Wood from coniferous trees is an important source of renewable biomass. It can contribute to provision of carbon neutral energy, biomaterials and housing for a growing population. Softwood is mainly composed of cellulose, galactoglucomannan, xylan and lignin. This thesis focuses on the biosynthesis and function of Glucuronic acid (GlcA) decorations on softwood xylan. Results demonstrate that this GUX (GlucUronic acid substitution of Xylan)-dependent xylan branching is critical for the maintenance of biomass recalcitrance in a model vascular plant Arabidopsis thaliana. Experiments employing in vitro and in planta activity assays show that conifer transcriptomes encode at least two distinct GUX enzymes which are active glucuronosyltransferases. Interestingly, these enzymes have different specific activities, with one adding evenly spaced GlcA branches and the other one being able to add consecutive decorations. It is possible that these different patterns of xylan branching may have an impact on ability of xylan to interact with cellulose fibrils. To investigate the role for xylan binding to cellulose, Arabidopsis mutant plants in which this interaction is lost were evaluated alongside transgenic mutant lines in which the interaction may be restored. Results of this analysis indicate that the presence of cellulose-bound xylan might have an influence on plant vasculature integrity and thus it may have an effect on plant growth and biomass properties. Moreover, further results indicate that some xylan cellulose interaction is likely to occur in cell wall macrofibrils which can be detected in softwood. Taken together, this thesis provides insights into the process of conifer xylan glucuronidation and the possible role these branches may be playing in the maintenance of softwood recalcitrance and mechanical properties. In addition to identifying potential mutagenesis targets for improving softwood processing, this work is a proof of concept for the use of GUX enzymes for in vivo and in vitro biosynthesis of novel xylan structures with potential industrial application.

Cost effective production of biofuel from plant biomass (second generation biofuels) is currently a key challenge. To achieve this, accessibility of plant cell wall polysaccharides to chemical, enzymatic and microbial digestion could be improved by altering lignin structure and composition or by reducing lignin content, as lignin is one cell wall component that has already been shown to contribute to biomass recalcitrance. Therefore, this thesis reports the genetic manipulation of lignin biosynthesis through down-regulation of cinnamyl alcohol dehydrogenase (CAD) genes in barley (Hordeum vulgare L.). Barley has been chosen as the target plant for lignin manipulation for a few reasons: it is a major cereal crop that produces large amounts of lignocellulosic plant biomass that can potentially be used as animal feed or to produce second generation biofuels and also because it is a model grass for other bioenergy crops. CAD, as the final enzyme in the lignin pathway, is a perfect target for lignin manipulation. Characterised CAD mutants and transgenics have shown that down-regulation of CAD improves digestibility and does not influence plant growth and fertility. Due to the difficulty and complexity of transformation of monocot species, there are only a few reports describing down-regulation of CAD in monocots, and none in barley. Here, in this thesis, lignin was altered by down-regulating CAD genes using an RNAi construct with part of the HvCAD2 gene, the gene which has the highest expression level of all CAD genes. Transgenic barley plants showed reduced enzyme activity in the T0 generation (31% compared to EV plants) and enzyme activity was reduced even more in the T1 (to 3%) and T2 (to 2%) generations. The HvCAD2 RNAi barley lines had similar or slightly reduced Klason total lignin contents relative to control plants, but lignin structure and composition were altered. The RNAi plants had lower thioacidolysis yields, S/G ratio was reduced (1.59 in the empty vector controls versus 0.96–1.21 in the transgenic barley plants), the relative frequency of S units was reduced by 11–20%, the proportion of G units was increased by 17–32%, there was increased sinapaldehyde accumulation in lignin and ferulic acid abundance was reduced relative to control plants. Analysed transgenic barley plants had an orange stem phenotype. Growth season and conditions hugely affected the intensity of the phenotype. Because lignin plays a major role in culm strength and pathogen resistance, the influence of down-regulation of CAD on these features was characterised. The changed physicochemical nature of cell walls in HvCAD2 RNAi lines does not decrease the strength of the straw and does not decrease the resistance to the biotrophic Blumeria graminis and to the hemibiotrophic Rhynchosporium commune pathogens. The modified cell walls in the HvCAD2 RNAi lines had moderately improved sugar release for biofuel production. This study proves that it is possible to down-regulate CAD in cereal crops in order to change lignin structure and composition in plants without a negative impact on plant growth, fertility or pathogen resistance.

abstract: Carbon dioxide (CO2) levels in the atmosphere have reached unprecedented levels due to increasing anthropogenic emissions and increasing energy demand. CO2 capture and utilization can aid in stabilizing atmospheric CO2 levels and producing carbon-neutral fuels. Utilizing hollow fiber membranes (HFMs) for microalgal cultivation accomplishes that via bubbleless gas-transfer, preventing CO2 loss to the atmosphere. Various lengths and geometries of HFMs were used to deliver CO2 to a sodium carbonate solution. A model was developed to calculate CO2 flux, mass-transfer coefficient (KL), and volumetric mass-transfer coefficient (KLa) based on carbonate equilibrium and the alkalinity of the solution. The model was also applied to a sparging system, whose performance was compared with that of the HFMs. Typically, HFMs are operated in closed-end mode or open-end mode. The former is characterized by a high transfer efficiency, while the latter provides the advantage of a high transfer rate. HFMs were evaluated for both modes of operation and a varying inlet CO2 concentration to determine the effect of inert gas and water vapor accumulation on transfer rates. For pure CO2, a closed-end module operated as efficiently as an open-end module. Closed-end modules perform significantly worse when CO2-enriched air was supplied. This was shown by the KLa values calculated using the model. Finally, a mass-balance model was constructed for the lumen of the membranes in order to provide insight into the gas-concentration profiles inside the fiber lumen. For dilute CO2 inlet streams, accumulation of inert gases -- nitrogen (N2), oxygen (O2), and water vapor (H2O) -- significantly affected module performance by reducing the average CO2 partial pressure in the membrane and diminishing the amount of interfacial mass-transfer area available for CO2 transfer. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2018

Global warming is becoming more concerned by the public. The escalating atmospheric CO₂ level has introduced the intensification of greenhouse effect which brought enormous impact to the environment and climate. Among different methods for CO₂ mitigation, biological treatment on CO₂ emission using microalgae is regarded to be more economical and beneficial. On the other hand, countries around the world are projected to face water scarcity in the coming decades. Therefore alternatives to the finite fresh water resources for consumption have to be explored. Seawater has been introduced for toilet flushing in Hong Kong since 1950s. The flushing water is mixed with the consumed fresh water in the sewage treatment process to give a sewage effluent with high salinity, which still contains nutrients to support the growth of algal cells. Using sewage effluent as an algal culture medium not only can have a lower operation cost while saving fresh water for food crops, but also the effluent can be purified before discharging. Besides Hong Kong, there is an increase in numbers of cities incorporating seawater in toilet flushing systems as an economical and sustainable solution to fresh water scarcity. / Taking sustainable development into consideration, the use of algal cell for the fixation of CO₂ in saline sewage effluent is proposed, which should be an effective mitigation for CO₂ emissions, removal of nutrient in sewage effluent as well as production of useful products such as biofuel feedstock. In order to find out a suitable algal species for CO₂ the bioremediation, commercially available algal strains were compared against the locally isolated species on the growth and CO₂ removal efficiency in saline sewage effluent. Chlorogonium capillatum, the algal strain isolated from a local fish pond, was found to be the best candidate for CO₂ sequestration and nutrient removal in the non-sterile saline sewage effluent since it could grow much better in the presence of other microorganisms, comparing with the majority of other algal species could not grow under this situation. / The effect of CO₂ enhancement on algal photosynthetic rate and growth was studied in terms of the change in cell number, biomass and lipid production, and the fatty acid profile. The optimisation of CO₂ mitigation was achieved by deploying the response surface methodology (RSM) approach with a model describing the change in CO₂ consumption rate being developed. In the minimal run resolution V (MR5) screening test, it was found that salinity, pH, CO₂ and PO₄³⁻-P levels were influential to CO₂ removal by C. capillatum in non-sterile sewage effluent. Further optimisation of the CO₂ consumption rate was performed using the Box-Behnken design. The results of study showed that C. capillatum was able to deliver its maximum CO₂ consumption rate at 58.96 mg L⁻¹ d⁻¹ at the optimal condition, which was very close to the ordinary condition in the average sewage. In addition, the lipid content of C. capillatum could reach 24.26±2.67% with fatty acid profile conforming to typical biodiesel composition, delivering a high potential for biofuel feedstock production. Together with a high nutrient removal rate, C. capillatum could be used to produce a promising waste-recycling oriented simultaneous treatment system. / Since the CO₂ consumption rate was not dependent to light intensity, the spectral effects on the light-enhanced algal growth and carbon sequestration were investigated to find the best culture condition and how the carbon sequestration process was being influenced. Five LED light spectra were chosen for the analysis and it was found that growth parameters and cell compositions were influenced by the colour of the light very differently. The results indicated that under the irradiation of white LED light, C. capillatum had the highest CO₂ consumption rate and lipid content. Red LED light induced the highest amount of cellular protein as well as the chlorophyll a content. However, the performance of the light dependent reaction of the red LED light culture did not show apparent improvement. Regarding the CO₂ fixation enzyme, the spectral effect on RuBisCO content was marginal and there was no obvious relationship between the light induced CO₂ consumption and solely the light induced RuBisCO content change. Chemical analysis on the algal biomass indicated the C. capillatum culture would be a suitable microbial system to mitigate CO₂ emission, remove nutrients from saline sewage effluent and produce biomass suitable for biofuel production. / This study delivers a bioremediation system which is capable of simultaneous CO₂ mitigation, nutrient removal and biofuel feedstock production with a newly isolated algal species in a waste recycling manner. The findings of this study are not limited to the application locally in Hong Kong, but hopefully all these can also be useful in similar works in other places to help with the sustainable development. / 全球暖化正越來越受到公眾關注，大氣中不斷上升的二氧化碳水平已經加劇了溫室效應，並對環境和氣候帶來了巨大的影響。在不同的二氧化碳緩減方法中，利用生物方法以微藻處理二氧化碳排放被認為是更具效率和回報價值。另一方面，世界各地已經預計會在未來幾十年面臨水荒，因此是有必須要為有限的淡水資源尋找代替品。自五十年代起，香港便引入了海水作沖廁用途。沖廁用水會在污水處理過程中混合經使用過的淡水而產生高鹽度的污水，而污水當中仍含有營養物質，以支持藻類細胞生長。使用污水作為藻類培養介質不但可以降低營運成本，並有助於節省淡水用以耕種糧食作物，而且可以在污水排出前進行純化。除香港以外，一些引入海水用於沖廁系統的城市數目正在增加。 / 考慮到可持續發展，我們提出利用微藻細胞在帶鹽污水中進行二氧化碳固定。這應該是一個有效的緩解二氧化碳排放，清除污水中養份，以及產生有價值產品的綜合方案。為了找出一種合適的微藻進行生物整治，我們比較了市面上買到的品種和於本地環境分離出來的藻種於帶鹽污水中生長和去除二氧化碳的表現。經過實驗後我們確定一種從魚塘中分離出來的綠梭藻(Chlorogonium capillatum)是能夠在未經消毒的帶鹽污水中進行碳封存和去除養份的最佳選擇，因為綠梭藻能夠在其他微生物存在下仍可以良好地生長，反觀其他大多數的藻種就不能在這情況生長。 / 我們從細胞數、生物量、脂質和脂肪酸譜的變化方面研究了二氧化碳量增強對微藻光合速率和生長的影響。我們利用了反應曲面法(Response Surface Methodology)對微藻的二氧化碳緩減進行最佳化，並將二氧化碳緩減率的變化製成模型。在條件篩選實驗，我們找出鹽度、酸鹼、二氧化碳和磷質水平是會影響二氧化碳緩減率。在隨後的二氧化碳緩減率的最佳化後，綠梭藻的最大二氧化碳緩減率為58.69微克每公升每天。研究發現綠梭藻能夠於接近平常環境條件下達成最高效的二氧化碳緩減。除此之外，綠梭藻的脂質含量可達24.26±2.67百份比。加上脂肪酸分佈符合典型的生物柴油成份和具有高度養份去除率，綠梭藻可以用來創造以廢物回收作主導的多功能的生物修復系統。 / 由於二氧化碳緩減率並不依賴於光的強度，我們進行了光譜對微藻生長和碳封存的影響的研究，以找出最佳的培養條件和了解碳吸收的過程如何被影響。我們分析了五種發光二極管光譜，發現燈光顏色對微藻的生長參數和細胞組合物有明顯的影響。結果顯示，在白色燈的照射下，綠梭藻有最高的二氧化碳緩減率和脂質含量。紅色燈引發了最高的細胞蛋白質，以及葉綠素a含量。然而，紅光並沒有明顯提升光依賴反應。關於固定二氧化碳的酶，光譜對羧化/加氧酶的數量變化效果細微。另外，羧化/加氧酶跟二氧化碳緩減率之間沒有明顯關係。從微藻生物質中的化學分析，我們認為綠梭藻是一個合適的微生物系統以達成二氧化碳的排放緩減，去除帶鹽污水中的養份和生產適用於生物燃料製造的生物質。 / 這項研究提供了一個生物修復系統，它能夠以廢品回收方式同時減少二氧化碳排放、去除營養和生產製作生物燃料的原料。這項研究的結果並不只限於香港使用，我們希望這些東西也可以應在其地方類似的工程上，為可持續發展出力。 / Lee, Kwan Yin. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 115-134). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, January, 2017). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Esta dissertação apresenta uma análise comparativa dos aspectos sociais e econômicos decorrentes da produção dos biocombustíveis, etanol e biodiesel, nos principais países produtores e consumidores da atualidade Brasil, Estados Unidos e Alemanha. Para tal, os biocombustíveis foram analisados por intermédio dos dados das matrizes energéticas, da produção de matéria-prima e do biocombustível, do consumo interno, das políticas de suporte, barreiras tarifárias e não tarifárias, bem como dos gargalos existentes. Nos aspectos econômicos foram abordados indicadores da macroeconomia, indicadores específicos para biocombustíveis, a logística e a presença de subsídios. Nos aspectos sociais, a situação do trabalhador rural foi analisada enfatizando o trabalhador temporário imigrante na agricultura. O histórico das políticas possibilitou verificar a associação entre os estímulos políticos e a variação na produção. A construção dos cenários permitiu estimar a área necessária para expansão da produção de biocombustíveis. Neste grupo heterogêneo de países, em que as características sociais e econômicas são distintas, verificou-se que a legislação apresenta peculiaridades conforme a realidade de cada país. Com relação aos aspectos sociais, a situação dos trabalhadores temporários imigrantes na agricultura é semelhante, apresentando como características em comum a faixa etária, o grau de escolaridade e a remuneração. No que diz respeito aos biocombustíveis, etanol brasileiro é competitivo com o combustível fóssil correspondente, sem ajuda de subsídios. Apenas recentemente, o biodiesel apresentou aumento significativo na produção, após políticas de incentivo nos três países estudados. A produção e o consumo de biocombustíveis têm avançado nos três países. Entretanto, o Brasil apresenta a maior aptidão para produção de etanol, da atualidade, devido à possibilidade de expansão agrícola e ao tipo de matéria-prima utilizada que possui um balanço energético melhor. Nos Estados Unidos e na Alemanha, o custo de produção e a menor disponibilidade de terras agriculturáveis são vetores desfavoráveis à produção de biocombustíveis. / This study aims at a comparative analysis of the social and economic aspects, as a result of biofuels production, ethanol and biodiesel, in the three main producing and consuming countries The United States, Germany, and Brazil. To that end, there were analyses of biofuels production in several data fields such as; energy matrix, feedstock and biofuels production, local consumption, supportive policies, tariff and non-tariff barriers and existing bottlenecks. In the economic aspects, it was analyzed some macroeconomic indicators, biofuels indicators, logistics and subsidies. In respect of social aspects, the situation of the rural worker was analyzed, emphasizing the rural temporary foreign worker. The historical review of biofuels policies enabled to verify the association between the biofuels policies support and the biofuels production variation. It was possible to estimate the amount of land required for the expansion of biofuels production by the construction of scenarios. In this heterogenic group of countries with distinct social and economic characteristics, it was observed that the legislation development process follows each countrys reality. Despite of social aspects, the condition of the rural temporary foreign worker is alike, presenting common characteristics such as; age average, educational level and wage. Regarding the biofuels involved in this study, the Brazilian ethanol is more competitive with the regular fossil fuel even without subsidies. Only recently, there was a significant increase in the biodiesel production, after support policies in the three countries. Lately, the production and consumption of biofuels have been increasing in the three countries. Nevertheless, currently, Brazil has the major possibility to enhance biofuels production due its land availability and the use of a feedstock with the highest energy balance. In The United States and Germany the production costs and the lower quantity of agricultural land availability are disadvantages to their biofuels production.

In recent years, there has been increasing concern with respect to the large dependence across the globe on nonrenewable energy sources, such as fossil fuels. Ethanol has been explored, however, in alleviating this problem; cellulose, a polymer of glucose molecules, is a precursor to this potentially useful biofuel. However, the strength and rigidity of the cellulose structure has proven to be a difficult obstacle to overcome in this multistep synthesis. Harsh conditions are required, often including concentrated sulfuric acid and extremely high temperatures, to complete hydrolysis to a useful extent. In this work, the hydrolysis of cellulose was performed with acidic hydrogen molybdenum bronze in the form of XPell™ R by Xplosafe in place of sulfuric acid. By analyzing total organic carbon present in hydrolyzed samples (after 2 hours) using persulfate oxidation and colorimetric measurements, results were obtained showing that hydrogen molybdenum bronze is successful in catalyzing the hydrolysis of cellulose in comparison to hydrolysis completed in water alone. The values that were obtained in this analysis are as follows: 160 ± 20 ppt/mol at 40 °C, 180 ± 20 ppt/mol at 60 °C, 180 ± 30 ppt/mol at 80 °C, and 280 ± 40 ppt/mol at 100 °C. This determination shows that the catalytic ability of this acid increases with increasing temperature. Hydrogen molybdenum bronze is a useful candidate to explore in biofuel synthesis from cellulose. Comparison to sulfuric acid will be completed in future tests. This method is currently being used to pursue conversion of hydrolyzed cellulose to ethanol using yeast.

The use of biomass as a potential feedstock for the production of liquid hydrocarbon fuels has been under investigation in the last few decades. This paper discusses a preliminary design and a feasibility study of producing liquid hydrocarbon fuels from biomass through a combined biochemical and thermochemical route. The process involves anaerobic digestion (AD) of the biodegradable portion of the biomass to produce methane rich gas. The methane rich biogas stream is purified by removing contaminants and upgraded to liquid hydrocarbon fuel in a gas to liquid facility (GTL) via thermochemical conversion route. The biogas conversion involves two major steps: tri-reforming step to produce syngas (a mixture of CO and H2), and Fischer-Tropsch Synthesis (FTS) step to convert the syngas to a spectrum of hydrocarbons. Separation and upgrading of the produced hydrocarbon mixture allows production of synthetic transportation fuels. AD is ranked as one of the best waste management options as it allows for: energy recovery, nutrient recovery, and reduction in greenhouse gases emission. A detailed process modeling of the process was carried out using ASPEN Plus process design software package. Data for the process was based on literature on AD combined with laboratory results on the biogas to liquid conversion process. The composition of the final liquid hydrocarbon from the ASPEN model has been compared to the composition of commercial diesel fuel, and results have shown good agreement. As a result, the most current commercial diesel prices were used to evaluate the potential revenue from selling the product in the open market. The total capital investment to construct the plant with a capacity of handling 100,000 ton per year of wet biomass is $16.2 million with a potential of producing 2.60 million gallons of diesel. The base case feedstock is corn stover. The annual operating cost to run the plant is estimated to be $8.81 million. An annual revenue from selling the diesel product is estimated to be $14.6 million taking into account a green energy incentive of $3.00/gallon of diesel sold. The net present worth at the end of the plant life is $8.76 million with a discounted cash flow of return of 26.2%. The breakeven cost of diesel is determined to be $4.34/gallon assuming no tipping fees are charged for handling the waste. Sensitivity analyses results concluded that the profitability of the process is most sensitive to variation in diesel selling price. Based on these results, it can be concluded that the process is profitable only if incentives are provided for renewable fuels due to the current low prices of fossil fuels.

With the first commitment of the Kyoto Protocol commencing in 2008, many signatory governments have identified bio fuels as a favoured response to the problem of fulfilling their countries' pledges to reduce total greenhouse gas emissions to 1990 levels. Despite the tendency for pressure over climate change to expedite the commercialisation process, detailed evaluation of the claimed benefits, likely efficacy or environmental impact of bio fuels in New Zealand in the form of the Bio fuels Sales Obligation policy (BSO): a mandate place on the Oil Companies to supply a percentage of bio fuel. Systems thinking propound the notion of complex interrelatedness: a pivotal concept in our current understanding of the cumulative effects of greenhouse gas emissions and their relationship to climate change. It also recognizes that the multiple ways in which different stakeholders perceive a contentious question are an integral feature of any problematical situation. By applying systems concepts to qualitative research, Soft Systems Methodology (SSM) is therefore particularly suitable for the analysis of multiple stakeholder discourse in this situation. The present study employs SSM to examine stakeholder opinion through the construction of conceptual models in the form of rich pictures and root definitions. The researcher invited diverse stakeholders to ‘see what they were thinking’ and reflect upon the beliefs and assumptions that informed their views with respect to New Zealand bio fuels development. With reference to official documentation arising from the policy development process and through a series of interviews and a focus group, the research examines perceptions of the consultation process on bio fuels development and its contribution to informed decision-making. The study data indicates that the scope of enquiry tended to be restricted by early presuppositions regarding the consultation process and its desired outcomes, which consequently shifted the focus of consultation the enquiry from an assessment of the desirability of bio fuels to an appraisal of the feasibility of government policy. However inadvertently, communication was distorted. The research examines the basic assumptions that shaped the discourse and enabled already established opinions to prevail. Furthermore, the thesis explores how using SSM to surface tacit assumptions and challenge proposed models of intervention can help improve the reflexivity of discourse and decision-making. By ensuring open communication remains at the centre of discourse, SSM could provide a means of establishing productive conditions for learning and co-creative dialogue. Consequently the study has important implications for how the ‘health’ of public discourse in New Zealand might be sustained when addressing other similarly complex issues.

The demand for biofuels continues to increase due in part to government standards and promotion as well as the ambitious goals set by various companies and industries. Camelina is considered to be an ideal energy crop because of its low input requirements, suitability for marginal soils, and naturally competitiveness with weeds. A partial equilibrium model with a break-even price approach is used to estimate the potential supply curves for Camelina in Idaho, Montana, Oregon, and Washington. The supply curves are used to determine if the 50 million gallon goal set by the "Farm to Fly" initiative can be met. Given the current price of Camelina, $0.15/lb, the estimated supply of Camelina in all 4 states is 1,756,076,887 lbs and 1,493,684 acres. This estimation assumes that if the wheat-Camelina rotation is more profitable than the current crop rotation, then all of the acres will be converted to a wheat-Camelina rotation. When a 5% adoption rate is applied to the low and the intermediate rainfall zones and a 1% to the high rainfall zones, the number of acres converted to Camelina decreases to 72,213. These results suggest that given current market conditions, the supply of Camelina in the Northwest is not enough to meet the biofuel goal without an increase in yield and government promotion. / Graduation date: 2013

Switchgrass (Panicum virgatum), a summer perennial grass native to North America, is currently being explored for its potential use in the production of biofuels. With these interests, genetic manipulation of switchgrass to produce plants that are easier to digest, have an increased resistance to diseases and stresses, and maintain viability longer in the field are required. Therefore, it was necessary to develop a reliable and efficient tissue culture system for the transformation of switchgrass. Current switchgrass tissue culture requires months for regeneration of transformants with relatively poor transformation efficiencies and are limited to derivatives of a single variety, Alamo. We have developed a tissue culture system, utilizing a novel media, LP9, which has demonstrated decreased time to the production of whole transgenic plants and with an increased efficiency. LP9 is not an MSO-based tissue culture system. It is comprised of both N6 macroelements and B5 microelements with the auxin, 2,4-D and does not include any cytokinin. After just 1 month on LP9 media, callus can be selected and used for Agrobacterium tumefaciens-mediated transformation or particle bombardment, and plants can be regenerated within 3 weeks of callus initiation. Our system is unique to previously explored MSO-based systems in that it is optimized for the production of type II callus, which has been shown to produce higher transformation efficiencies in other monocots. We have increased the transformation efficiency of switchgrass from to up to 4% to 34% efficiency by selecting for this type of callus.