Biomass potential of novel interspecific and intergeneric hybrids of Saccharum grown in sub-tropical climates

Sugarcane-derived biomass is a promising source of renewable energy to meet the growing demands for biofuel. Currently, modern sugarcane cultivars are unable to provide enough biomass due to their narrow genetic base and susceptibility to abiotic and biotic stresses. We have evaluated total of 23 hybrids derived from diverse genetic backgrounds of different Saccharum spp. and allied genera, one inbred and compared with commercial checks. Intergeneric hybrids (IGHs) KGS 99-100 and GU 04-432, produced significantly higher biomass (43.37 t ha−1 and 35.24 t ha−1, respectively) than commercial sugarcane have genes derived from Erianthus arundinaceus. Interspecific hybrids (ISHs) GU 07-3704 and 99-489, also produced significantly higher amounts of biomass (37.24 t ha−1 and 33.25 t ha−1, respectively) than commercial checks have genes from S. officinarum and S. spontaneum backgrounds. ISHs recorded significantly higher biomass yield, number of stalks and total dry matter percentage whereas, IGH group recorded significantly higher fibre percent. Furthermore, the clones resistant to red rot and sugarcane borers were identified. The estimated energy value for seven hybrid clones was found to be very high. Cluster analysis of genetic traits revealed two major clusters in traits improving biomass. Our study has revealed that the genetic diversity present in these hybrids could be used for improving biomass production and tolerance to abiotic and biotic stresses in cultivated sugarcanes.

biochar and soil carbon sequestration 1 . Most of these technologies, however, are currently only in the demonstration stages, and large-scale efforts are still required to offset the current levels of CO 2 emissions produced by humans. Since sugarcane is a high biomass producing crop, it could be used to convert CO 2 into soil carbon; one study shows that sugarcane plantations can convert more CO 2 into soil carbon than matured or secondary forests 9 . It has been found that sugarcane yields in Australia increased by 8% and 4% with weak climate change and moderate climate change, respectively, but were reduced by 10% with strong climate change 10 . Sugarcane based biomass (bioenergy) is a low cost energy production system 5 and a versatile source of renewable energy as it allows four to five times harvest of ratoon without replanting 6 .
To date, sugarcane is among the most efficient crops in the world along with other C 4 grasses when it comes to conversion of solar energy into stored chemical energy and biomass accumulation 11 . It has been estimated that more than 700 Mha of land being used to grow cereal crops produced a total of only 2400 mt year −1 of biomass, whereas only 21 Mha of sugarcane crops alone produced 1750 mt year −1 of biomass 12 . Therefore, sugarcane may provide a vital solution to many environmental problems without reducing economic benefits at the global level.
Conventionally, in the sugar industry, the "bagasse" that remains after juice extraction through crushing is an important by-product used to generate electricity and produce fertilizers 13 . More recently, it has been used for biofuel production 14,15 . However, the production cost for bioconversion of sugarcane biomass into biofuel is still considered to be relatively high, which makes it difficult to commercialize on a large scale 16 . Therefore, it is essential to develop improved sugarcane varieties with high biomass yield and fibre content, which also have better biomass degradability; in addition, we also need better enzyme digestion technologies to maximize the efficiency of conversion of sugarcane biomass into biofuel 17 .
Brazil is the first country to have successfully launched an ethanol-based fuel program (ProAlcooL), which produces alcohol on a large scale from sugarcane 18 ; in 2014, Brazil produced around 23.4 billion litres of ethanol from sugarcane biomass (Renewable Fuel Association, 2015). However, India has also successfully exploited the energy potential of sugarcane to produce ethanol and electricity 19 . To enhance Indian's energy security, the Government of India (GoI) aimed to increase ethanol blending percentages in gasoline fuels from 5 to 20% by 2017.
Since usage of biofuels now plays a significant role in drafting the energy policies of many countries, several studies have shown that producing biomass for use as biofuels can be significantly increased 20 . Unfortunately, at present, the cane varieties cultivated in India have only 13-15% fibre, and the development of high biomass producing cultivars has enormous potential to meet the energy demand in the country 21 .
In India, sugarcane is grown under both tropical and subtropical areas. The sub-tropical region faces climatic vagaries throughout the year, such as flooding, extreme winter and summer temperatures, drought, and salinity, all of which are major constraints for crop production. Therefore, in order to be able to grow high biomass producing sugarcane crops under subtropical regions, we need to develop high biomass-type clones which are also tolerant to several different types of environmental stresses 22 .
There is tremendous genetic variation found within the Saccharum species and allied genera of Miscanthus, and Erianthus species that can be exploited for high biomass and fibre in sugarcane breeding programs 23 . However, in the recent past, although the genetic diversity of S. officinarum has been exploited towards this end, the genetic diversities of S. spontaneum 24 , S. robustum and other allied genera have not been explored 25 .
The ICAR-Sugarcane Breeding Institute, Coimbatore, has been working to generate intergeneric (IGH) and interspecific (ISH) hybrids of sugarcane with high biomass production potential using S. spontaneum, S. robustum, S. barberi, and Erianthus arundinaceus 26 . In this study, we evaluate the potential of IGHs and ISHs and we have generated as potential energy and fuel producers.

Results
A high degree of variation among the clones under study (Table 1) for all the traits in both the years was observed. The interaction term for year and clone (year × clone) identity was significant for all traits except numbers of millable canes (NMC), which indicates that the performance of the clones varied between the two years of growth. Further, it was assumed that the interaction term genotypes x environment (G × E) plays a significant role in the performance of these hybrids over several years. However, the interaction between year and replication was found to be non-significant, indicating that replications over different years do not have a significant impact on the traits studied. The results of the two-way analysis of variance (ANOVA) for the eight biomass related traits for two years are shown in Table 2.
Biomass production and traits affecting biomass production. The characteristics of ISHs and IGHs were compared with either commercial sugarcane or population means to identify clones with better-performing traits in biomass production. Out of the 28 clones evaluated, five ISHs and two IGHs recorded significantly higher dry biomass yields as compared to the population mean of 29 www.nature.com/scientificreports/ commercial check. In overall experiment, ISHs group have recorded significantly higher biomass yield, number of stalks, and total dry matter percentage compared to other group whereas, IGHs group have recorded significantly higher fibre percent than ISHs (Table 3).
Cane growth and quality parameters. None of the IGHs and ISHs performed better than the commercial cultivars with respect to juice quality traits such as Brix% in juice, sucrose% in juice and purity% ( Resistance to insects and pests. All the ISH and IGH clones of sugarcane with four standard varieties of cane were evaluated to observe their resistance against major insect pests, namely, the early shoot borer (ESB), top borer (TB), stalk borer (SB), and root borer (RB). All the clones were least susceptible (LS) to ESB and TB (< 15% and 10%, respectively). Twenty-three clones were found to be LS (infestation index < 2), while one clone, GU 07-3730, was moderately susceptible (MS) to SB (infestation index = 2.1-5.00). In case of the RB, 22 clones were LS (< 15%) and two clones were MS (15.1-30%) to the RB (Table 4).
Resistance to red rot. The resistance of ISHs and IGHs to red rot were recorded using mixed inocula of two virulent races of C. falcatum (CF08 and CF09) prevalent in subtropical India. Out of seventeen ISHs screened, three clones exhibited resistance (R), nine were moderately resistant (MR), two were moderately susceptible (MS), and three were susceptible (S) to this disease ( Table 4). The ISH clones 99-81, 99-132, 99-438, and 99-488  Cluster analysis. Two-way cluster analysis separated the ISH and IGH clones into two major groups based on their traits (Fig. 2)

Discussion
The genetic variation found in Saccharum germplasm may play a very significant role in improving sugarcane for biomass production through breeding and biotechnological approaches. Sugarcane intergeneric and interspecific species like Miscanthus, Erianthus, S. officinarum, and S. spontaneum have a lot of allelic diversity and can be exploited further for improving sugarcane biomass yield 17,32,33 . It was possible to develop high dry biomass hybrid clones when the existing commercial cultivars are combined with high biomass type clone 22,34,35 . Miocque (1999) 34 , who investigated sugarcane growth and biomass for ten crops in Sao Paulo State (Brazil) reports that the highest biomass accumulation occurs when sugarcane has a longer growth period. Further, the potential for crude energy production of sugarcane clones can be enhanced by using fibre-rich species. One of the important findings of this study is that the early generation hybrids of S. spontaneum (F 1 and BC 1 ) and E. arundinaceus (F 1 ) had higher biomass yields than the later generation clones. This indicates a limited variation in modern cultivars (commercial canes) and necessitates the utilization of wild relatives in improving biomass in sugarcane (Table 3).
Two-way analysis broadly separated the accessions based on trait variation. Group A contains sugarcane hybrids with higher single cane weights, juice brix percentages, and numbers of stalks. This group contains mostly commercial cultivars and other hybrids of diverse origin. It is also indicated that these potential performers may be considered as candidate donors for further exploiting traits variation for genetic improvement of the sugarcane. www.nature.com/scientificreports/ Since trait improvement in sugarcane through marker assisted selection (MAS) and genomic selection at the molecular level is difficult due to its genomic complexity (large genome size and high polyploidy level) [36][37][38] , efforts were made in this study to introgress genes for improvement of biomass yield from allied genera and other species of Saccharum. Out of 28 hybrid clones that were evaluated in this study, five clones of ISHs-2004-186, GU 07-3704, KGS 2004-13, KGS 2004-60 and 99-489-showed significantly higher dry biomass yields as compared to the population mean indicating their superiority over the existing commercial cultivars. Since the ISHs have S. officinarum, S. spontaneum, S. robustum, S. barberi, or S. sinense as one of the parents in their backgrounds, it is likely that these species have contributed traits that have significantly improved morphology, good ratooning, and tolerance to various biotic and abiotic stresses 39,40 . In addition, since these species are ecologically well adapted to subtropical Indian conditions, their hybrid clones can grow well even in marginal lands. ISHs-GU 07-3704, GU 07-3730, GU 07-3764, GU 07-3784, and GU 07-3849-showed significantly superior winter sprouting over www.nature.com/scientificreports/ the best standard CoS 767 (supplementary Fig. S1), because of these hybrids background contains contribution of the genome from population improved S. officinarum and S. spontaneum. Screening against red rot from the wild germplasm of sugarcane, allows new sources of resistant and red rot resistant can be transferred into cultivated species through interspecific and intergeneric crosses 41 . In the study, ISHs clones-derived from population improved officinarum, S. barberi x S. sinense and S. officinarum × S. spontaneum, were reported as resistant against red rot. Among the species, S. spontaneum originated clones have high level of resistance against red rot 42 , and effectiveness of S. spontaneum in sugarcane breeding had been evident since the development of first interspecific hybrid 'Co 205' in sugarcane. IGH clones-derived form E. arundinaceous with commercial cane and S. robustum with E. arundinaceous produced resistant clones against red rot. Use of Erianthus spp. as potential source for diversifying the genetic base and red rot resistance in sugarcane 43 . Furthermore, the majority of clones studied were least susceptible for early shoot and root borers due to their wider genetic makeup, hence showed resistance reactions against the major insets prevailing in subtropics.
Our results show that ISH and IGH clones performed well in the field conditions. Sugarcanes, Miscanthus species (Miscanthus giganteus), Erianthus species (E. arundinaceus Retz.), and switchgrass (Panicum virgatum) are known to be efficient biomass accumulators and converters of solar energy into chemical energy 11,44 . These traits were also found in the IGHs KGS 99-100 and GU 04-432, which were significantly better than the best commercial cultivar Co 0238 in producing dry biomass. Similarly, in terms of estimated energy, IGH clone KGS 99-100-a hybrid derivatives of the commercial cane and E. arundinaceous, and clone GU04-432-a hybrid derivatives of S. robustum and E. arundinaceous, had significantly higher energy value than the existing commercial varieties (supplementary Fig. S2). Therefore, these hybrids could be good sources for improving biomass and bioenergy traits in popular cultivars of sugarcane. Erianthus spp. due its higher biomass accumulation and calorific value, good ratoon ability and exceptional adaptability to biotic and abiotic stresses, is considered as potential breeding material for use in future sugarcane breeding programs 45 . Intergeneric material is powerful tools for widening the genetic base in polyploidy crop breeding. Harvey et al. (1994) 46 also suggested the use of Miscanthus and Erianthus species for broadening genetic bases in sugarcane breeding programs. Furthermore, the use of ISH and IGH clones in sugarcane hybridization can tap into many relevant genetic variations in the germplasm for better biomass production traits while also broadening the genetic base 47 .
Though biomass improvement in sugarcane can be challenging due to several issues such as the small gene pool in currently available sugarcane cultivars, long breeding periods and selection cycles, poor synchronization and fertility of flowers in parental lines, and genome complexity associated with this 48,49 . Since wild sugarcane species have more genetic variability than domesticated sugarcane cultivars, it makes sense to use ISHs and IGHs clones to improve biomass traits in sugarcane. In addition, breeding efficiencies can be improved by selecting for parental strains with wide genetic backgrounds coupled with introgression of genes for improving biotic and abiotic stress tolerance.
In conclusion, our study shows that the utilization of the Saccharum and Erianthus species in sugarcane breeding programs has the potential for generating clones with enhanced biomass accumulation and yields. The tremendous genetic diversity present in these species could be used as a significant genetic reservoir for improving biomass and bioenergy production traits in commercial canes which have narrow gene pools. Currently, long breeding cycles, non-synchronous flowering, and poor fertility among the desired parents are major bottlenecks for the efficient improvement of sugarcane for biomass contributing traits. These hurdle's can be overcomes by utilizing ISH and IGH clones in sugarcane breeding programs to develop superior clones of high biomass potential and high tolerance for abiotic and biotic stresses. Generated clones with increased biomass and bioenergy traits in this study will be good genetic material for further utilization in sugarcane breeding programs in improving biomass-energy traits. Furthermore, these clones could be grown in lands other than prime agricultural land (thereby avoiding competition for land with food industries) to meet the growing demands for energy.

Methods
Plant material and experimental site. All experimental were carried out on 28 clones obtained from 17 ISHs and six IGHs (IGH), one inbred, and four commercial sugarcane checks (two early-Co 0238, CoJ 64, and two mid-late-CoS 767, CoS 8436 checks (Table 1). These clones were evaluated for their biomass production potential and other related traits under subtropical conditions in India. The population-improved S. officinarum (PIO), S. spontaneum (SIP), and S. robustum (PIR) clones used in this study were the product of intra population improvement programs for their respective species.
The experimental site was located at the ICAR-Sugarcane Breeding Institute, Regional Centre, Karnal (Haryana) India (29.1°-29.5° N and 76.3°-77.1° E), which has a subtropical climate. The location stands at an elevation of 243 m above mean sea level and receives an average rainfall of ~ 744 mm per year. The maximum temperatures here range from 34-45 °C in summer, and minimum temperatures range from 5-8 °C in winter. The maximum and minimum temperatures, evaporation rates, relative humidity, and rainfall during 2013-14 and 2014-15 are shown in Fig. 3. The soil in this area ranges from clay-loamy to loam, with a pH range from 8.0-8.5; the site is irrigated from a bore well source.
Experimental design. The experiment was conducted in a randomized block design with three replicates for two years (during 2013-14 and 2014-15 spring seasons). The plots used were 2 m × 6 m with 0.9 m spacing in size. Recommended crop production practices were followed for raising the experimental crop. Data on the numbers of millable canes (NMC), brix% in cane, single cane weight (SCW), dry cane weight (DCW), total dry weight (TDM), and fresh biomass weight (FBW) were recorded for each plot from five randomly tagged canes at a crop age of 10 months. Five canes from each plot were used for estimating the percentage of fibre; a subsample of 250 g of the shredded canes were crushed in a rapipol machine and oven dried. The fresh weights and dry www.nature.com/scientificreports/ weights of these samples were recorded. Fibre percentage was estimated using the rapipol extraction method and calculated as per the method explained in Thangavelu and Rao (1982) 50 .
where A is the dry weight of bag + bagasse after drying (g), B is the dry weight of bag alone (g), C is the fresh weight of cane (g). Juice brix and sucrose percentages were measured from extracted cane juice. Brix percentage in cane and dry cane weight were estimated in the 12th month of the crop, i.e., the harvest stage. Fresh biomass yield was worked out from single cane weight and NMC per plot. CCS percentage at the 12th month was computed as per the method explained in Chen and Chou (1993) 51 . To estimate the dry matter percentage, brix percentage in cane, and dry biomass yield, the following formulae were used.
where WSB is the weight of the sample before crushing (g), WSA is the weight of the sample after crushing i.e. Bagasse (g) and DMB is the dry matter content in bagasse (%).
Fresh biomass yield (t ha −1 ) was calculated from the number of millable canes/ha × weight of a single cane (kg) with top attached. Dry biomass yield was calculated in t ha −1 using following formula described by Mohanraj et al., 2014 21 .
To identify clones with better ratoonability when harvested during the peak winter period in subtropical climates (i.e., January when temperatures fall below 5 °C), sprouting of ISH/IGH clones was quantified. This was done by harvesting the plant crop (half of the row/replicate (3 m row length), and counting the numbers of sprouted clumps per row and recording the average numbers of shoots/sprouted clumps in February; using these, the WSI was calculated 52 . The number of stubbles sprouted per plot and number of shoots formed per clump during winter was counted on the 45th day after ratooning. To ascertain winter sprouting potential of sugarcane clones, a winter sprouting index (WSI) was used as per Bakshi Ram et al. (2017) 52 ; the WSI is calculated as follows: Red rot screening. To identify their resistance to red rot, all ISH and IGH clones were screened against the most prevalent and virulent pathotype of red rot (Colletotricum falcatum), namely, Cf 08 and Cf 09 under field conditions. A mixed inoculum of both races was inoculated into sugarcane plants by plug and nodal methods during the month of September in the 7-month old crops 53 . www.nature.com/scientificreports/ Energy calculation. The energy content of plant mass mainly depends on its composition; fats and proteins have higher energy contents than simple carbohydrates. Sugarcane is mainly composed of carbohydrates (sugar and lignocellulose) that have an energy content of (~ 15.9 MJ/kg) 54 . Energy content was calculated by multiplying the total dry biomass by 15.9 and expressed as Gigajoule/ha/year (Gj/ha/year).

Statistical analysis.
All data were subjected to analysis of variance (ANOVA) tests. Means, standard deviations, and coefficients of variance for different traits were computed using the statistical package SAS 9.3 software (SAS Institute Inc., Cary, USA). Cluster analysis was performed using the JMP pro 10.0 version to elucidate the differences between IGHs, ISHs, and commercial varieties of sugarcane. Analysis of means (ANOM) was done for comparing energy values and winter sprouting index (WSI) values of the different ISHs, IGHs, and commercial sugarcanes.

Data availability
The data generated during and/ or analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.