Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131

Review Article

A Review on the Sorghum for biofuel and microRNAs

Syeda Hafsa Ali1, Muhammad Younas khan Barozai1*, Muhammad Din1 and Ahmad Naseer Aziz2 1. Department of Botany, University of Balochistan, Quetta-Pakistan 2. Department of Agriculture and Environmental Sciences, Tennessee State University, 3500 John A. Merritt Boulevard Nashville, TN, United States of America *Corresponding author’s email: [email protected] Citation Syeda Hafsa Ali, Muhammad Younas khan Barozai, Muhammad Din and Ahmad Naseer Aziz. A Review on the Sorghum for biofuel and microRNAs. Pure and Applied Biology. Vol. 5, Issue 4, pp1064-1100. http://dx.doi.org/10.19045/bspab.2016.50131 Received: 04/07/2016 Revised: 04/08/2016 Accepted: 08/08/2016 Online First: 20/08/2016 Abstract Energy is lifeline in development and progress of a country, with pivotal use in industrial and agricultural sectors. While fossil fuels are primary source of energy, its rapid depletion is major concerns of the world today. Developed countries are finding alternates to strengthen its economic backbone by resolving limited supply of energy issues and meeting their enormous demands. Plants can offer better alternative as biofuels to address the energy crisis and meet projected energy demand. Recently, sorghum based research remained center of attention due to its exceptional properties to grow under dry, and hot environmental conditions with limited water requirement. All parts of sorghum have economic values with usage in syrup, sugar, fuel, alcohol, bedding and paper production. Whereas, Sorghum stalks are enriched source of carbohydrates with 16-18% of fermentable sugar that makes it potential candidate for bioethanol production. Bioethanol is considered environmental friendly as it reduces greenhouse gases and replaces MTBE (Methyl tert-butyl ether) pollutants in air. Yet, the benefits of sorghum as biofuels comes with a challenge of rapid degradation of sugar, therefore immediate harvest after maturity can ensure high sugar content. This review covers scope and recent research on sorghum in Pakistan and indicates its usage as an ideal feedstock to meet present energy crisis. Currently, developed countries are exploiting sorghum in bioethanol production due to its high tolerance to drought and salt, and improved sugar content in lieu of using sugarcane and maize. Moreover, studies involving high-tech research and role of microRNA in high yield and improved sugar content in biofuel production of sorghum are also addressed in this review. Keywords: Sorghum; Biofuels; miRNA; Bioethanol; Economic and environmental concerns Introduction called as ‘Jawar.’ It is widely used as staple Sorghum based research in Pakistan food for humans besides its usage as fodder Pakistan is an agriculture based country with for animals. Sorghum is warm-weather crop variety of important cereal crops, including and adapted to wide range of soil and sorghum. Sorghum ranks fifth among climate conditions [1]. Its grain comprises of important cereal crops of the world, and is 70% carbohydrates, 10-12% protein and 3% top summer grass of Pakistan and commonly fats, while fodder is highly enriched with

Published by Bolan Society for Pure and Applied Biology 1064 Hafsa Ali et al. prussic acid and oxalic acid, therefore grains RAPD analysis, and found 78.94% can be introduced in feeding programs for polymorphism. Through their work sorghum poultry and cattle [2]. Around 2.35 million varieties, YSS-9 and 84G01 showed distant hectares of land, contributing in 12% of the relationship, while RARI-S3 and RARI-S-4 cultivated land is cropped area for fodder were found to be closely related [6]. production in Pakistan (Agricultural Sorghum has diverse genome, therefore, Statistics of Pakistan, 2005). Though genetic fingerprinting provides an efficient production of good quality forage requires marker system. In 2010, another genetic well organized livestock industry, food crop diversity study on 29 sorghum varieties was land cannot be compensated for the fodder conducted among exotic, and approved local cultivation. Therefore, legumes are inter- lines using RAPD, revealing 95% cultured with cereal and forage crops to polymorphism among the varieties. They increase fodder supply. Although cereal found that the genotypes of exotic lines K- crops give high yield, their protein content is A-113 and Indian III exhibited maximum lower than legumes [3]. Yield of sorghum is similarity, whereas F-606 and F-601 showed higher even from small farm area as distinctive features [7]. compared to other plants however, the Till date genotypes of many crops are forecasted values of sorghum production evaluated using physiological markers, as revealed decrease in cultivation trend in near recent study [8] evaluated drought tolerance future. This decrease in cultivation trend is in sorghum (80 accession) using due to improper use of sorghum for physiological parameters revealing industrial purpose and use of low yielding significant differences in all accessions varieties [4]. Nevertheless, the research on under stress. Osmotic pressure was sorghum has been increased in recent years considered potential trait for drought while advancing from physio-morphological tolerance, while five accessions (80265, characteristics. Previously, germplasm 80114, SS-95-4, SS-97-7 and 80377) were characterization was performed using found widely tolerant to water stress [8]. physiological, morphological or isozyme Furthermore, a study conducted on role of markers. Recently, DNA finger printing has exogenous salicylic acid in salinity tolerance removed the barriers in germplasm on post germination seedlings (PARI-S-4 characterization by providing enough and YSS-9) revealed that low level of genetic information that was unavailable at salicylic acid reverse the impact of salinity phenotypic level. Tabbasum and her in seedlings but at high salt concentration coworkers [5] distinguished two sorghum (50 mg L-1): beyond threshold causing high hybrids while identifying 78.98% genetic level of Na+/ K+ ratio, salicylic acid was resemblance among them using random found ineffective in reducing salinity [9]. amplified polymorphic DNA (RAPD) [5]. Another study on determining effect of Advancement in molecular techniques has exogenous proline in reducing impact of helped identifying genetic diversity among salinity in sorghum cultivars revealed that crops and thus use of agronomic, low concentration (50 mM) of proline boost physiological and morphological traits have physiological characteristics of sorghum, been improvised to the level of biochemical whereas as high concentration (100 mM) and molecular markers. A study conducted together with high salt concentration by Mehmood et al. [6] approached alleviated the adverse effect caused by salt phylogenetic relationship and genetic stress [10]. Akram et al. [11] determined diversity of 10 sorghum varieties through genetic variability for drought tolerance in

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20 genotypes of sorghum cultivars using 10 applications. Potash fertilizer caused RAPD primers. Maximum similarity of significant differences in all parameters 95.5% was noted between YSS-17 and except for plant height, thus, highest grain PARC-SS-1 genotypes, while minimum yield was recorded in maize (8014 kgha-1) similarity of 51.5% was observed for DS- with three-split dose of 120 kgha-1, as 97-1 genotype. Furthermore, maximum compared to control with minimum grain similarity was observed between YSS-10 yield. Therefore, recommended application (C) and YSS-18, YSS-98 and SV-10, CSV- of 120 kgha-1 of potash fertilizer with 3 15 and Rasili, VI-1 and RS-29 [11]. In splits can help improve grain yield for both another study, 17 of sorghum landraces were crops [15]. evaluated for grain yield and drought In Pakistan, salinity is among major tolerance using physio-morphological challenges that adversely affect the yield and markers at seedling and post flowering growth of crops to various extents. stages, revealing high genetic difference in Therefore, cultivation of salinity tolerant water stress tolerance for higher grain yield lines can bring solution to obtain economical [12]. Hussain et al. [13] evaluated eight yield. Kausar et al. [16] screened genotypes of sorghum of Potohar region for physiological parameters of sorghum lines various grain and associated traits, showing to identify salinity tolerance. Among all significant difference in grain yield, plant sorghum lines, Sandalbar and JS-2002 were height, stalk yield, days to 50% flowering considered tolerant, while JS-263 and and maturity. Among tested varieties of Hegari had medium tolerance. Sorghum line sorghum, SPV-462 was stalk producing Noor was conceived as medium sensitive, variety with higher grain yield, whereas whereas, PSV-4 and FJ-115 were found Johar and CSP-15 SPV were high grain sensitive to salinity [16]. Another study for producing varieties, while SPV and YSS effects of heavy metal (NiCl) on were high stalk producing varieties. Among morphological characteristics of sorghum the sorghum varieties tested, PARC-SS-2 demonstrated that increasing concentration took minimum days to 50% flowering, up to 90 ppm (of NiCl) can adversely reduce whereas, CSV-15 and YSS-9 took longest dry weight of sorghum while 30ppm was time to 50% flowering [13]. Sher et al. [14] considered minimum threshold level [17]. investigated the effect of harvest time Seed priming technique widely used to associated with P and S fertilization on yield stimulate the seed emergence and seedling and quality of forage sorghum (Sorghum growth has been studied by Shehzad et al. bicolor (L.) Moench). They concluded that [18] to evaluate effect of these different harvesting at maturity stage along with P techniques such as: un-soaked seed and S fertilization increases forage yield and (control), Halopriming with KNO3 and other quality related traits as: 30% decrease CaCl2 (1% solution), Hydro-priming in leaf hydrocyanic acid content, and 50% (soaked with distill water) etc. On increase in stalk soluble-solid content germination and seedling growth of three widely used as an indicator of forage sorghum varieties (JS-2002, Hegari, and JS- juiciness and palatability [14]. Furthermore, 263). All priming techniques accelerated a study [15] on effect of potash fertilizer germination rate by 50%, while Halopriming dose on sorghum hybrid (Pioneer MR- with KNO3 and CaCl2 helped improving Buster) and maize hybrid (Pioneer 3062) seedling growth and sorghum emergence showed high grain yield with increase in [18]. Genetic potential of 20 sorghum potash level and number of split genotypes was evaluated for drought

1066 Hafsa Ali et al. tolerant traits (heritable and measurable) growth of rice and weeds: (Dactyloctenium after artificially creating water stress via aegyptium, Trianthema portulacastrum and PEG treatment and five (80353, 80365, Eleusine indica.Sorghum) and it was found 80199, 80204 and 80319) were found that sunflower water extract caused high superior to be used in drought tolerant inhibitory effects by reducing 50% time breeding programs. Therefore, crops at early required for seed emergence compared to growth stages can be screened using the powder treatments [22]. different heritable morphological parameters During 2011, a new sorghum hybrid variety for drought stress [19]. Seven advanced was introduced for general cultivation in lines of sorghum, i.e., Noor, Hegari, F- Pakistan. This dual purpose sorghum hybrid 9917, F-207, F-214, JS-2002 and PC-1 were Sorghum 2011 was developed by crossing evaluated for nutritive profile as well as dry Australian No.7 (exotic) with Sugrorib matter and forage material, and Hegari (local) through pedigree breeding method in genotype was found superior in terms of year 1998-99. During 2002-03 to 2009-10 forage material as well as nutritive dry homozygous progenies no. F6-6019 were matter due to high leaf area [20]. F-9917 and bulked to evaluate various traits for high F-214 displayed poor performance in terms yield and uniform fodder, and Sorghum- of ash content, crude protein and fiber 2011 hybrid showed exceptional needed to be served for forage purpose. performance compared to Hegari, JS-263 These finding on new genotypes revaled that and JS-2002 existing cultivars, in terms of genetic variation among sorghum genotypes high nutritional value, low fertilizer can contribute in potential traits for forage requirement, improved water efficiency, purpose and therefore, can be exploited [20]. high grain yield, robust growth, wide Although hydrogen cyanide (HCN) is adaptability and green fodder with minimum associated with it, sorghum is considered HCN content [23]. Akhtar et al. [24] main forage crop for livestock. Zahid et al. demonstrated impact on quality of forage [21] determined the effects of HCN content sorghum when these were sown individually at different growth stages (of plant, i.e., 3rd or together with forage legumes. Quality and leaf (GS1), pre-booting (GS2) and 50% quantity of forage sorghum intercropped heading stage (GS3) and post cutting) for with forage legumes, was significantly local sorghum cultivars: including JS-2002, higher as there was better mixed green and Chakwal sorghum. Sorghum JS-2002 forage yield obtained when forage sorghum was considered safe for forage purpose due was grown in 30 cm apart rows and cluster to high crude protein percentage, lowest bean was in between the rows [24]. HCN content and, higher crude fiber at pre- Another study was done on response of booting stage. Moreover, though the content sorghum varieties JS-263 (old cultivar), JS- of HCN was found high in young and early 2002 and Chakwal Sorghum (recent growth stages, yet it decreased and crude cultivars) on different levels of soil moisture fiber increased in mature and advanced using physiological and morphological growth stages. Furthermore JS-2002 traits. At high soil moisture JS-2002 showed cultivar, at GS2 stage, presented lowest higher potential than Chakwal Sorghum content of HCN after 18 hrs of post cutting while JS-263—old genotype showed [21]. Individual and combined allelopathic insufficiency to face drought. Therefore, JS- effects of root/shoot powder and water 2002 and Chakwal Sorghum holds extract of sunflower and sorghum applied on promising future in areas subjected to dry soil were evaluated for seed emergence and spells [25]. Hayyat et al. [26] demonstrated

1067 Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131 the effects of viable textile effluent tolerance at seedling stage. Among all traits concentrations, on physiology of Sorghum studied in the young seedlings; shoot related vulgare Pers CV-5000. They concluded, traits were found highly sensitive to water that the plant growth was adversely affected stress. Thus accession No.1749 was by maximum concentration of textile considered drought tolerant, while F-2007 effluents which was found injurious in and F-2008 were found drought susceptible comparison to control [26]. Six sorghum [30]. Kausar et al. [31] explored variation in parent varieties (V-1, SV-6, CVS-13, SPV- physiological parameters due to salinity 462, RARI-S-10, TSS-9) and nine crosses stress related to nitrogen metabolism of were evaluated for fodder yield by heterosis sorghum (Table 1). The sorghum genotypes and combining ability, revealing significant having efficient N-metabolism were difference for all the traits under assessment. considered tolerant due to high biomass This study [27] indicated that a significant production under saline stress. Salinity improvement in fodder yield of crosses is influence was less in sorghum lines JS- 2002 possible due to better and robust and Sandalbar and thus were categorized as performance of these genotypes than parent tolerant, while Noor was found to be heterosis along with good general combiner medium sensitive and FJ-115 was labeled as parents (V1, CVS-13). One of such sorghum sensitive one [31]. line TSS-9 was selected for number of tillers Recently, a new pathogen Curvularia lunata per plant, while V-1 presented highest GCA (Wakk.) was identified from the leaves of for plant height, and CVS-13 was noted for sorghum in Punjab Province, Pakistan. Early stem thickness, fresh weight per plant, and symptom indictae small reddish brown dry weight per plant [27]. In a study eight lesion that further increases in size and sorghum varieties (JS-2002, JS-263, MR- merges to form oblong lesion with chlorotic Sorghum-2011, Hegari, Pak-China-1, Sandal centers and affects the entire field. This Bar, F-7017 and F-114) compared for yield pathogenic strain showed 99% of 584-bp and other quality related attributes, showed sequence resembles with C. lunata strain significant difference in dry matter yield, pingxiang a causative agent of leaf spots of morphological traits, forage yield, and lotus particularly in China. Curvularia quality parameters (Table 1). Pak-China 1 species are known for causing leaf spot had difference in maximum number of traits, disease in various grass species, yet while F-114 differed in minimum traits. F- identification C. lunata on S. bicolor in 7017 and Sandal bar showed maximum Pakistan is among primitive reports [32]. crude protein and crude fiber compared to Kandhro et al. [33] demonstrated other varieties [28]. Raza and Naheed [29] economical, effective and eco-friendly weed determined correlation of high yield and its management strategy by intercropping related biometric characteristics among sorghum dwarf variety MR-Buster, sorghum hybrids improving yield. Thier sunflower hybrid Hysun-39 and cotton seeds results concluded that biometric variety Sindh-1. Their results showed that characteristics: such as yield and plant intercropping of these three seeds, i.e cotton, population; number of green leaves and sorghum and sunflower can significantly plant population; plant height and yield were enhance yield and reduce 59.6% of weeds. positively correlated [29]. Moreover, twice inter-culturing can decrease In another study sorghum accessions (10) 67.5 % of weeds whereas application of were evaluated for drought tolerance and Dual Gold 960 EC controlled 53.4 % of showed great variability for water stress weed, while significant strengthening the

1068 Hafsa Ali et al. cottonseed yield. The analysis of variance traits for their resistance toward drought showed that intercropping of sorghum or stress, together with impact of drought on sunflower with cotton suppressed weeds fodder yield and quality. Three different significantly by 59.6 %, respectively and levels of water stresses (100%, 75%, and significantly enhance growth, while inter- 50%) provided to sorghum genotypes culturing twice reduced further weeds and revealed variable results. Sorghum-11 and gave significantly higher cottonseed yield. NARC-11 were reported to be drought Among all the strategies, total crop tolerant, while F-114 was found drought productivity was highest through sensitive [36]. Sorghum genotypes differed intercropping of sunflower and sorghum from each other on the basis of with cotton as compared to Dual Gold 960 morphological traits, through a study usage in term of cottonseed yield [33]. conducted by Ghani et al. [37] that evaluated A study conducted by Mehmood et al. [34] grain and other quality associated traits in evaluated impact of mulching (no mulch sorghum. Their study reported following: application, poultry manure mulch, and YSS-17, YSS-10 (Cream), and YSS-9 were wheat straw mulch) and tillage application high grain producing, while YSS-10 (zero tillage, reduced tillage and (Cream) and YSS-9 also showed high fodder conventional tillage) on soil fertility and yield. YSS-98 (control) was reported for sorghum grain productivity. Poultry 50% flowering in minimum 79 days, mulching enhanced soil fertility and whereas, YSS-10 (Cream) took maximum sorghum grain yield significantly higher 86 days to 50% flowering. Among the tested than other mulching practices, while tillage genotypes, the study showed YSS-9 as dual- affected all soil properties except organic purpose variety with grain having no tannin matter, and soil pH, whereas conventional contents and high grain and fodder yields tillage was found superior for grain yield. [37]. Twenty four genotypes of sorghum Their study concluded that limited poultry including SS 97-2 (S1), PARC-SS-1, No. manure mulching and tillage can improve 1500, No. 1692, T-3-DADU, 1572-T, S-98- soil properties and sorghum grain yield [34]. 6, SS-95-4, No. 1761, No. 1542, No. 1620, A field experiment [35] determined growth No. 1728, SP-1832, SS-97-10, SS 98-3, and efficient uptake of nitrogen (N), No.1828 (2001), F-9806, R-19, No. 1623, potassium (K) and phosphorus (P) in BR-123, PARC-SV-2, SS-95-RG, SS-95- sorghum from different organic components RG and BMR-RED UNI) were tested for [Humic acid (HA), Farmyard manure morphological traits and yield to determine (FYM), and Press mud (PM) and its heritability, genetic variability, and genetic compost from rock phosphate] in sorghum. advance. The results revealed high The grain and dry mass yield, as well as variability for all the studied characteristics, plant height and uptake of nutrients whereas phenotypic co-efficient of variation significantly increased due to organic was slightly higher than genotypic co- composts in saline effected soil. Therefore, efficient of variation. This study indicated different types of organic compounds have that expression of physiological and potential to improve growth by increasing morphological traits were highly heritable in nutrient uptake in saline effected soils [35]. sorghum population and can be exploited to Crop breeding is an excellent strategy to improve crop productivity in breeding increase plant yield and producing stress program [38]. Another study on fodder yield resistant varieties. Ten sorghum genotypes potential of nine sorghum varieties through were tested through physio-morphological evaluating morphological characteristics

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showed, significant differences in fodder suggested that application of sorghum water yield, number of leaves and leaf area, yet extract in combination with Dual Gold plant height was found insignificant trait. reduced 66.6% weeds with improved yield Number of leaves was highly variable of cottonseed allowing maximum income among all varieties, yet variety-Local benefits, followed by sunflower combined Tandojam gave highest yield, whereas with Dual Gold that lowered 65.5% weeds, variety Giza-3 had the lowest yield [39]. while lastly sole application of Dual Gold A study was conducted on phyto-extraction reduced weeds by 55.9% with low yield of of heavy metals: like lead, chromium, and cottonseed. Nevertheless, inter-culturing cadmium (along with EDTA as chelating twice exponentially suppressed 67.7% of agent) having different concentrations at weeds, yet had low income benefits as various physiological levels in sorghum. The compared to sorghum and sunflower water study revealed that the heavy metal extracts in combination with Dual Gold. accumulation of cadmium, chromium, lead, Therefore, sorghum water extract with Dual and EDTA adversely affected shoot length, Gold showed maximum benefits in terms of fresh weight and dry weight of S. bicolor. net income, yield and low weed [41]. The metal uptake and accumulation also Another study conducted on allelopathic increased by increasing metal concentration, effect of sunflower and sorghum powder via whereas 5mM EDTA enhanced metal soil incorporation and water extracts, uptake [40]. Sorghum and sunflower showed substantial reduction of germination contains several allelochemicals inhibitory and morphological traits of cotton seedlings. metabolites now used as natural and cost Allelopathic effects of sorghum powder on effective tool for weed suppression. A study growth and morphological traits of cotton tested impact of inter-culturing:, Dual Gold seedlings were found superior compared to 960, sorghum and sunflower water extracts water extracts. Therefore, utilizing the different concentrations and intervals, as allelopathic potential of sorghum and well as sorghum and sunflower water sunflower can be effective strategy for weed extracts in combination with Dual Gold 960, management [42]. for controlling weeds (Table 1). The results

Table 1. Sorghum based research in Pakistan S. No. Sorghum variety Purpose Result Refs. 1. Two hybrids of Sorghum RAPD analysis to 8 (40%) primers detected [5] find DNA polymorphism between the polymorphisms hybrids, 33% were polymorphic 2. 10 Sorghum varieties: DS-97-1, 84- Molecular (RARI-S3 and RARI-S-4 are [6] Y-00, RARI-S-3, RARI-S-4, Mr. characterization closest; YSS-9 and 84G01 Buster, 86-G-87, 84-Y-01, 85-G-83, show distant relationship) PARC-SS-1, YSS-9 3. 29 Sorghum: Hegari Noor, JS-2002 Genetic divergence Highest similarity between [7] F-507 F-603 F-505 F-215 FJ-9601 F- among genotypes, Indian III and K-A-113 (both 9809 F-502 F-9917 F-2012 F-9606 using RAPD exotic lines), while the F-601 F-2022 F-214 F-2020 F-2018 F-9706 markers and F-606 were most diverse F-9603 F-604 F-601 F-508 F-606 genotypes Indian III, Australian-7 K-A-113 Australian-6 China small A82L-36

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4. Sorghum Seed of 80 Sorghum Drought tolerance Five accessions (80265, [8] accessions through 80114, SS-95-4, SS-97-7 & physiological 80377) were tolerant markers 5. Two Sorghum lines, viz., PARI-S-4 Role of exogenous Application of 25 ppm SA was [9] and YSS-9 salicylic acid in effective in salt tolerance salinity tolerance 6. Two cultivars of Sorghum Salt tolerance by Proline alleviated the adverse [10] exogenous effects of salt stress application of proline 7. Rari-S-4, SPV-462, Evaluation of SPV-462, CSP-15 and Johar [13] CSV-15, RS-29, PARC-SV-10, YSS- physiological produced higher grain yield, 9, PARC-SS-2, Johar parameters YSS-9 and SPV-462 produced higher stalk yield, YSS-9 and CSV-15 took maximum days to 50% flowering while variety PARC-SS-2 took minimum days to 50% flowering. PARC-SS-2 and Johar showed earliness to mature, while YSS-9 matured late 8. Seeds of 20 Sorghum (RASILI, VI-1, Drought tolerance DS-97-1, SPV- 462, YSS- [11] SPV-462, CSV-13, DS-97-1, PARC- (using 10 RAPD 10(R), YSS-19, JV-2002, SS-1, RARI-S-3, RARI-S-4, YSS-9, primers PARC-SS-2 and CSV-13 were RS-29, PARC-SS-2, YSS-98, YSS- found genetically diverse 10 (Red), YSS-10 (Cream), YSS-17, (genotypes are an important YSS-18, YSS-19, SV-10, JV-2002, source for drought tolerance) CSV-15) 9. Chakwal Sorghum (var Moench) effect of harvest Quality traits are improved [14] time associated when plants were harvested at with P and S a more advanced maturity fertilization on yield and quality of forage 10. Seventeen Sorghum landraces Exploited to Morpho-physiological markers [12] establish morpho- can be exploited for drought physiological tolerance in breeding criteria for drought programme tolerance and higher grain yield in sorghum at seedling and post flowering stages 11. Maize hybrid (Pioneer 3062) and Effect of split In sorghum and maize [15] Sorghum hybrid (Pioneer MR- doses of potash maximum grain yield was Buster) fertilizer on maize recorded with the application and Sorghum of 120 kgha-1 of Potash with three splits. 12. JS-2002, JS-263, Hegari-Sorghum, Salinity tolerance JS-2002 and Sandalbar [16] PSV- 4, Sandalbar, Noor, FJ-115 (tolerant), Hegari- sorghum

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and JS-263 (medium tolerant), Noor (medium sensitive), FJ- 115 and PSV-4 (sensitive) 13. Twenty accessions of Sorghum ( var Drought tolerance promising drought tolerant [19] Moench) 80353, 80365, 80199, accessions (80353, 80365, 80204, 80319, SS-97-7, 80174, SS- 80199, 80204 and 80319) 95-4, 80077, 80265, 80369, 80374, 80381, 80236, 80203, 80376, 80158, 80364, 80114, 80214 14. Three Sorghum genotypes: Hegari, Effect of Seed seed priming accelerates [18] JS-263 and JS-2002 priming techniques emergence of sorghum on seed emergence and seedling growth 15. Seven lines of Sorghum: Hegari, Performance of Hegari was preferred for [20] Noor, F-214, JS-2002, F-207, PC-1 forage material forage purpose and F- 9917 and nutritional profile of dry matter 16. JS-2002, Chakwal Identify the JS-2002 has the lowest HCN [21] Sorghum and local cultivar suitable cultivars content having low HCN content Stress disrupts normal growth and contribute toward increased HCN toxicity. Common cause of HCN in sorghums is drought 17. Sorghum (Sorghum bicolor L.) Effect of NiCl Morphological parameters [17] (salt) effected at high concentration 18. Sorghum and Sunflower Allelopathic sorghum with sunflower water [22] effects of water extracts showed more extracts of inhibitory effects on sorghum and germination sunflower alone and in combination on the germination and seedling growth of rice and weeds 19. Sorghum-2011: a cross Sugrorib Develop dual Sorghum-2011 superior than [23] (local) × Australian No.7 (exotic), purpose sorghum other cultivars Sorghum-2011, JS-2002, JS-263, cultivar Hegari 20. Sorghum bicolor L. Agro-qualitative Sorghum intercropped with [24] response of forage cluster bean produced sorghum (Sorghum significantly higher mixed bicolor L.) sown green forage yield alone and in mixture with

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forage legumes 21. JS-2002, Chakwal Sorghum and JS- Growth response (JS-2002 and Chakwal [25] 263 in drought Sorghum) 22. Sorghum vulgare Pers CV-5000 Effects of textile Textile effluents are injurious [26] effluent at high concentrations. 23. Nine Sorghum crosses and their six Estimation of Heterosis in fodder yield is [27] parents (V-1, SV-6, CVS-13, heterosis for feasible SPV-462, RARI-S-10, TSS-9) fodder yield Better combiner parents (V1, CVS-13) 24. Ten different accessions of Sorghum: Drought tolerance Promising drought tolerant [30] F.C-26-I, NO.1749, F.S.9902, F- at seedling stage accession (NO.1749) and 2007, AK-113, NOOR, F-2-2007, F- drought susceptible (F-2007 2008, M.R-SORG- 2011, F-114. and F-2008) 25. Eight forage Sorghum cultivars: JS- Comparative Maximum physiological value [28] 2002, JS-263, MR- Sorghum-2011, performance of recorded in Pak-China-1 Hegari, Pak-China-1, Sandal Bar, F- different sorghum followed by F-114, and 7017 and F-114 forage cultivars minimum in Sandal-Bar regarding yield Crude protein and crude fiber and quality were more in Sandal bar and attributes F-7017 26. Two tolerant (JS-2002 and Salinity induced Salinity influenced Noor as [31] Sandalbar) and two sensitive (Noor changes in medium sensitive and FJ-115 and FJ- 115) Sorghum genotypes nitrogen sensitive. Sorghum lines JS- metabolism of 2002 and Sandalbar were sorghum tolerant, Noor medium sensitive and FJ-115 as sensitive 27. Seeds of cotton: variety Sindh-1, Weed management Intercropping of both sorghum [33] Sorghum dwarf variety: MR-Buster, through and sunflower in cotton was Sunflower hybrid: Hysun-39 intercropping effective in weed management 28. Sorghum hybrid Find correlation Yield and plant population; [29] (46 genotypes) among yield of number of green leaves and sorghum hybrid plant population; plant height and its iometrical and yield were correlated characters 29. Leaves of Sorghum Leaf spot disease Pathogen identified [32] as Curvularia lunata 30. Sorghum variety JS-2002 Effect of tillage Reduced tillage operations and [34] and mulching poultry manure mulching is practices on grain suitable for improving soil yield and soil properties and grain yield of fertility sorghum 31. Sorghum Influence if use of different organic [35] organic materials materials improves sorghum on growth and growth phosphorus uptake 32. Eight Sorghum genotypes: YSS-9, Grain yield and YSS-9 proved as a dual- [37] YSS-10, YSS-17, YSS-10, YSS-98, related traits purpose variety with YSS-18, YSS-19, reasonable grain and fodder YSS-32 yields 33. Ten genotypes of Sorghum: FS-08 Drought tolerance NARC-11 and Sorgh-11 were [36] FSD-11 NOOR F-114 F-113 drought tolerant NARC-11 AARI-10 AARI-08 FA-08 F-114 was drought sensitive

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Sorgh-11 34. SS 97-2 (S1), PARC-SS-1, No. 1500, Yield and No. 1623 showed higher stalk [38] No. 1692, T-3-DADU, 1572-T, S-98- morphological yield while SS 97-2 (S1) give 6, SS-95-4, No. 1761, No. 1542, No. traits highest grain yield 1620, No. 1728, SP-1832, SS-97-10, SS 98-3, No.1828 (2001), F-9806, R- 19, No. 1623, BR-123, PARC-SV-2, SS-95-RG, SS-95-RG and BMR- RED UNI 35. Nine sorghum varieties: Local Green fodder yield Local Tandojam produced [39] Tandojam, F-9902, F-9917, No. potential highest green fodder yield 1863, JS-263, F-9909, Js-2002, Local while Quetta, Local D. I. Khan, Giza-3 Giza-3 produced lowest yield

36. Sorghum Phyto-remediation Application of 5mM EDTA [40] of Lead, enhanced the uptake of heavy Chromium and metal Cadmium 37. Sorghum or sunflower water extracts Weed management Dual Gold (herbicide) in [41] combination with sorghum or sunflower water extracts is effective weedicide 38. Sorghum and sunflower Phytotoxic Sorghum showed superiority [42] potential on cotton over sunflower in allelopathic seedlings efficiency

Research on biofuel production from Mg ha-1, whereas Hawaiian island Sorghum worldwide contributed highest yield of sugar with 12 Sweet sorghum is an emerging, potential Mg ha-1. Furthermore, per these sugar candidate to serve for biofuel production due yields theoretical ethanol production was to its vast adaptability, easy cultivation, and ranged between 2129 L ha-1 to 5696 L ha-1. high yield potential. Intensive exploitation Sweet sorghum are high adaptable as of its diverse germplasm in various breeding compared to other tropical plants that makes programs has led to improved syrup, grain them superior and exceptional source of and forage yield. Monk et al. [43] performed fermentable carbohydrates in diverse regions study on increasing yield of sorghum hybrid [44]. Smith and Buxton [45] determined in USA. The study demonstrated that both impact of various factors such as: nitrogen stalk and grain of this crops can contribute fertilizers, as well as irrigated and non- in energy sector such as 60 Mg ha−1 of irrigated conditions of USA on sugar yield fresh biomass can produce 5000 liters ha−1 of sweet sorghum. Addition of Nitrogen of ethanol [43]. Another study explored fertilizers had minute effect on improving potential of sorghum as a renewable energy fermentable sugar production. Their finding resource, as six sorghum cultivars were showed average of 3100 to 5235 liters ha−1 assessed for fermentable sugar production of ethanol yield for 2 year, suggesting by determining agronomic traits, potential use of sweet sorghum as an energy physiological characteristics and yields in crop [45]. nine different locations of USA [44]. Total Biofuels hold promising future in energy sugar yield was variable for different and transport sector to supply electricity and locations, ranging from 4 Mg ha-1 to 10.7 liquid fuels without any modifications in

1074 Hafsa Ali et al. current infrastructure. Woods [46] studied accumulation in stem, grain and leave that the potential of sweet sorghum under contribute in maximizing energy yields [48]. available condition and resources of They further identified QTLs responsible for Zimbabwe. The finding suggested 60 tons of high yield, with altered composition of stem total fresh weight of sorghum yielding 45 sugar and grain with no pleiotropic effects. tons of fresh stems contributed in 3000l/h of For example, a QTL on chromosome 3 was anhydrous ethanol and 12.6 GJ of electricity. responsible for 25% genetics variance for Furthermore, he suggested that sweet stem sugar concentration while remained sorghum can contribute in region’s disassociated with any grain QTL. This electricity and liquid fuel by utilizing only finding suggested that non-structural yield 1% of the land for growth and processing of can be improvised by altering genetic crop [46]. Monti and Venturi [47] evaluated potential of grain and stalk by opting for performance of four monocultures of QTLs responsible for sweet and high grain sorghum and wheat cultivars at variable traits. This strategy can develop feedstock doses of nitrogen fertilizers to determine net with qualitative traits and high yield to energy, and energy use efficiency. Sweet counter energy and food crisis [49]. sorghum could contribute maximum 50% of Zhao et al. [50] evaluated carbohydrates, net energy than wheat and fiber sorghum biomass and calculated ethanol yield (CEY) when grown in climatic conditions of Italy of five sorghum cultivars in Beijing, China (Figure 1). These finding suggested that from the day of anthesis to 40 days after ethanol production can be maximized to anthesis (DAA). Although, all cultivars 90% if dry matter and bagasse of crop is differed significantly in dry biomass, and also used; otherwise contributed net energy carbohydrate content; yet, hybrid as ratio was low. Moreover, highest ethanol feedstock for bioethanol conversion was production is obtained from straw, and comparatively higher performer than other resulting in low percentage of ethanol if cultivars. The environmental conditions and straw is not processed [47]. However, harvest time also influenced carbohydrate structural carbohydrates makes up content and biomass production that lignocellulosic biomass on digestion and ultimately affects the ethanol yield. thus fermentation has high potential to However, the CEY increased with increase produce energy per hector as compared to in crop cultivation duration and after nonstructural carbohydrates that include anthesis period due to elevated accumulation sugar and starch. Murray et al. [48] of carbohydrates [50]. Miller and Ottman demonstrated that energy yields can be [51] determined the effect of frequent maximized in sweet sorghum if grain and irrigation on sorghum growth and ethanol biomass related traits are targeted for yield. They depleted plant available soil improvement. They evaluated genetic basis water during pre-anthesis and post-anthesis of 31 traits involved in biomass yield in F1 stage of sorghum by 65% in Tucson, AZ, generation of grain sorghum ‘BTx623’ and USA (Figure 1). The result suggested that ‘Rio’ sorghum line, and identified 110 frequent irrigation showed negligible impact quantitative trait loci. Many structural and on biomass and ethanol yield, whereas, non-structural carbohydrate related QTLs water stress with 50% depletion reduced were found co-localized with flowering biomass per unit of water with no increase in time, height, and stand density–tillering. sugar concentration or accumulation at They identified separate genetic control for harvest of sweet sorghum [51]. structural carbohydrates and for protein

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Wortmann and workfellows [52] evaluated performance of F1 generation comprised of sweet sorghum, corn, and grain sorghum for high brix and sucrose content (due to Parent energy use efficacy, along with greenhouse 2 having high sugar percentage). In F1 gases (GHG) emissions and ethanol yield at generation: stalk, juice yield and plant seven different locations in Nebraska, USA. height were marked as over dominant traits. Grain crops showed 21% to 33% more In F2 generation sucrose percentage and calculated ethanol yield and net energy yield Brix were found polygenic traits, although, than sweet sorghum, although mean net juice and stalk were marked as oligogenic. energy yield of an earlier-maturing sweet These dominant and over-dominant traits sorghum cultivar was equivalent to grain were utilized in hybrid breeding programs to crops. The total energy utilized to convert enhance production of bioethanol [54]. grain crops to ethanol was 23% lower than Sweet sorghum hybrids vary with respect to that utilized on sweet sorghum. However, biomass yield and variable level of nitrogen sweet sorghum and grain crops reduced that ultimately affect quality of extracted GHG emissions by 69% than gasoline. The juice. Mosalia et al. [55] determined the byproducts of grain crops during ethanol impact of varying levels of nitrogen production were utilized efficiently, while fertilizers on biomass, juice yield, and sweet sorghum bagasse were returned to forage and bagasse quality in USA. field as soil fertilizers. Sweet sorghum Sorghum varieties harvested and evaluated cultivars are competitive candidates for at soft dough stage for required traits, producing biofuel, but not as liquid showed increase in forage and juice yield transportation fuel as grain crops performed due to fertilizers [55]. Guigou, et al. [56] better [52]. Economou et al. [53] studied effects of post-harvest conditions on demonstrated conversion of sweet sorghum three sweet sorghum varieties (M81, Topper into biodiesel via oleaginous fungus M. and Theis) used for bioethanol production. isabellina in Greece. The fungus effectively Juices were obtained by milling whole plant, converted sugar and nitrogen in sweet stalk and plant without panicle. Linear sorghum directly into storage lipid. The relationship was found between Brix degree process was performed on semi-solid and fermentable sugar concentration. fermentation that had certain advantages like Fermentable juice of sweet sorghum differed high quality oil—compared to liquid and with respect to extraction treatments, yet solid state fermentation. Their findings showed similarity in fermentation efficiency concluded that lipid accumulation is and sugar concentrations. Theis and Topper possible due to nitrogen limitation allowing varieties were superior in terms of ethanol biomass growth and oil synthesis, whereas yield, concentration and fermentation semi-solid state fermentation effectively efficiency. Furthermore, post-harvest extracted sugar from stem of sweet sorghum techniques and selection of desirable variety and lead to lipid accumulation [53]. played critical role in bioethanol production Audilakshmi et al. [54] facilitated breeding from sorghum [56]. of sweet sorghum for bioethanol purpose in Sweet sorghum is widely cultivated as relation to presence of high sugar content. summer crop due to high resistance to They crossed 27 B × BJ 204 (a Chinese line) salinity, while being productive through and 27 B × kellar (a US sweet sorghum line) drought and during limited water resources. to find generation mean and frequency Vasilakoglou et al. [57] conducted a distribution of useful traits during 2006 and research in Greece to access the productivity 2007 in India. They reported that mean of grass sorghum Susu as well as, sweet

1076 Hafsa Ali et al. sorghum varieties, Sugar graze, M-81E, Urja in USA. The abiotic stress had negligible and Topper-76-6, alpng with grain sorghum impact on glucose levels from grains as cultivar KN-30 in terms of biomass, total compared to control, yet an increase in fermentable sugar, juice and ethanol yield. ethanol production, up to 4.5%, was The cultivars were grown in high saline observed in flowering and seed-setting conditions with low to intermediate water stage. However, the heat stress significantly for irrigation. Sugar graze and Urja cultivars reduced glucose content and bioethanol were high ethanol producing varieties with production by 9% in different seed filling high juice content, for ethanol production stages. Thus, abiotic stresses adversely when grown in saline soil and 50-75% affected phenological stages of sorghum; irrigation [57]. Xu et al. [58] demonstrated ultimately reducing ethanol yield [60]. photosensitive sorghum (PS) grown in fields Sorghum is widely adapted around the world of USA, has great potential for bioethanol and serves as the most attractive feedstocks production due to high yield, drought for liquid fuels, due to efficient conversion tolerance, better sugar content and high of CO2 into sugar units. A study conducted biomass. Variable concentrations (0.5 to in USA evaluated use of sweet sorghum in 1.5%) of diluted sulfuric acid were used as biofuel production [61]. Their research pretreatment to hydrolyze cellulose for reported that sweet sorghum comprised of ethanol yield. and highest glucose yield of 37% juice, 36% bagasse, 19% leaf and 8% 80.3% was obtained with 1.0% of acid. The seed head, all of which can be utilized in efficiency of hydrolysis increased up to producing bioethanol via Saccharomyces 94.4% as the acid concentration rose up to cerevisiae. Around 229.8 Kg of 1.5% due to high degradation of available carbohydrates from 1ton of sorghum can cellulose. Their findings reported 74.5% of produce 59 kg of ethanol [61]. Qureshi et al. ethanol yield due to above mentioned [62] evaluated production of ethanol from processing conditions, where 1g of sorghum sweet sorghum grains and thereby sugar produced 0.2g of ethanol [58]. Similar study tolerance in Saccharomyces cerevisiae in conducted by Zhang et al. [59] produced Pakistan. Sorghum varieties were butanol from sweet sorghum bagasse via simultaneous saccharificated and fermented acetic acid treatment to hydrolyze to produce ethanol from starch grains. The hemicellulose-rich biomass using findings revealed that S. cerevisiae can Clostridium acetobutylicum ABE080—an tolerate 25% glucose and 11% ethanol. acetic acid resistant strain. Hemicellulose Separate hydrolysis and fermentation hydrolyzed by 2g/l of acetic acid at 121oC showed 95% fermentation efficiency in for 10 minutes produced various compounds ethanol production, whereas, simultaneous such as: carbohydrates, furfural, organic saccharification and fermentation showed acid, hydroxymethyl furfural (HMF) and high concentration of ethanol [62]. phenolics. Detoxification process with Erickson et al. [63] focused on optimizing laccase eliminated phenolic compounds that production of sweet sorghum using various were generated as by-products and served as management practices for bioenergy primary inhibitors in hydrolysate step purpose. The effect of N-fertilizers’ rate on resulted in 8.5 g/l of butanol [59]. Ananda et biomass, sugar yield, brix as well as N and P al. [60] investigated the effect of drought recovery were investigated for sweet and heat stress on glucose of sorghum grain sorghum in Florida, USA. N-fertilizers had at different phenological stages and also its no impact on biomass of sorghum, yet led to subsequent impact on bioethanol production greater N-recovery and minor gain in sugar

1077 Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131 yield. The results suggested that proper bioethanol from Keller and M81E was management of nutrients can contribute in greater than theoretically estimated ethanol optimizing sugar yield in sweet sorghum for and sugar yields. The findings reported low bio-based products [63]. Another study grain, but high starch content in Keller than conducted by Holou and Stevens [64] other cultivars being potential source for determined optimum rate of N-fertilizers in fermentable grain and sugar [66]. Martin et different soil materials (on silt loam, sandy al. [67] demonstrated effective, high loam, and clay soils) to produce high yield throughput method to screen feedstock sweet sorghum at Missouri, USA. N-rate toward biomass, cell wall digestibility, and and soil material significantly affected juice fermentable sugar at 10 days post-anthesis yield by increasing sugar content for biofuel production in Australia. They (particularly due to N-fertilizers). However, designed a mathematical method along with negative correlation existed between juice high throughput strategy using fourth yield and sugar content of sweet sorghum. internode of plant to assess soluble sugar, Sorghum displayed high sugar content when stalk biomass, stalk fermentable sugar, and grown in silt loam compared to that in clay. cell wall composition. The high throughput Addition of N-fertilizers in clay soil method using partial least squares (PLS) increased sugar yield of sweet sorghum, modeling measured soluble glucose, while in silt loam it displayed high sugar sucrose, and fructose in juice via Fourier content only if cultured after corn. transform infrared (FTIR) spectra. The Significant difference existed in juice yield findings showed sugar PLS correlated with ranging 2-9.9 Mg/ha due to N-rates, soil and brix and gas chromatography–mass other factors. Whereas, sorghum required spectrometry (GC-MS) [67]. low quantity of N-fertilizers if planted after Forage sorghum, Brown Mid-Rib (SBMR) cotton or soybean. In general, 67 kg of N- and non-BMR (SNBMR) types, and sunn fertilizers were enough to optimize sugar, hemp (Crotalaria juncea L.) are widely used juice and bagasse yield [64]. as fiber and forage crop in USA. Kamireddy Sambusiti et al. [65] demonstrated et al. [68] evaluated these crops for biofuel production of methane from five sorghum purpose. The feedstock were treated with varieties via alkaline pretreatment in France. dilute acid and further by enzymatic The samples were pretreated with NaOH in hydrolysis cellulase. SNBMR showed closed bottles for 12h at 55oC. The highest xylose yield with 95% wt of xylose, pretreatment reduced sorghum into lignin, than that of SBMR with 91 at combined cellulose, hemicellulose and galacturonic severity factor (CSF). while sunn hemp acids; thus further accelerating hydrolysis of showed maximum of 75% wt of xylose at cellulose and hemicelluloses. However, CSF. On harsh pretreatment conditions, the NaOH pretreatment was unsuccessful for amount of xylose decreases due to increasing methane yields [65]. Rutto et al. degradation. Similarly, after enzymatic [66] conducted a research on five sorghum hydrolysis glucan saccharification yield was varieties (Dale, M81E, Sugar Drip, Della highest in SNBMR with 90% wt, while and Keller) to evaluate quality of grain and SBMR showed 84% wt, and Sunn hemp juice as potential source of bioethanol in showed 68% wt at CSF. They concluded USA (see Figure no.1). Thus, stem weight that SBMR and SNBMR can serve as and juice volumes were correlated ranging potential candidate for biofuel production from 10 to 24 m3∙ha−1, though brix value due to less crystallinity than Sunn hemp ranged between 14-19%. Yet, production of [68].

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Recently, biofuel research has been Brazil. The cultivars were pretreated, then accelerated in Pakistan due to scarcity of fermented by S. cerevisiae, at 33oC for 6 resources (Figure 1). Lignocellulosic hours. Sorghum cultivars SF 15 showed biomass is pretreated to convert complex it highest bioethanol potential, followed by BR into simpler substances for hydrolysis and 506 [71]. fermentation. Sorghum bicolor straw Theuretzbacher et al. [72] evaluated three potential lignocellulosic feedstock was sorghum varieties for bioethanol and biogas evaluated for various pretreatment synthesis and compared the outcomes with a conditions (Acid concentration, Time and maize variety. One grain variety “C” Temperature) in Pakistan. Samples were (Chopper), and two sweet sorghum varieties analyzed using HPLC, where maximum “SG1” (Sugargraze I) and “SG2” sugar value was observed with 2% H2SO4 (Sugargraze II) were grown in eastern at 121oC for 10 minutes, suggesting that Austria. The varieties were analyzed for optimized pretreatment conditions were sugar content and composition to determine effective prerequisite for production of sorghum potential to produce biofuel. SG1 bioethanol [69]. In India, Rao et al. [70] and C varieties showed highest and compared sorghum hybrids with open improved energy output than maize. pollinated varieties (OPVs) for various Furthermore, biogas and bioethanol quality related traits to produce potential production was variable in among sorghum bioethanol and high yielding grains. Sixteen varieties due to difference in sugar and genotypes were evaluated for sugar and stalk starch. Variety C provided 54% bioethanol related traits. Stalk yield from sorghum and 46% methane, while SG1 contributed in hybrids and OPV ranged between 29.4 to 30% bioethanol and 70% methane [72]. 46.5 t ha-1, while grain yield was recorded Lignocellulosic biomass are converted into higher for hybrids than OPVs. SPSSV30 bioenergy based products via various showed highest total soluble sugar and juice pretreatment and fermentation strategies. A Brix. Among hybrid group genotypes,: study conducted by Vandenbrink et al. [73] SPSSH 24, SPSSH 27, PAC52093, whereas investigated crystallinity index trait, and among OPV genotypes: SPSSV 20, SPSSV feedstock composition of twenty sorghum 27 and SPSSV 15, showed superior varieties grown in USA. They performed bioethanol and total sugar yields. Hybrids enzymatic hydrolysis (using Aspergillus can support energy sector due to10-18% niger cellulases and Trichoderma viride) on high production of stalk biomass and sorghum to obtain high sugar yield. The bioethanol yields compared to OPV [70]. study illustrated positive correlation of Sugarcane has served as renewable hydrolysis yield potential with stem (due to alternative source to solve energy crisis in presence of lignin content) as well as developed countries, though, after paying negative correlation due to crystallinity heavy price of using agricultural land, index. Thus, sorghum leaf showed negative nutrients and water. Recently, the research correlation for hydrolysis due to absence of has been shifted toward use of sorghum lignin. However, each genotype efficacy cultivars as an alternative source of energy was variable with respect to pretreatments due to its high adaptability, resistance and (ammonium hydroxide and T. viride limited use of water making it an excellent cellulose). These pretreatment factors can be candidate. Dutra et al. [71] evaluated optimized to obtained bioethanol in large- bioethanol production from eight sorghum scale. Similarly, butanol synthesis were cultivars developed in state of Pernambuco, positively correlated with stem tissues, while

1079 Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131 suggesting that selection of genotype with constant water stress in Italy. They high ratio of stem to leaves can improve end determine its potential use as first and product yield [73]. second generation biofuel. The study Cotton et al. [74] studied biomass and concluded that duration of growth and cellulosic properties of forage sorghum cultivation period should depend on differed on basis of brown midrib trait (i.e., objective of use. Such as, if the target is bmr12) to produce ethanol under limited bioethanol production, then longer duration irrigation, while comparing to dry land (no for cultivation period was suitable, on the irrigation) conditions of Southern High contrary, flowering stage was considered Plains of the USA (see Figure no.1). The suitable harvest time to address optimal use Sorghum Partners 1990 produced highest of water [75]. Another study was performed biomass under irrigated and dry land in Guatemalan Pacific coastal plains on six conditions, yet, PaceSetter bmr showed varieties of sorghum to evaluate sugar highest cellulosic efficiency on simultaneous potential in stem for bioethanol and saccharification and fermentation to produce nutritional value from grains. Of all varieties bioethanol. The study demonstrated that Top 76-6 variety was found exceptional in irrigation can increase up to 49% of biomass both terms of grain nutrition and high sugar and 72% of cellulose for bioethanol value in stem with capacity to produce 2465 production compared to limited irrigation l ha-1 of ethanol. Grains had 83.2% of (had no effect on biomass and biofuel digestible proteins and low level of production). The findings concluded that polyphenols [76]. Sweet sorghum was low precipitation did not affect the cultivars, investigated as an alternate feedstock for while high precipitation increased cellulose bioethanol by Embrapa (Brazilian by 28% in sorghum Partners 1990 that Agricultural Research Corporation of Maize further amplified bioethanol production. The and Sorghum). Fernandes et al. [77] used results showed that precipitation and juices of 4 four sorghum varieties (BRS 506, irrigation were crucial factors that directly BRS 508, BRS 509, BRS 511 and BRS) and impacted biomass and biofuel production evaluated their nutritional contents [74]. Mehmood et al. [34] evaluated four including, sugar, starch and respective varieties of sorghum (84-Y-01, 85-G-86, theoretical ethanol yield. The study Mr. Buster and RARI S-3) to unleash their concluded that three varieties (BRS 508, use as ideal feedstock for bioethanol BRS 509 and BRS 511) were potential production in Pakistan. The straw of candidates for bioethanol as their maturity sorghum varieties were pretreated with exceeds 30 days. The nutritional value in diluted sulfuric acid and enzymatically sorghum varieties decreases with increase in hydrolyzed to ensure complete sugar cultivation duration. BRS 511 showed extraction and conversion to bioethanol. highest sugar production, whereas BRS 508 Their study assigned following sequence to yielded 90.5% theoretical bioethanol within sorghum varieties with respect to sugar 8 hours of fermentation, which is similar to yield: 85-G-86>Mr. Buster> 84-Y-01 > bioethanol produced from sugarcane juice RARI S-3. Nevertheless, variety (85- G-86) [77]. was considered ideal feedstock due to low Cuevas et al. [78] agronomically evaluated by-products, furans and high sugar yield 925 accessions (originally belonged to [34]. Similar study was conducted by Dalla different countries and compared them with Marta et al. [75] to examine sorghum growth accessions of USA) obtained from U.S. at different developmental stages under National Plant Germplasm System (NPGS).

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They evaluated physiological parameters, Sweet sorghum is considered analogous to morphological parameters, fermentable sugarcane due to sugar accumulation, Brix sugar, juice volume, brix values, and disease content, and lignocellulosic residues responses toward rust (Purcina purpurea) (pretreated to obtain sugar for ethanol). A and anthracnose (Colletotricum study was conducted in Kenya on 16 sweet sublineolum). The findings concluded that sorghum bagasse, which could be nine accessions belonging to Ethiopia, South sustainable source of biofuel [80]. Sweet Africa, Sudan, Zimbabwe showed high brix sorghum bagasses were dried and pretreated values than accessions belonging to USA. with alkaline hydrogen peroxide and Similarly, dry mass of accessions belonging phosphoric acid to yield 63.40% and 49.12% to Ethiopia and USA were higher than of glucose. Further hydrolysis and reference number of other countries. New fermentation of bagasse by Trichoderm sources of rust and anthracnose disease reesei via cellulose enzyme produced 88% resistance were identified, while accession (treatment with sodium hydroxide) and 77% from Tanzania was found resistant to both (treated with phosphoric acid) of sugar diseases. The findings showed that NPGS yield. The study suggested that bioethanol sorghum collection consisted of valuable can be produced from sorghum bagasse to germplasm that can be exploited in breeding widen the scope and strength of bioenergy programs to fulfill biofuel purpose paving sector [80]. Similarly, Khalil and his integration of such germplasm in genetic colleagues [81] analyzed productivity and diversity and new varieties for bio-based biofuel related traits from bagasse of five products [78]. Mocoeur et al. [79] crossed sweet sorghum varieties in Egypt. Tested sweet and grain kaoliang sorghum to obtain varieties differed in juice content, bagasse recombinant inbred lines to find genetically and stalk yield. Extracted juice was stable biomass, as well as morphological fermented by Zymomonas mobilis ATCC and biofuel related traits in field trials 29191 and Saccharomyces cerevisiae ATCC conducted in Denmark and China. They 7754 to produce bioethanol. Three sorghum determined 53 QTLs using PAV markers varieties were squeezed directly to obtain (presence and absence variant) from which juice while Ramada, Mn-1054, and SS-301 18 QTLs were common for both countries. rich in fiber had to be pretreated with 2% However, out of 15 traits only eight were H2SO4 (98%) at 120oC to extract juice genetically stable and heritable. Plant height from sugar rich filtrate for bioethanol was positively correlated with biomass, production. The finding showed that mixed- whereas juice and brix content were cultured treatment can help to obtain highest negatively correlated. In Denmark trials juice such as: 1 Kg of SS-301 variety sheading stage was correlated with biomass produced 160mL of bioethanol from juice and morphological traits. QTLs for maturity and bagasse [81]. identified on two chromosome of SBI01 and Biofuel from crops not only reduces our SBI02 were associated with early chilling dependency on depleting fossil fuel but also tolerance; suggesting that accelerating lowers CO2 emission in environment. maturity built the tolerance to low Currently, bioethanols are commercially temperature. The finding also suggested that available and used in certain percentages selection of sorghum on basis of early with gasoline in developed countries like maturity, plant height, and high Brix content USA, and Brazil. Therefore, production of can contribute in bioenergy and biofuel bioethanol depends on agricultural land, alternative for Northern Europe [79]. crop production, resources, as well as

1081 Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131 governmental and social policies. Xuan et al. USA (see Figure no.1). N-fertilizers failed to [82] evaluated optimum time required for show significant impact on brix content; yet, sowing sorghum seeds and determined other parameters were highly influential in suitable cultivation condition required to both varieties, though. highest ethanol yields obtain maximum yield for biofuel was obtained with 168 kg ha−1 of N- production in Vietnam. Suitable cultivation fertilizers. Whereas low temperature and conditions to obtain >5000 L ha−1 yield of high precipitation levels were also important ethanol were 5.5 pH, along with Al and Zn factors to contribute in ethanol yields [83]. content ranging between 39.4 and 0.6 g Mengistu et al. [84] determined water use kg−1. pH higher than 6.0 can reduce efficiency for sweet sorghum and its impact biomass and bioethanol yield through on biofuel production under two different altering Zn content, however, other minerals climatic conditions of South Africa (Hatfield like P, N, K, Organic C, and Fe are less and Ukulinga research farm). Theoretical likely to contribute in biomass. Their finding ethanol production was calculated through recommended that cultivar 4A can highly Brix % produced as well as stalk yield. The contribute in ethanol production due to it findings concluded that sweet sorghum has high resistance toward peat diseases variable water use efficiency (WUE) under especially cut worm (Agrotis spp) [82]. different climatic conditions, such as Maw et al. [83] tested the effect of N- Hatfield produced maximum of 0.70 L-m-3 fertilizers on dry mass, stem juice yield, of ethanol, which was comparatively higher sugar yield, theoretical ethanol juice, than that from Ukulinga research farm. lignocellulosic ethanol yield, and total Therefore, WUE of sweet sorghum was ethanol yield from two sorghum varieties considered sensitive to plant density [84]. (Dale and Top 76-6) grown in Missouri,

Research on Sorghum based Biofuel production worldwide

USA South Africa Italy China Greece India Uruguay Pakistan Austria Brazil Australia France Guatemalan Pacific lowlands Vietnam Kenya Egypt

Figure 1. Biofuel production from sorghum worldwide

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Role of microRNA in biofuel production role in biological, metabolic processes and in different crops plant development. Whereas gene ontology MicroRNA (miRNA) plays important role in showed that miRNA has crucial role in 527 development and stress resistance of plants biological processes on the basis of these via post transcriptional gene regulation. targets. Among these processes, 23 miRNA are small RNAs that are usually 21- processes are involved in metabolic 22 nt long, and produced in series from activities of carbohydrates, lipids and xylem splicing action of RNAse-III like enzyme formation. The findings analyzed 118 Dicer on its precursors with short hairpin- metabolic pathway networks involved in loop structures [85]. miRNA are negative oxidative stress response, hormone regulators of gene expression and act by regulation, carbon fixation, metabolism of splicing or inhibiting the target mRNA for fat and sucrose and other secondary protein translation [86]. Keeping function of metabolites [88]. miRNA in view, many novel miRNAs has Zanca et al. [89] reported 19 sugarcane been identified and further identification is a miRNA precursors responsible for high key to unravel the mechanism of stress bioethanol yield in sugarcane and found tolerance. similar to sorghum at nucleotide and Cultivation of Brachypodium, Miscanthus secondary structure levels (Table 2). Mature and switchgrass are emerging with broad miRNA can be accumulated in specific scope in bioenergy field. Switchgrass is tissues and organs of sugarcane. The center of attention worldwide due to its wide findings determined 46 potential targets for adaptability to grow, and tolerate drought 19 miRNA of sugarcane several targets of spells. However, the basis of genome conversed miRNA were involved in plant responsible for such traits is still unexplored. development as transcription factors. The Recent research shows that microRNA are findings classified 19 miRNA precursors in responsible for gene expression in growth, sugarcane and one miRNA precursor in development and adaptation to wide range sorghum into 14 families. Comparative of circumstances (Table 2). Matts et al. [87] analysis of sugarcane with sorghum showed identified miRNA families in switchgrass common homologous miRNA and their responsible for growth and development targets in genome of these two species. with tissue-specific expression, and Hence, such conservation may help to ubiquitous expression in some species. The clarify specific aspects of miRNA regulation findings showed expression of miR395 and and evolution in the polypoid sugarcane miR399 under optimal level of sulfate and [89]. phosphate, and slightly altered under Foxtail millet (Setaria italica) belongs to phosphate and sulfate deficit conditions. poaccace family and is widely used as food, They suggested 4 target mRNA like NAC fodder and as model crop for biofuel domain containing transcription factor, grasses. Zhang et al. [90] drafted genome apetala 2-like, Squamosa promoter binding- onto nine chromosomes and annotated like factor, and HD-Zip homologs and 37 38,801 genes. The findings demonstrated genes as targets for miRNA [87]. Similar that reshuffling events of foxtail, sorghum study performed by Xie et al. [88] identified and rice contributed in divergence of the 121 miRNA in switchgrass belonging to 44 crops. Two reshuffling events of key families using comparative genomics chromosomes were identified via approach. These miRNA targeted 839 collinearity between 2 and 9 chromosome of proteins coding genes and played important foxtail millet with 3, 7, 9, and 10

1083 Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131 chromosomes of rice that occurred after drought stress was dose-dependent, and divergence of foxtail and rice. Single upregulated the gene expression by 0.9 folds reshuffling event occurred between foxtail and downregulated by 0.7 folds. Though, millet chromosome 3 with rice chromosome miRNA under both stresses showed similar 5 and 12, after divergence of sorghum and range of expression yet, miRNA were more millet [90]. Recent study showed genetic sensitive in drought stress, as miR156 and improvement of switchgrass biomass to miR162 displayed significant expression, develop emerging bioenergy crops. miR156 suggesting role of miRNA to cope up with precursor was overexpressed in switchgrass drought stress (Table 2). Therefore, and its effect on squamosa promoter binding transgenic lines can improve bioenergy protein like (SPL) genes were determined crops for biofuel production [92]. Drought via microarray and RT-PCR. The findings spells are widely known to reduce the yield characterized biomass yield, forage of majority crops. Certain genes are digestibility, saccharification efficiency and expressed to plant cope up with drought morphological alterations. miR156 stress and manage water, yet this mechanism overexpression suppresses SPL gene and is remains unexplored in majority of crops. associated with apical dominance and Ferreira et al. [93] conducted a study to transition in flowering time; while, low explore expression of miRNA in two expression of miR156 was sufficient to cultivars of sugarcane under drought stress. increase biomass with normal flowering Sugarcane cultivar RB855536 known for time, and disruption of apical dominance. low drought tolerance and RB867515 Moderate expression of miR156 improved cultivar with high tolerance were grown for biomass, yet inhibits flowering. Thus, low 3 months and subjected to drought stress for and moderate expressions contribute in 2-8 days. The results revealed identification 58%-101% high biomass yield, though high of 18 miRNA families via deep sequencing, miRNA expression result causes stunted out of which 7 miRNA were differentially growth (Table 2). Consequently, the degree expressed in drought stress. Furthermore, of morphological alterations depends on differential expression of miRNAs depends level miR156 expression. High expression on duration of stress, such as 6 miRNAs enhances solubilized sugar yield, forage were differentially expressed after 2 days digestibility and biomass yield by increasing and 5 miRNA were expressed at 4 days of tiller number [91]. stress [93]. Switchgrass dedicated biofuel crop is Brachypodium distachyon belongs to broadly cultured for its high adaptability for Poaceae family and known to produce marginal lands and high biomass yield. biofuel. Jeong et al. [94] sequenced 17 small However, limited knowledge is available on RNA libraries and identified miRNA basic mechanism of its gene expression populations representing diverse stress under stressful conditions. Study conducted conditions and tissues. The study identified by Sun et al. [92] demonstrated expression 115 miRNAs using computational tools, of regulatory miRNA on physiological which included conserved, non-conserved parameters under salt and drought stress. and novel miRNA. PARE (Parallel Analysis The finding indicated that 1% salt stress of RNA Ends) based identification of adversely effected germination rate and miRNA cleavage function were used to growth, whereas, drought stress showed construct PARE libraries. The PARE slight impact on germination. The miRNA libraries constructed from key tissues expression of switchgrass under salt and resulted in ~70 million raw sequences and

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~5 million genome matched sequences. improvement. Muthamilarasan et al. [97] PARE data analysis of miRNA mediated studied foxtail millet and identified gene cleavage represented 250 sites involved in families responsible for monolignol miRNA targets. Furthermore, biosynthesis (PAL, C4H, 4CL, HCT, C3H, characterization of target sequences and CCoAOMT, F5H, COMT, CCR, CAD), miRNA in Brachypodium were involved in callose (Gsl) and cellulose (CesA/Csl). tissue-preferential control of miRNA family Comparative analysis of lignocellulose members, responsible for differential target biosynthesis genes of foxtail millet with cleavage. The PARA data from other crops genome of C4 crops showed high and Brachypodium provided insight on role resemblance with switchgrass followed by of miRNA and its target regulation to sorghum and maize. Whereas, expression exploit various features to fulfill biofuel profiling revealed that lignocellulosic gene purpose [94]. Another study performed by was differentially expressed under abiotic Green [95] identified genome wide targets stresses and hormonal treatments. The of miRNA in Arabidopsis using PARE results suggested that monolignol analysis and applied PARE further to biosynthesis proteins were highly diverse, Brachypodium distachyon genome. The while Gsl and CesA/Csl proteins were findings suggested identification of new homologous to Oryza sativa and miRNA and regulations with respect to Arabidopsis thaliana [97]. Furthermore, environmental stresses and type of tissue. lignocellulosic crops faces economic barrier 260 targets of miRNA were identified using during biofuel production due to cell wall PARE sequences were responsible for recalcitrance. Many researchers are actively precise miRNA guided cleavage. PARA working to explore genes that can offer analysis revealed that differentially solution to fix this problem via genome wide expressed miRNA guide is responsible for study. miRNAs are known to be involved in specific target RNA cleavage in tissue- all biological, developmental and metabolic preferential manner miRNA-target RNA processes due to broad functions of their regulation. Therefore, specific miRNA targets. Alteration in miRNA expression can targets RNA association with known lead to pleiotropic effects. Such as miRNA physiological functions, providing insight on regulates physiological and biological traits gene regulation and response under such as low expression of miR156 increases environmental stresses in different tissues biomass and reduces recalcitrance, while [96]. Recently, genomic tools are used to high expression of miRNA in bioenergy exploit lignocellulosic synthesis genes to switchgrass and poplar reduces lignin harness high yield of biomass, however, the content, increase biomass and flowering lack of complete genome in majority of time with improved responses toward harsh bioenergy grasses hinders the study on crop environment [98].

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Table 2. Role of miRNA in crops for biofuel production S No. Crop No. of miRNA miRNA Refs. 1. Switchgrass 20 conserved miRNA miR156a,b, miR156e, miR156f, miR156k, [87] (Panicum virgatum families. 37 genes were miR159b, miR160, miR164a, miR164c, L.) predicted as targets for miR166, miR167b, miR168, miR169a, miRNAs and 4 target miR169c, miR169d, miR169b, miR169k, mRNAs miR171g, miR172a, miR172c, miR172b, miR172d, miR319, miR393, miR394, miR395n, miR396n, miR397, miR398n, miR399, miR408, miR437, miR444, miR528 2. Switchgrass 121 potential miRNAs, [88] belonging to 44 families 3. Sugarcane 19 miRNA precursors miR156a, miR159a, miR167a, miR168a, [89] (Saccharum spp.) a total of 46 potential miR169, miR396a, miR827, miR408a, targets for 19 sugarcane miR437, miR444, miR528, miR1128, miRNAs miR1432, miR319b 4. Foxtail millet Seven homologues of miR169g [90] (Setaria italica) miRNA 5. Switchgrass miR156b precursor miR156b [91] overexpression improved biomass, solubilized sugar yield and forage digestibility 6. Switchgrass miR156 and miR162, miR156, miR157, miR159, miR162, miR167, [92] showed significant change miR169, miR172, miR395, miR396, miR397, in expression miR398, miR399 7. Sugarcane miRNAs differentially miR156, miR160, miR164, miR166, iR167, [93] (Saccharum spp.) expressed under drought miR169, miR171, miR172, miR319, iR390, stress miR393, miR394, miR396, miR397, iR399, miR528, miR529, miR1432 8. Wild grass 116 miRNAs conserved miR5163b-3p, miR5181e, miR5185l-3p.2, [94] Brachypodium and non-conserved miR7731, miR7738-3p, miR7754-3p, distachyon miRNAs miR9480ab, miR9481a, miR9481b, iR9482, miR9483ab, miR9484, miR9485, miR9486a, miR9486a, miR9486b, miR9487, miR9488, miR9489, miR9490, miR9491, miR9492, miR9493, miR9494, miR9495, miR9496, miR9497, miR9498, miR9499 9. Wild grass 80 new miRNA precursors miR166, miR156, miR529, miR5163b-3p [96] (conserved and non- conserved) 260 targets of new and known miRNAs with PARE sequences at the precise cleavage site were identified and characterized 10. Foxtail millet Potential miRNAs target miR156d-1, miR156d-2, miR395b, miR114- [97] (Setaria italica L.) few genes for post- npr, miRn29 transcriptional gene silencing 11. Wild grass Metabolism, development, miR156, miR160, miR167, miR169, miR172, [98] stress response miR397, miR398, miR414, miR5200

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12. Cassava (Manihot Metabolism, development, miR156, miR160, miR164, miR172, miR395, [98] esculenta) stress response miR482

13. Jatropha curcas Metabolism, development, miR004, miR156, miR172, miR395, miR398, [98] stress response miR5201, 14. Maize (Zea spp.) development, stress miR156, miR160, miR164, miR166, miR167, [98] response miR169, miR172, miR319 15. Poplar (Populus Metabolism, development, miR156, miR159, miR160, miR164, miR166, [98] spp) stress response, Pathogen miR169, miR172, miR319, miR472, response, Cold response miR1445, miR1446 16. Sugarcane development, abiotic stress miR156, miR159, miR164, miR397, miR399, [98] (Saccharum Spp.) response miR528 17. Switchgrass Metabolism, development, miR156, miR164, miR166, miR167, miR172, [98] biofuel yield, abiotic stress miR398, miR414, miR444, miR477, miR528, response miR531, miR854, miR1535, miR1848, miR2102, miR2118

Role of identified miRNAs in Sorghum precursors, secondary structure of miRNA Sorghum bicolor is well known staple crop and stem length. The findings suggested two in developing world and closely related to classes of miRNA plant precursors. The maize. It is desired crop due to high abundant precursor class has strong adaptability to wide range of climatic conserved blocks that are corresponding to conditions, short production time, salinity mature miRNA and its complementary and drought tolerance making it potential sequence, while less frequent precursor class candidate for cultivation for biofuel has two additional conserved blocks with production. Bedell ety al. [99] suggested that long stem region and extensive secondary 95% of sorghum genes are sequenced tagged structure that includes miR159/319 and with 65% coverage across its length, miR394 families [100]. miRNA are wide generated sequence through hypomethylated spread in all organism. Some miRNAs are portion of genome using methylation highly conserved in plant species and linked filtration (MF) technology. They studied to common ancestors in early evolution, functional parts of sorghum genome using with minute difference or absence of MF technology to obtain information on nucleotide substitution among plant species. miRNA, promoters, single sequence repeats, Whereas, others potential miRNA are exons and introns, while minimizing expressed and regulated under biotic and interspersed repeats. MF based sequence is abiotic environmental stresses. Some powerful source for comparative genomics miRNA are tissue specific and expressed with grasses and other important crops [99]. under developmental switching. Previously, Previously, 286 miRNA genes clustered into 75 miRNA belonging to 14 families were 43 family has been identified in identified. Zhang et al. [101] suggested that Arabidopsis, maize, and rice. Furthermore, EST analysis can identify new miRNA, and Dezulian et al. [100] reported identification its targets (Table 3). They predicted RNA of 200 miRNA genes belonging to 43 secondary structure of 812 EST sequence families in genome of sorghum, maize, and identified 338 new miRNA in 60 plant medick and poplar and expression of 37 species. miRNA are present among plant miRNA precursors of sugarcane and species in abundance and involved in variety soybean via computation tools. The study of developmental, signal transduction and analyzed plant precursors such as: conserved

1087 Pure Appl. Biol., 5(4): 1064-1100, December, 2016 http://dx.doi.org/10.19045/bspab.2016.50131 environmental stresses that stimulate sorghum genome is genetically recombined miRNA synthesis and regulation [101]. having gene density and gene order similar Zhang et al. [102] identified 481 miRNA of to rice. About 75% of sorghum genome is 37 families from 71 different plants via EST larger than rice genome because of retro- analysis. The research revealed that miRNA transposon accumulation in in some plants were clustered together recombinationally recalcitrant polycistron suggesting similar expression heterochromatin. However, repetitive DNA and transcription. Number of miRNA and genes were preserved since 70 million identified were related to available EST year ago, yet prior to sorghum rice instead of evolutionary relatedness to A. divergence most of the duplicated genes had thaliana, indicating that conserved and lost one member. Sorghum genome phylogenetic relation are linked to presence comprises 7% sorghum-specific genes and and absence of miRNA. However, miRNA 24% grass-related genes. Therefore, expression existed in early evolutionary duplicated miRNA genes contribute in stages of plant and remains functional for drought tolerance of sorghum [104]. Zhang 425 million years. Many miRNA families and coworkers [58] sequenced small RNA are conserved among major plant lineages library by identifying miRNAs in Sorghum. like: monocots, gymnosperms, mosses, and The findings revealed 13 novel miRNA, 7 eudicots [102] (Table 3). Zhang et al. [103] conserved miRNA and 29 miRNA families performed genome wide analysis of miRNA similar to monocots. Differential expression genes in maize, and characterized their of conserved and novel miRNA indicated structure, evolutionary relatedness and diverse role of 125 genes. These miRNAs expression. Using computational homology were involved in diverse processes, and and secondary structure modeling approach provides insights on miRNA controlled about 150 genes of 26 miRNA families were process that can be manipulated to improve identified and validated. Moreover, 68 biomass and stress tolerance in sorghum miRNA precursors belonging to 18 families [58]. were highly conserved among several Majority of biofuel producing crops like: species of plants, depicting this class of sorghum, Miscanthus, switchgrass, genes have splice variation and similar sites sugarcane and corn belong to tribe of for alterative transcription. Analysis of Andropogoneae. Sorghum has simple sequence variation in diverse maize inbred genome as compared to other species due to lines teosinte accessions suggested that lack of additional rounds of whole genome evolution of flanking sequences are similar duplication events, and contribute in to other genes. Additionally, the research possibility to generate high quality genome suggested that, duplicated miRNA genes sequence of sorghum. The research undergo gene lost like protein coding genes characterized small RNA from grains, stems with only 35% ancestral sites retained; of sweet sorghum and F2 generation though the retained number is higher than (derived from cross segregated for flowering protein coding genes [103]. time and sugar contents). Variation in Paterson et al. [104] determined genome- expression of miR395 and miR172 were wide sequence of sorghum via shotgun correlated with flowering time, whereas sequencing. About 98% genes in variation in expression of miR169 was chromosomal context were validated by correlated with sugar content of stems. physical genetic, and syntenic information. Furthermore, due to genotypic differences in The study revealed that one-third of miR395* were expressed equivalently as

1088 Hafsa Ali et al. abundant as miR395 in sweet sorghum physiological and molecular basis of though lacked expression in grain sorghum. sorghum genotype during drought response The research predicted 7 new miRNA and in “dry-down” experiment, where stress was 27 known miRNA from expression of 25 determined by water potential in 4-leaf-old miRNA families in sorghum genome. The plants. 1205 genes were up-regulated due to study validated small RNA sequences from drought stress and involved in carbon the stem of sweet sorghum for previously metabolism (NADP-ME), detoxification predicted miRNA, depicting its role in (CYPs, GST, AKRs), signal transduction flowering time and stem sugar accumulation (phosphoesterases, kinases, phosphatases), [105]. Katiyar et al. [106] identified miRNA osmoprotection mechanisms (P5CS), and its target genes from 1379 unique and regulation of transcription (bZIPs, MYBs, known miRNA of 33 different crops via HOXs), and stability of protein membranes Genomic Survey Sequence (GSS) and EST (DHN1, LEA, HSPs). The findings using computational tools. They determined suggested differentially expressed genes 37 miRNA of 10 miRNA families with 72 responsible for upregulation of 4 miRNA potential target genes. These predicted families, whereas one family was targets lay foundation to understand miRNA downregulated during early phase of function and involved in plant development drought stress [108]. In another similar and growth [106] (Table 3). study, Katiyar et al. [109] constructed small MicroRNA are involved in gene expression RNA libraries to identify tasi-RNAs and and plays crucial role in environmental drought-responsive miRNAs via next stresses and development. Thiebaut et al. generation sequencing on drought [107] predicted small RNA via deep susceptible (C43) and drought tolerant sequencing and discovered novel miRNA (M35-1) sorghum lines. Research showed using bioinformatics tools that regulated, that around 241 miRNA has been identified pathogen infections, drought and salt in sorghum and deposited in the miRBase. stresses in sugarcane. Using miRNA The research identified 526 novel miRNA precursors in sorghum genome 623 new and 97 conserved miRNA belonging to mature miRNAs were identified in unique miRNA families from sorghum, out sugarcane, from which 44 miRNA highly of which 97 were regulated by drought unique in their biological function. Sets of stress (49 down regulated, 32 up-regulated, miRNA in sugarcane were known to target and 14 miRNA depicted contrasting zinc finger protein, Myb, and expression between genotypes). A serine/threonine kinases. RPP2B protein and maximum of 18 miRNA were differentially MADS-box transcription factor were regulated in tolerant and sensitive genotypes involved in disease resistant protein and under drought stress condition. Genotype plant development, regulated by miRNA dependent miRNA regulation under drought cleavage and DNA methylation. stress contributed in differential drought Comparative analysis of miRNA between tolerance of sorghum genotypes. Around monocots provides valuable insight on 1300 target genes related to conserved and conservative miRNA and their targets in novel miRNA were identified with specific unidentified genome sequence of plants role related to biological, cellular, metabolic, [107]. and development processes. Stress Sorghum is C4 plant with high water responsive miRNA, tasi-RNA and their efficacy and high adaptability to semi-arid respective targets identified via genome- regions. Pasini et al. [108] evaluated wide search can unravel genetic and

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molecular mechanism responsible to cope Analysis Pipeline for Isoform Sequencing up with drought stress, thus providing a key (TAPIS) (tool used for iso-sequencing data role to improve stress response and biomass analysis) to identify APA sites and full- production in sorghum [109]. length splice isoforms. The findings Recently, Sanousi et al. [110] studied suggested transcriptome-wide full-length regulatory mechanism of miRNA related to isoforms over 11,000 novel splice isoforms drought and salinity in sorghum. They and discovered more than 2,100 novel genes studied expression profiling of small and ~ 11,000 expressed genes of APA [111] regulatory RNAs in pre- and post-flowering (Table 3). stages of six sorghum genotypes under Small RNAs are responsible for various drought and salinity stress using RT-qPCR. developmental processes, whereas miRNA The findings suggested that miRNA expression is regulated by environmental expression profile was dependent on dosage, stresses in plant species. Hamza et al. [112] whereas each miRNA exhibited different determined the expression profile of 8 expression in response to stress in all down-regulated miRNA in 11 elite sorghum genotypes. Furthermore, the expression of genotypes under drought stress and watered following miRNAs: miR156, miR167, conditions. Expression level of miR396 and miR168 and miR399 were variable as miR398 was highest in all sorghum compared to previous studies, which genotypes, yet high deregulation was suggested adaptation of sorghum toward observed in, miR166, miR167, miR168, stresses. Therefore, using transgenic miR393, miR396, miR397-5p. The study technology sorghum can be exploited to suggested that high gene expression profile produce improved varieties [110]. in grain sorghum HSD3226 in well-watered Alternative polyadenylation (APA) and conditions; shifted the expression profile alternative splicing of pre-miRNA under drought stress. N98 and Atlas—forage contribute in diversity of transcriptome, accession showed contrasting expression expression regulation and coding capacity of profile of miR397-5p under drought stress genome in eukaryotes. These transcriptomes depicting active mechanism to tolerate are analyzed by second-generation drought stress. Moreover, the research sequencing technologies. Abdel-Ghany et al. provides knowledge on miRNA with [111] sequenced sorghum transcriptome and potential to improve drought stress in other developed pipeline—Transcriptome cereal crops [112].

Table 3. Role of miRNA in Sorghum miRNA S. NGS/Bioinformatics Identified miRNAs’ families associated(biotic/abiotic Refs. No approach used ) traits 1. 44 (85%) miRNAs identified in sorghum DREB1-like proteins Methylation Filtration (MF) [99] Technology, bioinformatics 2. sbi-miR156a,b,c,d, miR157, miR159a,b, Biotic or abiotic Bioinformatics tool (EST [101] miR165, miR166, miR168, miR169, environmental stresses analysis) miR170, miR171, miR172 and hormone signaling 3. sbi-miR156a,b,c,d, miR159, conserved sequence Bioinformatics tools [100] miR160a,b,c,d,e, miR164, miR164b, blocks miR166a,b,c,d,e,f, miR167a,b,c,d,e,f,g, miR168, miR169a,b,c,d,e,f,g,h,i, miR171a,b,c,d,, miR172a,b,c,d,e,

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miR319, miR393, miR394a,b, miR395a,b,c,d,e, miR396a,b,c, miR399a,b,c,d,e,f 4. A total of 481 miRNAs, evolutionarily conserved Bioinformatics [102] miR156, miR172, miR319, miR396 miRNA families 5. miR156, miR159, miR160, miR162, Involved in secondary Next generation sequencing [103] miR164, miR166, miR167, miR168, metabolic processes, Bioinformatics miR169, miR171, miR172, miR319, biological processes miR390, miR393, miR394, miR395, including gene expression miR396, miR397, miR398, miR399, and response to stimuli miR408, miR482, miR528, miR529, miR827, miR1432 6. miR156a,b,c,d,e, miR159,b, Rice miRNA 169g, Genome sequencing [104] miR160a,b,c,d,e, miR162, miR164b,c, upregulated during Integration of shotgun miR166a,b,c,d,e,f,g, miR167a,b,c,d,e,f,g, drought stress assembly with genetic and miR168, miR169a,b,c,d,f,g,i, Cytochrome P450 physical maps miR171a,b,c,d,e,f, miR172a,b,c,e, domain-containing genes, -Bioinformatics (BLAST) miR319, miR390, miR393, miR394, often involved in miR395a,b,d,e,f, miR396a,b,c, miR397, scavenging toxins such as miR399a, b,c,d,e,f,g,h,i, miR408, those accumulated in miR437, miR444, miR528, miR529, response to stress miR821, miR1432, miR1435, miR1436, miR1439 7. 25 miRNA families:miR395, miR169d, Regulation biological SOLiD next generation [105] miR172c, miR437g , miR169, miR172, processes other than the sequencing system, miR169a,b,c,d,e,f,g,h,i, miR172a, b,c,d,e, sulfur metabolism Bioinformatics tools miR395a,b,c,d,e,f,g,h, miR5381, miR5382, miR5383, miR5384, miR5385, miR5386, miR5387, miR5388, miR5389 8. miR156, miR159 miR160, miR164, Drought tolerance, Bioinformatics tools [59] miR165, 166, miR167, miR168, miR169, physiological processes (prediction of miRNA miR170/171, miR172, miR319, miR390, and biotic and abiotic targets, BLAST) (5′RACE) miR393, miR394, miR395, miR396, stress responses assay miR397, miR398, miR408, miR437, The predicted targets are miR444, miR528, miR529, miR530, pre- dominantly miR827, miR894, miR1126, miR1318, transcription factors: miR1436, miR2118, miR2910, miR156 is predicted to tasiRNA3a, tasiRNA3b, miR5564a, target 7 Squamosa miR5566, miR5568, miR5387b, promoter binding miR5569, miR5570, miR5565, c, d, e, f, transcription factors; miR5564b, miR5567 miR159, 4 MYB transcription factors; miR160, 6 auxin response factors 9. sbi-miR156j,k,l,m, miR166l, In leaf development, and Bioinformatics tools (used [106] miR166m,n,o,p,q,r,s,t, miR167j,k, evolutionarily conserved ESTs and GSS to predict miR168b,c, miR171l,m, miR390b, in all land plants. miRNAs) miR396f,g,h,i,j,k, miR398b, miR399l, Regulates expression miR444a,b,c of the HD-ZIP III (class- III homeodomain-leucin zipper) miR444 family members are conserved only in monocot species

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10. sbi-miR164,b,c, miR395a,f,k,i, Drought affected gene Transcriptome analysis [108] miR399a,b, expression. Regulation using a high density of transcription (bZIPs, microarray MYBs, HOXs), signal Bioinformatics tools transduction (analysis of drought-related (phosphoesterases, genes and stay green QTLs) kinases, phosphatases), carbon metabolism (NADP-ME), detoxification (CYPs, GST, AKRs), osmoprotection mechanisms (P5CS) and stability of protein membranes (DHN1, LEA, HSPs) 11. sbi-miR44, miR94, miR105a,b, miR108, Drought stress Next-generation [109] miR114, miR131, miR141, miR149, expression sequencing, miR182a,b, miR191, miR192, miR205, miRNAs were found to Small RNA Library miR221a-h, miR224, miR229, miR255, be involved in cellular, Construction and miR263a-c, miR269, miR313, miR322, metabolic, response to Sequencing, Bioinformatics miR324, miR337, miR383 stimulus, biological Analysis of sRNA down-regulated under drought regulation, and Sequences, Differential miR169d-l, miR529, miR57, miR111, developmental processes Expression Analysis of miR211, miR245, miR266, miR339, miRNAs miR387 up-regulated in M35but down regulated in C43 miR160a, miR396b-c, miR396d-e, miR5385, miR4, miR6, miR19, miR26, miR41, miR46, miR48, miR76a-d, miR82, miR87, miR119, miR138, miR144, miR151, miR164, miR259, miR176, miR178a-b, miR180a-c, miR212, miR240, miR285, miR287, miR292, miR304, miR310a-e, miR314a- c, miR316, miR335, miR336, miR340, miR351, miR368, miR373, miR385, miR390, miR391, miR392a-c, miR412, miR413, miR416 down-regulated in M35 but up regulated in C43 miR2118e, miR2275, miR36, miR59a-c, miR64, miR120a-b, miR200, miR215, miR223, miR227a-c, miR256, miR268, miR295, miR297a-c, miR301, miR344, miR359, miR360a-c, miR376 12. miR156d, miR156, miR159a, Regulation of salinity RT-qPCR [110] miR167c,g,f, miR168, miR393a,b, stress and drought stress miR160f, miR166a-d, miR166f, miR168, miR171, miR399b, miR1435b 13. sbi-miR171h, miR5567, miR171d, Sequenced the sorghum Bioinformatics tool [111] miR528, miR166j, miR167d, miR156d, transcriptome o identify (BLAST), RT-PCR, RNA-

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miR5568d, miR1432, miR5568f, full-length splice sequencing, Differential miR319a, miR5567, miR159a, miR408, isoforms and APA sites. gene expression analysis miR168, miR397, miR166d, miR396c, miR6225, miR169k, miR169l, miR5565g, miR5567, miR399h, miR156e, miR167c, miR166h 14. miR396, miR393, miR397-5p, miR166, Regulate drought and expression profiling [112] miR167, miR168 other abiotic stresses

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