Effects of Elevated Temperature and CO 2 on Biomass and Sucrose Accumulation of Selected Sugarcane Genotypes

Global warming cause due by increasing atmospheric CO 2 concentration, and the resulting increase in air temperature is a considerable challenge in crop production. Hence, the objectives of this study were to determine the: (a) responses of biomass and sucrose accumulation of sugarcane to elevated CO 2 (ECO 2 ) and elevated temperature (ET a ), both individually and together, and (b) genotypic variation of these responses. A three-factor factorial experiment considering the combination of CO 2 concentrations and temperatures as the main-plot factor and eight sugarcane varieties as the sub-plot factor arranged in a split-plot design in open-top chambers. Plots in open field conditions were the negative control. The main plot factor had four levels, combinations of ambient/elevated CO 2 concentrations (344-351/777-779 ppm) and ambient/elevated temperatures (34.9-35.6/36.6-38.4


INTRODUCTION
Increasing atmospheric CO2 concentration and resulting increase of air temperature (Ta) has variable impacts on the productivity of sugarcane depending on varieties and growing conditions (Marin et al., 2013(Marin et al., , 2014. Therefore, determining the impacts of climate change on the growth and productivity of sugarcane is vital to sustaining an adequate supply of sugar and its by products to meet their increasing demand in the future. Biomass and sucrose accumulation of sugarcane shows variable responses to elevated CO2 (ECO2) and temperature (ETa) depending on the specific growing conditions. For example, several studies conducted using potted plants grown in open-top chambers (OTCs) under well-watered conditions show that biomass accumulation of sugarcane increases at ECO2 (Vu et al., 2006;De Souza et al., 2008;Vu and Allen, 2009;Allen et al., 2011;Marin et al., 2013). Vu et al. (2006) has shown ECO2 increases sucrose accumulation as well. In contrast, Stokes and colleagues did not observe a stimulation of biomass accumulation in ECO2 under well-watered conditions (Stokes et al. (2016), which agrees with the majority of research findings on C4 species (Lawlor and Mitchell, 1991;Bowes, 1993;Kimball et al., 2002;Kim et al., 2006;Leakey et al., 2006Leakey et al., , 2009. Elevated CO2 has been shown to induce shifts in the carbon and nitrogen dynamics, and consequently, changes in the source-sink interactions within crops (Wolfe et al., 1998). Furthermore, the interactive effects of ECO2 and ETa on the physiology and growth of plants differ depending on their genetic make-up and ecological adaptation (Eller et al., 2013). Therefore, it is likely that the magnitude of the impacts of increasing atmospheric CO2 could be different on different crops grow under varying environmental and management conditions (Chaves and Pereira, 1992;Leakey et al., 2006). Vu and Allen (2009) show significant increases and genotypic variation in biomass accumulation of sugarcane in response to ETa. In addition, they observe amplification of the stimulation of biomass accumulation of sugarcane when ECO2 is combined with ETa. In contrast, Allen et al. (2011) show that the increasing trend of biomass and sucrose accumulation in sugarcane at ECO2 is downregulated when ECO2 is combined with ETa. Only limited work reported on the response of sugarcane growth and yields to climate change (Vu et al., 2006;De Souza et al., 2008;Vu and Allen, 2009;Allen et al., 2011;Stokes et al., 2016). Even such experiments have been conducted at the lower temperature range, where the daily mean temperature did not exceed 29 °C while being less than 25 °C for most of the crops' duration. The net assimilation rate of sugarcane shows a positive correlation with air and soil temperature (Venkataramana et al., 1984). However, increasing temperatures increase respiration rates (Atkin and Tjoelker, 2003) resulting in a decrease in growth and sucrose accumulation. Therefore, even though growth and sucrose accumulation may increase with ETa up to an optimum temperature, further temperature increases are likely to reverse this response. Accordingly, the objectives of this study were to determine responses, and genotypic variations of biomass and sucrose accumulation of sugarcane to ECO2 and ETa expected in the future.

Experimental location and period
The experiment was conducted in open top chambers (OTCs) installed in the research farm of the Sugarcane Research Institute (SRI), Uda Walawe, Sri Lanka (6 o 24'40"N latitude, 80 o 50'17"E longitude and 76 m altitude) from 12 th September 2017 to 25 th September 2018. The experimental site has an average annual rainfall of 1450 mm with a distinctly bimodal distribution. The average annual minimum and maximum temperatures and daily pan evaporation rates were 22 °C and 32 °C and 5 mm/day, respectively (Panabokke, 1996). The soil has been classified as Walawe Series of Reddish Brown Earth (RBE), in the great group of Rhodustalfs (order Alfisols, suborder Ustalfa) soils and has a sandy clay loam texture (De Silva and Dassanayake, 2010).

Design and construction of open top chambers
Twelve OTCs were constructed with ion bars covered with UV-treated 200-gauge polythene ( Figure 1). The frame of OTCs was approximately circular (3 m in diameter) with 12 sides fitted together. Its total height was 3 m and had an opentop (1.5 m in diameter). The vertical beams were bent, at a height of 2.5 m, inwards to form a frustum at the open top with a diameter of about 2 m. It maintains air temperature (Ta) within OTCs in near-natural conditions with a diurnal variation pattern similar to open field conditions (Norby et al., 1997;Welshofer et al., 2018).

Elevation of atmospheric CO2 and temperature
The methodology used in elevating, maintaining, and monitoring atmospheric CO2 and temperature is described in detail in De  and De . Air blowers (0.25 kW) were used to maintain the adequate air circulation in all OTCs. The CO2 level in designated OTCs elevated during the daylight hours (i.e. 0630 -1800 hours) | 69 by injecting pure CO2 through a gas regulator and a hose from 31 kg CO2 cylinders housed outside the chambers. The required CO2 concentration in the chamber was maintained by adjusting the pressure of release of CO2 from the cylinders. The pressure of CO2 release from the cylinder was set at 5 bars during the daylight hours when CO2 was being injected into the OTCs. The concentration of CO2 within the OTCs was monitored using the IEQ Chek environmental quality monitor (Bacharach Inc.). The Ta in designated OTCs elevated via heating coils installed in the air blowers. A heating unit had three 1 kW heaters. Each unit of heaters and air blower was manufactured as a single compound unit and was housed outside each chamber. The ambient or heated air was pumped to the chamber via an underground PVC pipe, connected to an adjustable polythene tube laid within the plot around the middle and perimeter of the OTC. The supply of CO2 with ambient or heated air was also circulated within the chamber using the same mechanism as the air supply. The height of the polythene tubes was raised gradually with time as the height of the canopy increased. A daytime mean temperature elevation of up to 3.5 °C above the prevailing ambient Ta achieved by a thermostat in the heating unit of the air blower and a temperature sensor installed at canopy level in the middle of the OTC. The thermostat was adjusted so that the heating unit switched off when Ta in the middle of the OTC as sensed by the temperature sensor exceeded 35 °C. The heating unit switched back on when Ta decreased below 35 °C. Elevation of CO2 and Ta in OTCs was done daily from 0630 to 1800 h to coincide with daylight hours following globally-adopted standard methodology (Norby et al., 1997;Welshofer et al., 2018). The open field plots, established in the same field but 20 m away from OTCs, also had the same dimensions as the plots within OTCs.
Eight commercial sugarcane (Saccharum hybrid spp.) varieties (i.e. Co775, SL7130, SL8306, SL88116, SL906237, SL924918, SL96128, and SL96328) were randomized separately within the two halves of each OTC and open field plots. Each variety was planted as 2 m rows spaced at 1.3 m. Therefore, each half of the OTC contained one row each from all eight varieties. As the eight varieties did not differ substantially in their plant and canopy architecture, the crop within each main plot (i.e. an OTC or an open field plot) was considered one contiguous population of sugarcane plants.

Crop establishment and management
Crop rows were established using single-budded seed-cane setts of the eight varieties. Each row consisted of four seed-cane setts of each variety. Hence, there were eight hills of each variety in a given OTC arranged in two rows with one row in each half of the OTC. Additional hills were established along the borders of the plots within OTCs to minimize the border effects. Crops were maintained with recommended fertilizer application and plant protection measures under well-watered conditions. Irrigation was provided at 0.15 m 3 of water per plot at 5-day intervals, while the soil water potential in the top 1 m was maintained above -0.05 MPa.

Monitoring environmental conditions
Micro-climatic conditions in OTCs were monitored continuously by installing sensors and data loggers (WatchDog1000 series micro stations model 1400, Spectrum Technologies, Inc. USA). Four temperature sensors (A, B, C, and D) were placed outside the OTC (A), and within it at 30 cm soil depth (B), at the soil surface (C), and in the air 2 m above the soil surface (D). The sensors monitored Ta continuously and recorded at 5-minute intervals. In addition, CO2, relative humidity (RH%), and Ta in the chambers were monitored using the IEQ Chek environmental quality monitor (Bacharach Inc., USA) while taking physiological measurements. During the experimental period, daily meteorological conditions in the experimental site, i.e. rainfall, minimum and maximum Ta and, relative humidity (RH%), were recorded at the SRI weather station located near the experimental field.

Estimation of biomass and sucrose accumulations
One sugarcane bush in each sub-plot was uprooted for biomass estimation on 77, 121, 156,187 and 370 days after planting (DAP). Roots, stems, leaves with the immature top of canes, and trash separated and oven-dried at 105 °C to a constant weight. The biomass of each portion of canes was estimated. The number of shoots, stem diameter, the height of all shoots at the leaf of a top visible dewlap (TVD), and the number of leaves in each sub-plot taken, and above-ground total biomass per stalk and bush, was estimated. Biochemical analysis of juice quality parameters such as Brix, pol, purity, pure obtainable cane sugar (POCS), and fiber percentages in all stalks in the subplots, was done in the laboratory after harvesting sugarcane at the end of the experiment i.e 370 DAP.

Statistical analysis
The significance of treatment effects on measured variables was tested by analysis of variance (ANOVA) in the general linear models procedure (SAS® Studio, SAS Institute Inc. 2021). When the CO2 × Ta × variety interaction was significant (p<0.05), the effects of CO2 and Ta on tested variables were evaluated for each variety separately. The following pre-planned mean contrasts were used to separately test the significance of the effects of elevated CO2 (ECO2), elevated Ta (ETa) and the combination of ECO2 and ETa in each variety: Effect of ECO2 = xCeTa -xCaTa Effect of ETa = xCaTe -xCaTa Combined effect of ECO2 and ETa = xCeTe -xCaTa where, xCeTa, xCaTa, XCaTe and xCeTe were the respective means of treatment combinations, elevated CO2 + ambient Ta (CeTa), ambient CO2 + ambient Ta (CaTa), ambient CO2 + elevated Ta (CaTe), elevated CO2 + elevated Ta, (CeTe). The 'chamber effect' (i.e. effect of the presence of the chamber) was estimated as the difference between the respective variable means of the CaTa and the open field treatment.

Environmental conditions in the treatments
During the daylight hours, CO2 enrichment increased CO2 in OTCs having the ECO2 treatment by 433 ± 2.6 and 435 ± 3.4 ppm, respectively in CeTa Relative humidity (RH%) inside all OTCs was greater than that in the open field throughout the day (Table 1). Compared to CaTa, ECO2, and ETa, both individually and in combination, RH% decreased within the OTCs. The highest depletion of RH% was in CaTe, whereas the lowest was in CeTa during the day and in CeTe during the night. The reduction of RH% was lower in the two ECO2 treatments compared to the CaTe treatment because the ECO2 retained more water vapor than ambient CO2. This data agrees with the atmospheric processes taking place on a global scale (Houghton, 2009).

Number of shoots per hill (Nsh)
Significant (p<0.05) effects of treatment, variety, and treatment × variety interaction on the number of shoots per hill (number of shoots emerged from a one-budded sett planted) were observed at 121, 156, and 187 DAP (Table 2, Figure 2). Separate analyses of variance for different varieties showed significant (p<0.05) treatment effects on Nsh in some of the varieties. In comparison to the ambient (CaTa), elevated Ta increased Nsh in the majority of varieties (e.g. Co775, SL7130, SL924918, and SL96328) at 121, 156, and 187 DAP. As such, the data suggest higher temperatures stimulated tillering and the population density of sugarcane. Similarly, Inman-Bamber (1994) showed that temperature is a major factor influencing the tiller and leaf appearance of sugarcane, and it is more important for efficient canopy light interception of sugarcane.
Elevated CO2 and the combination ECO2 and ETa increased Nsh relative to CaTa in a minority of varieties ( Figure 2). It showed that when elevated Ta combined with ECO2, the observed stimulation of tillering and population density have been further enhanced in some varieties (ex. SL7130 and SL96128) but not in others. However, encouraging good tillering is vital to building an adequate plant population in the field (Robertson et al., 1996;van Heerden et al., 2010), and it provides the crop with the sufficient number of stalks required for a good plant and ratoon cane yields (Vasantha et al., 2010;Matsuoka and Stolf, 2012;Bonnett, 2014.    Silva et al. (2022) Tropical Agricultural Research, 33(1): 67-79 | 73

Biomass in the individual stalks (Wst)
Biomass per stalk (dry weight basis) showed significant (p<0.05) treatment effects at 121, 156, and 370 DAP (Figure 3). The variety × treatment interaction effect on Wst was not significant (p>0.05). Elevated CO2 increased Wst by 27% relative to CaTa at 156 DAP. However, at 370 DAP, Wst was decreased by 7% and 6%, respectively, in ETa and the combination of ECO2 and ETa in comparison to CaTa. Elevated CO2 did not show significant (p>0.05) effects on Wst at 370 DAP. It could be due to the low response of C4 photosynthetic process to ECO2 (Long, 1999;Ghannoum et al., 2000;von Caemmerer and Furbank, 2003;Long et al., 2004;Leakey et al., 2009;Stokes et al., 2016;. Significant (p<0.05) genotypic variation was observed in the variation of Wst of sugarcane at different growth stages (Figure 4). Varieties Co775, SL7130, and SL8306 recorded higher values, and SL88116 and SL906237 recorded lower values of Wst on a majority of measurement days. Inman-Bamber et al. (2011) revealed that increasing biomass content in stalks through breeding and selection may not necessarily result in reduced sucrose content and increased fiber content.

Biomass per hill (Wh)
Biomass per hill (Wh) in the majority of days of measurement was not affected by ECO2 or ETa or their combination (Figure 5), despite the increase in the number of shoots (Nsh) in the majority of days of measurements, especially in ETa (Figure 2). Therefore, the absence of a response in the biomass per stalk (Figure 3) has had a dominant influence in controlling Wh under elevated CO2 and elevated Ta, which simulated future climates. In agreement with these results, previous studies of sugarcane (Stokes et al., 2016) and C4 species such as maize (Leakey et al., 2006) did not show stimulation of biomass at ECO2 under well-watered conditions. In contrast, at 156 DAP, the individual effects of both ECO2 and ETa and their combination caused a significant (p<0.05) increase in Wh relative to CaTa. In ECO2, this has occurred because of an increase in both Nsh and Wst. De Souza et al. (2008) and Allen et al. (2011) observed similar patterns, where sugarcane was grown at ECO2.

Each bar represents a mean of 30 data points across five treatments and six replicates. Error bars denote standard errors of mean
In response to ETa, the increase in Wh had occurred because of increased Nsh while Wst has remained unchanged. Similarly, Vu and Allen (2009) showed significant increases and genotypic variation in biomass accumulation of sugarcane in response to ETa. In contrast, Allen et al. (2011) showed that ETa caused a slight downward trend in sugarcane biomass accumulation regardless of genotypes or CO2 elevation.
Significant (p<0.05) genotypic variation was shown in the responses of Wh at different growth stages ( Figure 6). Varieties Co775 and SL96128 recorded higher Wh on a majority of measurement days. Notably, SL88116, which had higher Pol and POCS (Figure 7), recorded the lowest Wh than all other varieties at all measurement days ( Figure 6).

Sucrose accumulation and quality characters
Percentages of Pol and POCS in cane juice showed significant (p<0.05) effects of treatment, variety, and variety × treatment interactions at 370 DAP ( Figure 7). Separate analyses of showed significant (p<0.05) treatment effects on Pol and POCS in the majority of varieties.  (Vu et al., 2006;De Souza et al., 2008). They further showed that increased photosynthetic rates (+30%) and biomass accumulation (+40%) at ECO2 enhanced biomass partitioning to sucrose. Inman-Bamber et al.
(2011) and Lobo et al. (2015) showed that photosynthesis did not alter by a feedback mechanism induced by sucrose accumulation in stalks. Sink strength for sucrose storage in the upper internodes is stronger in both high fiber and high sucrose varieties. McCormick et al. (2006McCormick et al. ( , 2008McCormick et al. ( , and 2009) and Ribeiro et al. (2017) showed an increase in leaf photosynthetic capacity with a decrease in assimilating availability in young sink tissues.
Elevated Ta in the absence of ECO2 (CaTe) reduced Pol in SL7130, SL96128, and SL96328. The highest reduction of Pol and POCS by 20 and 19%, respectively, was recorded in SL96128. The increase in Ta may eliminate the benefits of the projected rise in CO2 on sugarcane as lower temperatures are essential for enhancing sucrose accumulation (Verma, 2004). Our findings agree with the previous results of Ebrahim et al. (1998) and Verma (2004) Vu and Allen (2009) have observed significant varietal variation on sucrose productivity of sugarcane at ECO2 and ETa. However, the increased respiration rates due to ETa (Hofstra and Hesketh, 1969;Atkin and Tjoelker, 2003;Heskel et al., 2016) could be the most probable reason for the reduction of sucrose accumulation in sugarcane. Notably, Pol and POCS in SL96328, which had higher respective values, were affected by the ECO2 and ETa individually and in combination. The significant (p<0.05) genotypic variation in the response of sucrose accumulation in sugarcane to ECO2 and ETa is further demonstrated by the observation of SL88116 not showing a significant (p>0.05) response in its already higher Pol and POCS to both ECO2 and ETa individually or in combination. Therefore, unresponsiveness of higher Pol and POCS in SL88116 to ECO2, ETa, and the combination of ECO2 and ETa could be vital in a future climate to ensure the stability of sugar recovery of sugarcane at milling.

CONCLUSIONS
As increasing atmospheric [CO2] and the resulting increase in air temperature (Ta) are clear features of future climate change, the current results provide indications and insights into the impacts of future changes in climate on sugarcane. Biomass production of sugarcane was not affected by ECO2 or ETa or their combination. The combinations of ECO2 and ETa reduced sucrose accumulation in 3 out of 8 varieties (Co775, SL8306 and SL96328) at the end of the growing season. However, sucrose accumulation in the variety SL88116, which had higher respective values at ambient and simulated future climatic conditions, was not affected by either ECO2 or ETa individually or in combination. The responses and the significant genotypic variation observed in sucrose accumulation to ECO2 and ETa, both individually and together could be utilized to maintain the stability of sugar production in CO2-rich warm climates.