Article pubs.acs.org/JAFC
Estimating Broad Sense Heritability and Investigating the Mechanism of Genetic Transmission of Cold Tolerance Using Mannitol as a Measure of Post-freeze Juice Degradation in Sugarcane and Energycane (Saccharum spp.) Anna L. Hale,*,† Ryan P. Viator,†,Δ Gillian Eggleston,‡ George Hodnett,§ David M. Stelly,§ Debbie Boykin,# and Donnie K. Miller⊥ †
Sugarcane Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 5883 USDA Road, Houma, Louisiana 70360, United States ‡ Commodity Utilization Research Unit, Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, Louisiana 70124, United States § Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas 77843, United States # Southeast Area, Agricultural Research Service, U.S. Department of Agriculture, 141 Experiment Station Road, Stoneville, Mississippi 38776, United States ⊥ AgCenter Northeast Research Station, Louisiana State University, 4589 Highway 605, St. Joseph, Louisiana 71366, United States ABSTRACT: In approximately 25% of the sugarcane-producing countries worldwide, conventional sugarcane (Saccharum spp. hybrids) is exposed to damaging freezes. A study was conducted during the 2009 and 2010 harvest seasons to compare lateseason freeze tolerance among three groups: commercial Louisiana sugarcane genotypes, early generation genotypes selected for cold tolerance in the U.S. Department of Agriculture sugarcane breeding programs at Houma, LA, and Canal Point, FL, and potential energycane genotypes selected for high total biomass per acre. Mannitol concentrations in cane juice following freezing temperatures were determined to evaluate levels of cold tolerance. Genotypes selected for cold tolerance in Houma, LA, had significantly more late-season freeze tolerance than commercial sugarcane genotypes and genotypes selected in Canal Point, FL. Genotypes showing the most cold tolerance were Ho02-146 and Ho02-152, and those that were most highly susceptible were US87-1006 and US87-1003 (early-generation breeding genotypes) and L99-233 (commercial genotype). Broad-sense heritability for late-season cold tolerance in the two-year study was estimated at g2 = 0.78. The enzymatic mannitol analysis successfully differentiated high-fiber energycane genotypes from those from other sources. KEYWORDS: energycane, freeze deterioration, Leuconostoc bacteria, mannitol, sugarcane
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INTRODUCTION
season freeze damages the cane to the point that the juice can no longer be processed.2 High juice acidity and the formation of impurities such as polysaccharides following a damaging freeze often result in slower processing rates, which leads to reduced sugar recovery and, thus, lower end-product yields (e.g., sucrose, ethanol, second-generation biofuels) and profits. Severe freezes can lead to cessation of factory activities.3 The nature and extent of damage to sugarcane depends on the intensity and duration of the freeze event. The weather af ter the freeze also affects the rate of deterioration. Critical temperatures for sugarcane freeze damage have been previously reported.2 Slight damage to the terminal buds occurs between 0.0 and −2.2 °C, and the apical meristem and auxiliary buds are killed between −2.8 and 3.9 °C. When temperatures reach −4.4 °C, lateral buds begin to weep, and if the temperature falls below −5.6 °C, the stalks are susceptible to cracking, allowing for the invasion of bacteria,
When sugarcane (Saccharum spp. hybrids) is grown in subtropical climates, early maturing cold- and freeze-tolerant genotypes are critical to the survival and expansion of the sugar and energycane industries. In most sugarcane-producing areas of the world, the climate is tropical and the crop is grown for at least 12 months before harvest; however, frequent early- and lateseason freezes in subtropical environments, for example, Louisiana in the United States, limit the growing seasons to 7− 9 months. Early harvest, therefore, begins before the sugarcane is physiologically mature, and this contributes to fewer ratoon crops and lower sugar yields than those seen in tropical production areas.1 In Louisiana, commercial sugarcane is planted in August, but fall and early winter frosts kill the above-ground leaf tissue. The crop must then re-emerge in the following spring. It is not unusual for above-ground plant tissue to be killed by cold temperatures and re-emerge, only to be repeatedly killed again until the first or second week of April. The processing season begins in late September or early October and continues until all of the cane is harvested (typically late December) or until a late© XXXX American Chemical Society
Received: August 4, 2015 Revised: January 26, 2016 Accepted: January 29, 2016
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DOI: 10.1021/acs.jafc.5b03803 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 1. Details of Saccharum Genotypes Assessed for Their Resistance to Post-freeze Juice Degradation Showing the Clonal Classification (E, Energy; Ho, Houma Cold Tolerant; Com, Commercial; and CP, Canal Point Cold Tolerant), Their Parentage and Recurrent Backcrossing History (Generation), and the Basic Germplasm from Which They Were Derived (Basic Soruce) genotype
female
US87-1003 US87-1004 US87-1005 US87-1006 US87-1007 US87-1008 US87-1010 US87-1013
US72-1151 US72-1289 CP79-1380 US72-1151 US78-1005 US72-1151 NCO 310 US78-1005
HoCP96-540 L97-128 L99-233
LCP86-454 LCP81-10 CP79-0348
Ho06-9001 Ho06-9002 US72-114 Ho00-961 Ho01-07
Ho02-149 Ho02-149 CP52-068 US94-01 CP83-0664
Ho02-144 Ho02-145 Ho02-146 Ho02-147 Ho02-148 Ho02-149 Ho02-150 Ho02-152 Ho02-153
SES234B SES234B SES234B SES234B SES234B SES234B SES234B SES234B SES234B
male Classification CP polycross polycross CP70-321 CP57-526 CP72-2086 CP57-526 CP70-321 CP72-2086 Classification Com LCP85-384 LCP85-384 HOCP91-552 Classification E HoCP96-540 HoCP96-540 US66-65-11 HoCP91-552 IND81-166 Classification Ho LCP85-384 LCP85-384 LCP85-384 LCP85-384 LCP85-384 LCP85-384 LCP85-384 LCP85-384 LCP85-384
generation
basic source
BC1 BC1 BC1 BC1 BC2 BC1 BC3/BC5 BC2
US56-15-8 US66-56-9 US66-56-4 US56-15-8 US66-56-4 US56-15-8 S. spontaneum/S. robustum US66-56-4
BC5 BC5 BC3
US56-15-8 US56-15-8 28NG251 (S. robustum)
BC1/BC6 BC1 BC1 BC2/BC4 F1
SES234B/US56-15-8 SES234B/US56-15-8 Gehra Bon SES231/SES205A IND81-166
F1/BC5 F1/BC5 F1/BC5 F1/BC5 F1/BC5 F1/BC5 F1/BC5 F1/BC5 F1/BC5
SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8 SES234B/US56-15-8
to °Brix ratio is used as the indicator of degradation. An ezymatic mannitol test is considered a fast and simple method to indicate whether sugarcane can be economically processed and is less expensive and simpler and the results are easier to interpret than tests for haze dextran.5,9,10 Cold tolerance is a major focus of the breeding program at USDA-ARS Sugarcane Unit in Houma, LA, because of the potential economic impact to the sugarcane industry. Another aim of the program is to develop energycane genotypes for release from the basic breeding program (e.g., Ho02-113).11 Commercial sugarcane genotypes produce high sugar yields and must have 14% fiber and are released solely for renewable energy production. This includes use as a feedstock for the production of ethanol or syngas.12 In the United States, potential energycane production areas are being considered outside the traditional sugarcane-growing regions on marginal soils and in more northern latitudes; thus, cold tolerance is a major consideration in the release of these genotypes. There have been no previously published reports of late-season freeze tolerance in energycane genotypes. Sugarcane is very genetically complex,13 and genotypes are derived from interspecific hybrids primarily involving Saccharum officinarum L. and Saccharum spontaneum with contributions from other Saccharum species such as Saccharum barberi, Saccharum sinense, and Saccharum robustum as well as other related genera such as Erianthus and Miscanthus.14,15 The crop is both aneuploid and polyploid, with genotypes containing
especially Leuconostic mesenteroides, and the subsequent conversion of sugar to metabolites such as dextran and mannitol.2,4 The frozen cane is particularly susceptible where warm and humid conditions prevail following the freeze.3,5,6 Once temperatures reach −5.6 °C, severe deterioration is observed within 1−2 weeks for most genotypes. Studies have shown there are clonal differences for plant response and the rate of deterioration following damaging freezes, and these differences between genotypes may be evident for ≥2 weeks following the freeze event.4,5 Because the Louisiana sugarcane industry is located in a subtropical area that is prone to damaging freezes, cold tolerance has, historically, been an important selection criterion in breeding programs. Selection for cold tolerance in sugarcane has been difficult because the biochemical and physical responses to low temperatures in some genotypes are still unknown. In addition, a practical and easy phenotypic evaluation method to assess juice cold tolerance is not available, making the routine evaluation of large numbers of parental genotypes difficult. Routine screening of small numbers of elite genotypes for freeze-damage resistance in the USDA-ARS Sugarcane Research Unit’s breeding program in Houma, LA, has historically been conducted by measuring changes in titratable acidity, juice pol, and dextran content as indicators of L. mesenteroides deterioration;3 however, more recent studies have shown that mannitol, a sugar alcohol, is a more sensitive indicator of freeze damage.7,8 Because genotypes have naturally varying levels of soluble solids (°Brix), when levels of mannitol in juice are evaluated, a mannitol B
DOI: 10.1021/acs.jafc.5b03803 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry variable numbers of chromosomes.16 When S. off icinarum and S. spontaneum are hybridized with S. off icinarum as the female parent, the progeny typically inherit 2n chromosomes from the S. off icinarum and n from the S. spontaneum.17 The 2n + n transmission of chromosomes also tends to happen when S. off icinarum is used as the female parent and crossed to early generation hybrids of S. spontaneum.18 When S. spontaneum is used in sugarcane hybridization, it is typically used as the male parent. The species is considered a noxious weed in most countries, and hybrids must be verified before planting in the field if the S. spontaneum is used as the female. The first objective of this study was to compare post-freeze juice degradation in commercial LA sugarcane genotypes, earlygeneration genotypes selected for cold tolerance at the USDAARS Sugarcane Research Unit in Houma, LA, and the USDAARS Sugarcane Field Station in Canal Point, FL, and energycane genotypes previously selected without regard to cold tolerance. The second objective was to determine the broad sense heritability of post-freeze juice degradation in these genotypes and assess the mode of transmission of cold tolerance genes in this diverse breeding population.
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Juice samples were processed as described above. Sample collection in 2010 was stopped at 16 DAF because excessive post-freeze degradation prevented accurate laboratory analysis of juice quality. Preparation of Reagents. Solutions were prepared according to the methods of Eggleston and Harper20 and Eggleston10 with minor modifications. Previously, the NAD solution was prepared daily, but for this study, it was stored for up to 1 week in a −20 °C freezer. Four mannitol standards (10, 100, 250, and 500 ppm) were prepared in triplicate by diluting a 500 ppm stock in deionized water. A new standard curve was generated for each new enzyme preparation. Factory Mannitol Enzymatic Method. Cane juice was thawed and diluted 1:1 v/v in glycine buffer with mannitol (2-fold). Ten milliliters of juice was then mixed with 0.5 g of Celite (diatomaceous earth) and filtered through a glass filter (25 mm diameter) attached to the end of a plastic syringe. The first 2 mL of filtrate was discarded, and 5 mL was used for the assay.10 A master-mix was prepared by combining a ratio of 1.4 mL of glycine buffer to 0.2 mL of NAD for all samples. For each test sample, 1.6 mL of master-mix was combined with 0.2 mL of juice and 0.2 mL of mannitol dehydrogenase (MDH) (10000-fold dilution), and for each control, 0.2 mL of deionized water was added in place of the MDH. A blank containing 1.6 mL of master-mix and 0.4 mL of water was used as a standard correction for the spectrophotometer. Mixtures were vortexed and transferred to a 10 mm quartz cuvette, and the absorbance was measured for the blank, control, and sample at 340 nm at 0 and 5 min on a Shimadzu UV-1201 spectrophotometer. The sample and control absorbance values were corrected with the blank at both sample times. Each sample was run in duplicate, and CVs were generally
