiological and Biochemical Characteristics Related to Cold Resistance in Sugarcane

In order to screen the cold tolerant sugarcane varieties, in Ziyuan County, one of the coldest counties having longest duration for frost and snow, in Guangxi province we comprehensively evaluated 21 sugarcane varieties using subordinate function method combined with correlation analyses based on six physiological and biochemical indexes relevant to cold tolerance under natural conditions. It was found that different sugarcane varieties had different responses to the long duration of continuous cold stress, which were dull for soluble protein, proline and malondialdehyde contents. Based on the analysis by subordinate function method, we divided the tested varieties into three groups of cold tolerance. The group I had four varieties with high cold tolerance, the group II had ten varieties with moderate cold tolerance, and the group III had seven varieties with low cold resistance. It is feasible to evaluate the cold resistance at processing maturing stage by using subordinate function method with multiple physiological and biochemical indexes, which can reveal the differences of cold hardiness in different sugarcane varieties, and provide good references for commercial sugarcane production.

Identification of a new nitrogen fixing endo-bacterium strain isolated from sugarcane stalk

A bacterial strain B8S, with nitrogen fixation ability, was isolated from the stalk of sugarcane variety RB867515 introduced from Brazil. This strain was identified based on morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The 16S rRNA gene sequence analysis showed that the fragment sequence length was 1403bp. When compared withAgrobacterium tumefaciens (AY51349, AB116668.1 and AJ1307119) in GenBank, the homology of the nucleotides was 100%. Certain physiological and biochemical characteristics of new bacterium strain B8S were almost the same as that ofAgrobacterium tumefaciens. Based on its nitrogen fixation ability, the strain B8S was named asAgrobacterium diazotrophicus.

Endogenous hormone levels at technical maturing stage of sugarcane

Ethylene evolution of leaf and contents of ABA and IAA in cane-tip were measured during late growth stage of sugarcane. The ethylene evolution of different leaves (the leaf with TVD was defined as leaf +1, other leaves were count downward) showed that the younger leaves had higher ethylene release rates and the older leaves lower. This indicated that the ethylene evolution in leaf was not associated with leaf ripening or senescence but possibly depended on the physiology of whole plants. There appeared ethylene evolution peaks at the onset of ripening process in all varieties examined. These ethylene peaks were more than triple of normal. The ABA accumulation characters varied from varieties. The ABA peak appeared at the onset of ripening, one or two week(s) before the ethylene peak presented, for Guitang15 and ROC16 but no discernible ABA peak for Guitang11. These results indicated that both ethylene and ABA would play roles in inducing ripening of sugarcane. And divers mechanisms of hormones action or interaction were suggested. IAA content increased at the early time of ripening stage, indicated that IAA did not play opposing role to ABA and ethylene in the ripening of sugarcane.

Intercropping in Sugarcane Cultivation Influenced the Soil Properties and Enhanced the Diversity of Vital Diazotrophic Bacteria

Soil survey was conducted at four different locations of Guangxi, China to investigate the effect of peanut and soybean inter-cropping in sugarcane cultivation on the soil properties and diazotrophic bacterial diversity. Principal component analysis result shown (PCA) showed difference impact of intercropping and monoculture on the basis of biological and chemical properties of soil. Two-way analysis of variance of soil properties showed a significant interactive effect with locations and cultivation systems. Microbial enumeration results demonstrated that intercropping system enhanced the diazotrophic population. After isolation of diazotrophs, 21 selected bacteria were characterized by siderophore, phosphate, indole acetic acid, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and nitrogenase assay. Among the identified bacteria, isolate MYSP104 showed maximum nitrogenase activity and isolate MYSS78 showed maximum ACC deaminase activity. Partial 16S rRNA gene sequencing results classified these diazotrophs in six phyla (Firmicutes, α, β, Υ-proteobacteria, actinobacteria and bacteroidetes). Diazotrophs such as Bacillus tequilensis, Variovorax paradoxus, Acidovorax facilis, Leucobacter aridicollis, Streptomyces fimicarius and Pseudomonas nitroreducens were reported for the first time from the sugarcane rhizosphere of Guangxi, China. Venn diagram explained that seven bacterial species (Brevibacterium, Burkholderia, Delftia, Leucobacter, Pseudomonas, Sinorhizobium and Variovorax) were recognized with soybean and sugarcane intercropping cultivation systems. This study concluded that intercropping system could enhance the population of N-fixers in soil. Soybean intercropping influenced the soil chemical and biological properties better than peanut. Moreover, the isolated diazotrophs need further characterization and they might be utilized as bio-inoculums for commercial sugarcane production in the future.

Combined application of NPK on yield quality of sugarcane applied through SSDI

Field experiments were conducted to study the effects of combined application of nitrogen (N), phosphorus (P) and potassium (K) on growth, yield quality and nutrient balance of sugarcane with subsurface drip irrigation by using the sugarcane variety ROC89/1626 and adopting “3414” project design. It was observed that the appropriate rate of N, P2O5 and K2O increased the plant height, cane yield and cane quality of sugarcane under the experimental conditions. The fertigation model recommended for highest cane yield was 367.2 kg N, 162.6 kg P2O5 and 423.3 kg K2O per ha. The highest cane yield recorded with this dose was 101.8 t/ha. The best fertigation model recommended was 340.1 kg N, 141.9 kg P2O5 and 401.1 kg K2O per ha for the cane yield of 101.6 t/ha.

Analysis of the genetic diversity in Saccharum spontaneum L. accessions from Guangxi Province of China with RAPD-PCR

Random amplified polymorphic DNA (RAPD)-PCR was employed to analyze the genetic diversity within 89 Saccharum spontaneum accessions collected from five different ecological regions of Guangxi Province, China. Of the 87 bands generated by nine RAPD primers, 96.6% were polymorphic. A substantial degree of genetic diversity was found among the locally collected S. spontaneum accessions. Pairwise genetic distance coefficients between accessions ranged from 8.6% (between accessions 87-10 and Yunge ③) to 74.2% (between accessions 87-31 and 85-53) with an average of 39.9%. Based on these genetic distance coefficients, the 89 S. spontaneum accessions were clustered into four groups irrespective of their ecological regions using the Cluster program of SAS (V8) software. Information from the present study would be useful for the conservation of locally collected S. spontaneum germplasm and exploitation of its genetic potential during sugarcane crossing program.

Genetic Diversity of Nitrogen-Fixing and Plant Growth Promoting Pseudomonas Species Isolated from Sugarcane Rhizosphere

The study was designed to isolate and characterize Pseudomonas spp. from sugarcane rhizosphere, and to evaluate their plant- growth- promoting (PGP) traits and nitrogenase activity. A biological nitrogen-fixing microbe has great potential to replace chemical fertilizers and be used as a targeted biofertilizer in a plant. A total of 100 isolates from sugarcane rhizosphere, belonging to different species, were isolated; from these, 30 isolates were selected on the basis of preliminary screening, for in vitro antagonistic activities against sugarcane pathogens and for various PGP traits, as well as nitrogenase activity. The production of IAA varied from 312.07 to 13.12 μg mL−1 in tryptophan supplemented medium, with higher production in AN15 and lower in CN20 strain. The estimation of ACC deaminase activity, strains CY4 and BA2 produced maximum and minimum activity of 77.0 and 15.13 μmoL mg−1 h−1. For nitrogenase activity among the studied strains, CoA6 fixed higher and AY1 fixed lower in amounts (108.30 and 6.16 μmoL C2H2 h−1 mL−1). All the strains were identified on the basis of 16S rRNA gene sequencing, and the phylogenetic diversity of the strains was analyzed. The results identified all strains as being similar to Pseudomonas spp. Polymerase chain reaction (PCR) amplification of nifH and antibiotic genes was suggestive that the amplified strains had the capability to fix nitrogen and possessed biocontrol activities. Genotypic comparisons of the strains were determined by BOX, ERIC, and REP PCR profile analysis. Out of all the screened isolates, CY4 (Pseudomonas koreensis) and CN11 (Pseudomonas entomophila) showed the most prominent PGP traits, as well as nitrogenase activity. Therefore, only these two strains were selected for further studies; Biolog profiling; colonization through green fluorescent protein (GFP)-tagged bacteria; and nifH gene expression using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The Biolog phenotypic profiling, which comprised utilization of C and N sources, and tolerance to osmolytes and pH, revealed the metabolic versatility of the selected strains. The colonization ability of the selected strains was evaluated by genetically tagging them with a constitutively expressing GFP-pPROBE-pTetr-OT plasmid. qRT-PCR results showed that both strains had the ability to express the nifH gene at 90 and 120 days, as compared to a control, in both sugarcane varieties GT11 and GXB9. Therefore, our isolated strains, P. koreensis and P. entomophila may be used as inoculums or in biofertilizer production for enhancing growth and nutrients, as well as for improving nitrogen levels, in sugarcane and other crops. The present study, to the best of our knowledge, is the first report on the diversity of Pseudomonas spp. associated with sugarcane in Guangxi, China.