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The transient gene expression of protoplasts in plant is a considerable tool for gene functional research that has been widely used in gene analysis and functional characterization. Therefore, the objectives of this study were to develop a protocol for the isolation and purification of sugarcane protoplasts (Saccharum spp. hybrids), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 × 106 protoplasts g−1 FW) and a survival rate of > 80.0% was achieved through enzymatic hydrolysis at constant temperature of 28 °C, 60–70 rpm min−1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG 4000 for 25 min and the ScD27 protein was localized in the chloroplasts. The localization of ScD27 protein in sugarcane protoplast demonstrated that the newly developed protocol was functionally effective. This optimized sugarcane protoplast isolation, purification, and transient expression protocol lays a foundation for future molecular biology research in sugarcane.

[This corrects the article DOI: 10.1371/journal.pone.0233752.].

Sugarcane (Saccharum L. spp. hybrids) is a globally important crop. While roots contribute to its net primary production, knowledge of sugarcane root growth is incomplete and limited in scope. The objective of this study was to determine cumulative root density (LA), root distribution, effective rooting depth (ERD), and root growth rate for four sugarcane genotypes across three crop cycles (plant cane [PC], first ratoon [FR], and second ratoon [SR]). The experiment was conducted in a rain shelter on four sugarcane genotypes (ROC22, YZ04-241, YZ05-194, and YZ05-51) using minirhizotron tubes to a soil profile depth of 1.08 m. Most roots in were found at 0–0.36 m-depth, and about 85.0% were within a 0–0.72 m depth in PC. In FR and SR, however, > 81.8% of roots were found at 0–0.54 m-depth regardless of genotype. The LA of ROC22 was often significantly higher than that of the other genotypes at depths < 0.54 m regardless of crop cycle. The genotype ROC22 had greater root length and faster root growth rate than YZ05-194 at 0.18–0.36 m in PC and FR, while SR YZ05-194 had slower root growth than the other genotypes at 0–0.18m. Ratooning ability of sugarcane genotypes may be related to these root growth attributes.

Sugarcane (Saccharum spp.) is an important economic crop, supplying up to 80% of the table sugar and ~60% of bio-ethanol worldwide. Due to population growth and dwindling fossil-fuel reserves, the demand for sugar and bio-ethanol requires significant improvement in sugarcane production. Breeding sugarcane cultivars with high-performance agronomic traits is undoubtedly the most efficient way to achieve this goal. Therefore, evaluating agronomic traits and dissecting underlying loci are critically important for this aim steps in providing genetic resources and molecular markers for selection. In this study, we assembled a diversity panel of 236 elite sugarcane germplasms originally collected from 12 countries. We evaluated 28 agronomic traits in the diversity panel with three replicates. The diversity panel was genotyped using amplified fragment length polymorphism markers, and a total of 1,359 markers were generated. Through the genome-wide association study, we identified three markers significantly associated with three traits evaluated at a stringent threshold (P < 0.05 after Bonferroni correction). The genotypes of the three associated markers grouped respective trait values into two distinct groups, supporting the reliability of these markers for breeding selection. Our study provides putative molecular markers linked to agronomic traits for breeding robust sugarcane cultivars. Additionally, this study emphasized the importance of sugarcane germplasm introduced from other countries and suggested that the use of these germplasms in breeding programs depends on local industrial needs.

Sugarcane is a major economic crop that is grown mostly where water availability is a dominant limitation to production. However, selection for cane yield under dry conditions is difficult because of variable rainfall and sometimes high experimental error variance. If measurements made under relative non-stress conditions could be used to predict relative yield of genotypes under dry environments it could improve effectiveness of selection, especially in early stage selection trials in breeding programs. In this study, field experiments were conducted to evaluate 31 sugarcane genotypes in rain-fed and irrigated treatments across multiple sites and years in Yunnan province, South China. The aim was to examine relationships between cane yield and several leaf functional traits and determine if measurements made in environments experiencing low water stress could be used to predict relative yield of genotypes in dry environments. The water treatments had a large impact on mean cane yield and all the measured leaf function traits. However, for cane yield genotype × water treatment interactions were relatively small relative to genotype main effects, except when water stress was severe. For the leaf functional traits in most cases, genotype main effects were highly statistically significant while genotype × date and genotype × water interactions were mostly not statistically significant despite contrasting conditions at different dates of measurement. It was found that a combination of high yield and low leaf conductance or high leaf temperature (both of which may be related to reduced rates of water use), all measured under non-stress conditions, was predictive of yield under the limited water treatments, with prediction levels better than either yield or leaf based measurements alone. This is suggestive of a potentially valuable role for this combination of measurements in optimal selection indices in early stages of selection in sugarcane breeding programs. High sampling and error variances and high labour requirements associated with measuring conductance or leaf temperature using traditional methods are a limitation to practical application. However, these may be overcome through emerging technologies enabling related measures through aerial imaging. Development and testing of these approaches is recommended to develop better selection methods in early stage selection trials in breeding programs.

Sugarcane is a high biomass crop grown mostly in rain-fed environments. Water is a key determinant of sugarcane productivity, and in most production regions, varying and sometimes severe water stress occurs. Understanding the extent of genetic variation in response to water stress may allow for better targeted breeding programs. In this study a set of field experiments evaluating 31 sugarcane clones under rain-fed and irrigated conditions was conducted in two sugarcane production areas in Yunnan province, South China. While the water treatments had a large impact on mean cane yield there were little or no genotype × water treatment interactions for cane yield or sugar content at harvest, or for dry biomass sampled about five months (early biomass) after the start of crop growth. The high genetic correlation between well-watered and water stress treatments is consistent with the hypothesis that clone performance under water stress is mostly associated with general vigour rather than traits acting specifically under water stress. From a practical breeding program perspective the results suggest that choice or management of water stress level in selection trials is not a critical consideration for sugarcane breeding programs, and that selection under reasonably well watered conditions will be effective for attaining satisfactory selection gains under at least moderate levels of water stress. Early biomass was positively correlated with yield at final harvest for all water treatments. Resolution of the physiological basis of the high genetic correlations between growth under well-watered and water limited conditions may assist in future breeding efforts.

One hundred and thirteen accessions of exotic sugarcane germplasms consisted of Q,VMC,FR,RB,CP and TCP were investigated in this study.Thirteen agronomic and quality traits including germination,tillering,ratoon germination,stalk height,stem diameter,number of millable stalk,stalk weight,cane yield,brix,sugar content,simple purity,fiber content and sugar yield were observed by principal component analysis and cluster analysis.The result of correlation analysis showed that sugar and cane yield was significantly positively related to height,millable stalks and single stem weight.Sucrose content was significantly positively related with brix and purity.Significant negative correlation was found between millable stalks and diameter.Ratoon sprout was closely linked to rate of germination,tillering percentage,and there was multiple co-linear relation among these traits.The result of principal component analysis on 13 traits showed 5 principal elements,which provided 75.16% cumulative variance.Based on the principal component scores,113 accessions of sugarcane germplasms were grouped into 7 clusters.ClusterⅠwas high in fiber content,low in yield.Cluster Ⅱ was weak in germination and tillering,low in yield.Cluster Ⅲ was strong in ratoon germination,high in cane yield and sugar content.Cluster Ⅳ could be used as parents for energy cane breeding.Cluster Ⅴwas high in cane yield,low in sugar content.Cluster Ⅵ was high in sugar content,low in cane yield.Cluster Ⅶ was high in cane yield,high in sugar content.

Thirty-one cross combinations consisted of thirteen ROC varieties including ROC1,ROC6,ROC8,ROC9,ROC10,ROC11,ROC20,ROC22,ROC23,ROC24,ROC25,ROC26 and ROC28 as female parents were made,and methods of family evaluation and ratooning ability(RA)were combined together for evaluating the ratooning traits of progeny populations under natural drought conditions.The results showed that the progenies were more significantly affected by ratoon crop than plant crop.Moreover,heritability of ratoon crop in cane yield,sugar yield and stalks were higher than that in plant crop,availing to breeding the strong ratoon offspring.Among the thirteen ROC female parents,the general combining ability(GCA)of main traits from ROC25,ROC11 and ROC24 were all higher in plant crop and ratoon crop.Furthermore,values of gca in cane yield,sugar yield and brix of ratoon crop were higher than that of plant crop.Thus these ROC parents could be excellent female parents with drought resistance and strong ratooning traits.The special combining ability(SCA)of main traits in plant crop and ratoon crop of ROC25×Dezhe 93-88 were higher than that of other cross and the ratooning ability of sugar yield and cane yield were 13.7% and 20.4% higher than those in ROC22 respectively.These ROC25×Dezhe 93-88 could be excellent cross for breeding the new varieties which could combat drought and have strong ratoon sugarcane.