Journal of Energy Bioscience 2025, Vol.16, No.3, 151-162 http://bioscipublisher.com/index.php/jeb 156 CRISPR/Cas9 doesn’t just edit one gene. It can adjust several genes at the same time. For example, scientists can simultaneously adjust genes related to biomass, straw digestibility, and stress resistance. This helps them create new maize varieties with a combination of desirable traits (Barriere et al., 2009). 6 Evaluation of the Biofuel Suitability of Maize Lines 6.1 Screening methods for starch yield and fermentability Screening maize lines suitable for biofuels is usually carried out by combining morphological and biochemical analyses. Morphological screening includes measuring plant height, ear characteristics, grain yield, etc., while biochemical analysis mainly detects the starch content of grains. By measuring the starch content of different maize inbred lines or hybrids, lines with high yield potential can be preliminarily screened (Pratikshya et al., 2025). Further fermentability evaluation is usually carried out in small-scale fermentation trials under laboratory conditions to measure the ethanol yield per unit mass of grain under standard enzymatic hydrolysis and fermentation conditions. In the breeding process, statistical methods such as principal component analysis (PCA) and correlation matrix are often used to screen and cluster maize lines with excellent biofuel characteristics, which can help identify lines with better performance in terms of starch content and fermentation efficiency. 6.2 Evaluation of lignocellulose degradability To evaluate the lignocellulose degradability of maize lines, it is first necessary to analyze the cellulose, hemicellulose and lignin content in the straw. The contents of cellulose (28.05%~37.05%), hemicellulose (7.06%~15.81%) and lignin (20.65%~28.35%) in the straw of different maize inbred lines and hybrids are significantly different (Pratikshya et al., 2025). Generally, strains with high cellulose and low lignin have better enzymatic hydrolysis efficiency and higher ethanol yield. Under laboratory conditions, commonly used evaluation methods include enzymatic saccharification rate determination and simulated fermentation test. Through standardized pretreatment and enzymatic hydrolysis, the releasable reducing sugar content and final ethanol yield in the straw can be determined (Choudhary et al., 2020; Pratikshya et al., 2025). 6.3 Comparison of laboratory scale and field scale evaluation The evaluation of the suitability of maize biofuels includes not only small-scale biochemical and fermentation tests in the laboratory, but also large-scale yield and quality determination in the field. Laboratory scale evaluation is simple to operate, short cycle and low cost, and is suitable for the initial screening of a large number of materials in the early breeding stage. By measuring starch content, cellulose and lignin content, enzymatic saccharification rate and other indicators in the laboratory, potential lines can be quickly screened out (Choudhary et al., 2020; Pratikshya et al., 2025). Laboratory results are often difficult to fully reflect the performance in the field environment, especially under the influence of climate, soil and management factors. Field-scale evaluation is closer to actual production conditions and can comprehensively examine the growth performance, yield, harvest index and biochemical characteristics of maize lines in different ecological environments (Serrano et al., 2014; Munaiz et al., 2021). Field trials can test the yield stability, straw yield and quality of different lines in multiple locations for many years, and can evaluate their adaptability to low-input conditions (such as reduced fertilization and irrigation) (Serrano et al., 2014). Field evaluation can also be combined with harvest time to monitor the dynamic changes of biomass, ash content, energy value and other indicators (Serrano et al., 2014; Wojcieszak et al., 2022). 6.4 The role of high-throughput phenotyping The high-throughput phenotyping platform can automatically measure the morphological, physiological and biochemical characteristics of a large number of maize samples in a short time (Choudhary et al., 2020). Through high-throughput imaging and near-infrared spectroscopy (NIRS) technology, the content of starch, cellulose and lignin in maize kernels and straw can be quickly determined (Choudhary et al., 2020). High-throughput
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