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2019 A Pipeline for Exogenous Gene Expression and Biomass Analysis in Bijay Bisunke Eastern Illinois University

Recommended Citation Bisunke, Bijay, "A Pipeline for Exogenous Gene Expression and Biomass Analysis in Plants" (2019). Masters Theses. 4417. https://thekeep.eiu.edu/theses/4417

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Printe N e Printed Name '&1 t>'l...C><\ \ML SCt~CES Graduate Degree Program Date

Please submit in duplicate. A PIPELINE FOR EXOGENOUS GENE EXPRESSION AND

BIOMASS ANALYSIS IN PLANTS

By

Bijay Bisunke

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGICAL SCIENCES IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 2019

I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE

~ R DATE DEPAR;ENT CHAIR DA TE

't-ltr.!t? THESJSCOMMITTEEMEMBER ~ATE THESIS COMMITTEE MEMBER DATE Abstract

Biological pretreatment of biomass is typically required for the production of liquid biofuels, which are viable alternatives to traditional fossil fuels. While traditional thermomechanical pretreatments are effective at helping to deconstruct lignocellulose, they are often expensive and tend to generate environmentally dangerous waste materials.

An alternative to traditional pretreatment strategies is biological pretreatment, which has focused on lignocellulose-degrading organisms, such as white-rot fungi. While direct pretreatment of biomass with white-rot fungi has shown great promise, directly expressing functional white-rot fungi genes in plants (i.e. in plan/a expression) has the potential to be even more efficient because the biomass itself would produce the deconstruction enzymes, which would decrease the amount of steps toward biofuel production. This thesis details an efficient pipeline for cloning and expressing genes in planta followed by protocols for assessing changes to carbohydrates and lignin chemistry. An In-Fusion cloning system was developed for Agrobacterium-based transformation into Arabidopsis thaliana using green fluorescent protein as a representative gene. Modified acid hydrolysis and thioacidolysis procedures were used to explore cellulose and lignin chemistry on pretreated (i.e. non-traditional) biomass.

Overall, this research generated a procedural work-flow for the rapid cloning and transformation of exogenous genes into Arabidopsis, followed by reliable carbohydrate analysis of the resulting biomass. This pipeline is expected to be used in future studies examining the expression of enzymes involved in biomass deconstruction from white-rot fungi into commercially-relevant biomass. TABLE OF CONTENTS

1. Introduction ...... 1

2. Research Objectives ...... 5

3. Materials and Methods ...... 6

3 .1. Agrobacterium-based Transformation of Arabidopsis thaliana ...... 6

3 .1.1 . Creation of Competent Agrobacterium cells ...... 6

3.1.2. Transformation of competent cells ...... 6

3 .1.3 . Plasmid Isolation and Transformation Validation ...... 7

3.1.4. Transformation of Arabidopsis thaliana wi th Agrobacterium tumefaciens .. 8

3.1.5. Seed Germination and Selection ...... 9

3 .2. Pretreatment and Analysis of Biomass ...... 9

3.2.1. Biomass Sources ...... 9

3 .2.2. Fungal Growth and Biomass Inoculation ...... 10

3.2.3 . Preparation of the Biomass for Chemical Analysis ...... 10

3.2.4. Acid Hydrolysis ...... 11

3.2.5. Thioacidolysis ...... 11

4. Results and Discussion ...... 12

4.1. Transformation Efficiency ...... 12

4.2 Lignocellulose Analysis ...... 13

5. Concl us ion ...... 15

6. References ...... 23 LIST OF FIGURES

Figure 1. Detailed structure of lignocellulose ...... 2

Figure 2. General outline of the research pipeline ...... 6

Figure 3. Map of pGFP ...... 7

Figure 4. Chemical structures of guaiacyl and syringyl lignin ...... 12

Figure 5. Total number of seeds sprouted in Arabidopsis thaliana ...... 16

Figure 6. Second generation of transformed A. thaliana ...... 17

Figure 7. Glucose content after acid hydrolysis of biomass ...... 18

Figure 8. Xylose content after acid hydrolysis of biomass ...... 19

Figure 9. Galactose content after acid hydrolysis of biomass ...... 20

Figure 10. Arabinose content after acid hydrolysis of biomass ...... 21

Figure 11. S/G ratio after thioacidolysis ...... 22 1. Introduction

With the world becoming more aware about the limited reserve of traditional fossil fuels in the planet and the adverse effects of their extensive use to all the living beings and the planet itself, we can see a clear shift of interest towards discovery and development of alternate sources of energy. Renewable energy is broadly defined as the energy harnessed from the renewable sources that can be naturally replenished within a human timescale.

Some examples of such sources include sunlight, tides, wind, geothermal, water and biomass (Painuly, 2001 ). Renewable energy and/or sustainable energy sources are becoming more and more popular as a plausible solution to the indubitable energy crisis that the world is headed towards. This thesis focuses on the production of bioenergy from biomass.

Plant biomass is a sustainable source of energy which can be used to generate a variety of energy forms like electricity and liquid fuels (U.S. E.I.A., 2010). The bulk of plant biomass is comprised of lignocellulose which is a structural complex of lignin, cellulose and hemicellulose linked through a variety of covalent and non-covalent interactions (Zhong and Ye, 2015). Liquid fuel can be produced by converting the lignocellulose to monosaccharides and then converting the monosaccharides to ethanol or similar compounds using microbes. But lignocellulose makes the plant cell wall biologically recalcitrant and resistant to enzymatic hydrolysis. The lignin is present as a physical barrier which surrounds the cellulose fibrils which limits accessibility, inhibits enzymatic activities and causes various non-specific molecular interactions (Zeng et al. ,

2014). This creates hurdles like increasing the loading of enzymes, time of hydrolysis and in most cases, requirement of other pretreatment procedures. To make the lignocellulosic

1 application economically viable, the cost for enzymes required in lignocellulosic fractionation and hydrolysis needs to be lowered substantially (Klein-Marcuschamer et al., 2012). / OH / OH

QOH QlOH o

Macrofibril

Plant cell

Macrof1bnl

wall~, O l1gnm Hem1cellulose

Crystalline cellulose

Figure 1. Detailed structure of lignocellulose (Potters et al., 2010).

Lignocellulose is structurally comprised of lignin, cellulose and hemicellulose, which are important in the formation of plant cell walls (Harris et al., 2010; Templeton et al., 2010). Cellulose is a polymer composed entirely of glucose that are linked in a ~-1 , 4 configuration (Figure 1) . Among the three components in lignocellulose, cellulose is present in highest percentage. Hemicellulose is a carbohydrate-rich compound present in plant fiber materials (Harris et al., 201 O; Wymelenberg et al. , 2011 ; Zhang et al. , 2017), but its structure and monosaccharide composition varies according to the species. The

2 cellulose and the hemicellulose are encapsulated by lignin, which is composed of phenyl­ propane units and linked through strong covalent bonds like ~-0-4 linkages

(Aleksandrova et al., 1998; Boerjan et al. , 2003).

In search of possible solutions to deconstruct plant biomass, a lot ofresearch and technology has been explored in the past to develop strategies for the pretreatment. These strategies can be classified into three main categories - chemical, physical and physiochemical. Chemical pretreatment can be wet oxidation, alkaline, acid or use of green solvents (Mosier et al. , 2005 ; Kumar et al. , 2010). The hydrolysis process is stimulated by using these chemicals. A combination of physical and chemical processes is used in the physiochemical pretreatment. This includes liquid hot water, steam explosion, pretreatment of superficial fluids with other methods (Hendriks and Zeeman,

2009; Kumar et al. , 2010). But these methods are highly energy intensive and may involve dealing with hazardous chemicals during the process or as waste products.

In recent years, biological pretreatment methods have raised an interest in the scientific community because of their ability to take advantage of innate biological process which require less energy and low chemical waste production (Canam et al,

2013a). One of the promising biological pretreatment agents are white-rot fungi which can degrade lignin, cellulose and hemicellulose at relatively equal rates (Canam et al. ,

2013b). The research on white-rot fungi is being focused on production ofbioenergy from biomass like bioethanol production. Using the white-rot fungi directly on biomass can be more efficient and cost effective approach as it removes the use of enzyme mixtures for biochemical management.

3 A lot of research involving white-rot fungi has been done with the members of the

Polyporales order, including carnosa, Phanerochaete chrysosporium and

Trametes versicolor (MacDonald et al., 2011; Canam et al., 2011; Wymelenberg et al. ,

2006; Kalinoski et al., 2017). Different omics-level studies have been done in the

Phanerochaete genus which shows great potential as pretreatment agents (MacDonald and Master, 2012; Sato et al., 2009; Wymelenberg et al. , 2010). Phanerochaete chrysosporium is regarded to be an important species of white-rot fungi for pretreatment as it is found to have the capacity to degrade aromatic lignin polymers with very little effect on cellulose content (Cullen et al., 2004). White-rot fungi like Phanerochaete chrysosporium are believed to be able to depolymerize lignin in order to gain access to cellulose (Wymelenberg et al. ,2006).

Similarly, Trametes versicolor has been found to improve total glucose yield and lignocellulosic deconstruction in waste canola straw making it an important biological pretreatment agent (Canam et al. , 2011). A study exploring the effect of wild-type strain

(52J) and cellobiose dehydrogenase-deficient strain (m4D) of T. versicolor on

Miscanthus and hardwood found that there was a significant increase in soluble lignin and decrease in insoluble lignin after hydrolysis with acid resulting in improved lignin extractability (Kalinoski et al., 2017). Other studies exploring the transcriptome response of P. chrysosporium and T. versicolor to maple and miscanthus discovered that the fungal response was different in each substrate type despite having similar lignocellulosic properties (Alanazi, 2018; Alaradi, 2017).

With the help of all these studies and research, we are only recently beginning to understand the role of different genes or enzymes expressed by the fungi to breakdown

4 plant biomass. By understanding these molecular mechanisms, we can engineer plants to selectively express the enzymes that could make lignocellulosic biomass less recalcitrant to deconstruction. The overall goal of this research was to develop a pipeline for in planta expression of novel genes from T. versicolor and P. chrysosporium into plants like

Arabidopsis thaliana.

A. thaliana is a member of the family Brassicaceae with a relatively small genome of about 13 5 Mbp. Its height may range from 5 cm to 3 0 cm. The life span of a typical plant completes in about 5-6 weeks. Because of the shorter life cycle and simple genome structure, A. thaliana has been extensively used in various plant research (Strand et al,

2000; Heyer et al, 2004; Tsai et al, 2012). A simple floral-dip method can be used to transform A. thaliana. When the plant is in flowering stage, it can be dipped into a solution made up of Agrobacterium tumefaciens cells, 5% sucrose and Sil wet L-77

(Clough, 1998). This Agrobacterium-mediated transformation process is relatively simple and highly efficient.

2. Research Objectives

The main objective of this research is to explore the development of a plasmid-based cloning system for Agrobacterium-based transformation of green fluorescent protein

(GFP) into a model plant, Arabidopsis thaliana. The research also expands to the optimization of analytical procedures for biomass (Miscanthus, Maple and Sunflower) that are destined for biofuel production.

5 PLASMID Agrobocterium­ PLANT BIOMASS ANALYSIS CONSTRUCTION TRANSFORMATION TRANSFORMATION

Figure 2. General outline of the research pipeline.

3. Materials and Methods

3.1. Agrobacterium-based transformation of Arabidopsis thaliana

3.1.1. Creation of Competent Agrobacterium cells

Agrobacterium tumefaciens (GV3101) was grown in 5 ml of YEP medium overnight at

28 °C with rotation of 250 rpm. A 2 ml culture was added to 50 ml YEP medium in a 250 ml flask and incubated at 28 °C with rotation of 250 rpm. When the optical density

(OD6oo) of the culture was 0.97, the culture was chilled on ice and centrifuged at 8000 rpm for 10 minutes at 4 °C. The supernatant was discarded and cells were resuspended in

1 ml of ice-cold 20 mM CaCh. It was then mixed gently by pipetting and 100 µl aliquots were dispensed into prechilled 1.5 ml tubes. Then the contents were snap-frozen on dry ice and stored at -80 °C.

3.1.2. Transformation of Competent Cells

Frozen cells were thawed slowly on ice. A total of 1 µl of plasmid DNA (pGFP; Figure

3) was added to the thawed cells and gently swirled with a pipette tip and incubated on ice for 15 minutes. The cells were heat shocked by placing them in 3 7 °C water bath for 5 minutes. 1 ml YEP medium was added and then incubated at 28 °C for 4 hours at 225 rpm. The tubes were centrifuged for 1 minute at maximum speed and supernatant was

6 discarded. The pellet was then resuspended in 100 µl of fresh YEP medium. The cells were cultured on a YEP agar plate with 50 µg/ml kanamycin. Plates were left to incubate overnight at 28 °C. Liquid cultures of individual colonies were prepared with YEP broth with 50 µg/ml kanamycin.

Hindlll - I - A'AGCT T Hindlll - 545 - A'AGCT_T Pstl - 557 - C TGCA'G Sall - 559 - G'TCGA_C

EcoRI-1315-G'AATT C EcoRl-1574-G'AATT C

RB - 1969 LB - 8228 pGFP 10403 bp

Figure 3. pGFP was created by inserting the gene for green fluorescent protein into

pCAMBIA1390 in a method described by Tsai et al. (2012).

3.1.3. Plasmid Isolation and Transformation Validation

The incubated culture (1.5 ml) was centrifuged at maximum speed for 3 min. The liquid was removed with a micropipette without disturbing the pellet. A total of 250 µl of P 1 buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris at pH 8.0) was added and the pellet was resuspended to obtain a homogenous solution. Then 250 µl of P2 buffer (1 % SDS/

0.2 N NaOH) was added and mixed by inverting the tube. It was left to sit at room

7 temperature for 3 min. Then, 350 µl of P3 buffer (3 M KO Ac at pH 6.0) was added and again mixed by inverting the tubes. It was then left to sit on ice for 3 min. The tubes were then centrifuged at maximum speed for 10 min. A portion of the liquid (400 µl) was transferred to a fresh tube and 400 µl of isopropanol was added and mixed well. It was then centrifuged at maximum speed for 10 min. All the liquid was discarded using a micropipette and a small white pellet (plasmid DNA) was left on the bottom of the tube.

A total of 200 µl of 95% ethanol was added and it was let to sit for 1 min at room temperature. All the liquid was discarded and the DNA pellet was air dried for about 5 min. An aliquot of 14 µl of nuclease-free sterile water was used to finally resuspend the

DNA. The pGFP plasmid was verified through restriction enzyme analysis.

3.1.4. Transformation of Arabidopsis tlutliana with Agrobacterium tumefaciens

Arabidopsis thaliana (Col-0) were transformed according to the floral-dip method by

Clough and Bent (1998), as was described previously (Tsai et al. 2012) using A. tumefaciens GV3101 containing pGFP (section 3.1.2). Briefly, immature flowers from A. thaliana were dipped in A. tumefaciens solution containing 5% sucrose and 0.05% Sil wet

L-77 for approximately 5 s. Treated plants were left to recover on their sides under plastic covers for approximately 24 h, and then returned to an upright position. The plants were then continued to be watered on a regular schedule and received 16 h of light per day.

For the current experiment, the height of the plants that were transformed was noted. The plants were divided into 3 different categories based on height of the tallest bolt: Small

(less than 5 cm), Medium (5-10 cm) and Tall (greater than 10 cm). After senescence, seeds were collected from seven different plants for each height category. The seeds were sterilized using chlorine gas according to standard procedures.

8 3.1.5. Seed Germination and Selection

The sterilized A. thaliana (Col-0) seeds were sprinkled on Petri dishes containing about

20 ml of Yz MS media containing hygromycin (50 µg/mL). Hygromycin is an antibiotic aminoglycoside that is produced by Streptomyces hygroscopicus. It can be used to select and maintain the cells that have hygromycin-resistant genes. The resistant genes inactivate hygromycin through phosphorylation (Rao et al., 1983). The number of seeds on each Petri dish was counted and noted. All the seeds were put in cold room at -4 °C.

After 3 days, the seeds were taken out and put under 16 h of light per day until germination. After about 12 days, the germinated seedlings on each plate were counted.

Then, the seedlings were transferred to Pro-Mix soil and were allowed to grow until full maturity. The seeds from those plants (T1 generation) were collected as before. Those seeds were also planted to obtain an additional generation of plants. In all cases, the total number of seeds plated and the seeds that sprouted on the antibiotic media were noted.

3.2. Pretreatment and Analysis of Biomass

3.2.1. Biomass Sources

The miscanthus (Miscanthus x giganteus cv. Illinois) used in the experiment was grown and harvested by a local farmer in Pesotum, IL, and acquired as chopped straw. The maple chips (Acer spp.) were purchased from J.C.'s Smoking Wood Products. The sunflower stems (Helianthus argophyllus) were harvested after one season of growth

Eastern Illinois University' s Thut Greenhouse. The dried stems were shredded to approximately 1 cm x l cm pieces using a biomass shredder (DEK CHI). All biomass was autoclave-sterilized prior to inoculation.

9 3.2.2. Fungal growth and Biomass Inoculation

P. chrysosporium and T. versicolor were grown on 30 Petri plates each containing malt extract agar for approximately 7 days until the media was covered by a layer of mycelia.

The 2 different types of fungal biomass was scraped from the surface of the corresponding plates and then combined in 2 different 1 L beakers separately. Then, with the help of a handheld mixer, the mycelia were macerated in 500 mL of malt extract broth each. The slurries were mixed with 2.5 L of malt extract broth in 5 L buckets. The broth with the macerated mycelia was then incubated at room temperature at 100 rpm.

After 4 days, the fungal biomass was filtered from the broth using a 0.45 µm mesh screen, then rinsed with 1 L of autoclaved water. The rinsed fungal biomass was gently mixed with 1L of water. From this mixture, about 100 mL was added to different containers containing maple chips, sunflower chips, or miscanthus straw. Twelve containers were prepared for each making a total of 24 containers. Small holes of

60 mm diameter were created on opposite sides of each container using a drill and sealed with 3M Micropore tape. The inoculum was mixed with the biomass by shaking the sealed containers and all the containers were kept in dark at room temperature for 5 weeks to allow the fungus to grow.

3.2.3. Preparation of the Biomass for Chemical Analysis

After 5 weeks, the inoculated biomass was dried at 105 °C for about 36 hours to stop the fungal growth. The dried samples were milled to pass a 20-mesh sieve (0.85 mm) using a

Wiley mill. A control sample of miscanthus straw, maple chips and sunflower straw untreated with any fungi was also dried and milled.

10 3.2.4. Acid Hydrolysis

The acid hydrolysis technique was according to Sluiter et al. (2008). Briefly, 3 ml of 72% sulphuric acid was added to 300 mg oven-dried sample in a pressure tube and macerated intermittently for 1 hour. The acid was then diluted to 4% by adding 84 ml of purified water and the tubes were autoclaved at 121 °C for 1 hour. The autoclaved hydrolysis suspension was then filtered using vacuum filtration and oven-dried Pyrex crucibles

(medium frit). An aliquot of filtered hydrolysate (approximately 10 ml) was collected and remaining solids were rinsed out of the pressure tube. The crucibles along with the retentate were put in an oven at 105 °C to dry overnight. Weight of the dried crucibles were measured after 16 hours. The hydrolysates were filtered through 0.22 µm filters and analyzed using HPLC according to Kalinoski et al. (2017).

3.2.5. Thioacidolysis

Approximately 2 mg of oven-dried samples were weighed in 4 ml scintillation vials. A 2 ml aliquot of reaction mixture (2.5% boron trifluoride etherate and 10% ethanethiol in dioxane) was added in each vial. The vials were then blanked with nitrogen gas and capped tightly. Then, they were incubated at 100 °C in a dry bath. After 4 hours, the vials were allowed to cool down at room temperature for 20-25 minutes. An internal standard

(5 mg/mL oftetracosane in methylene choride) and 120 µl of 0.4 M sodium bicarbonate were added in the vials. Then, 800 µl water and 400 µl of dichloromethane were added and mixed using a vortex shaker. The phases were left to settle overnight. Approximately

1 ml of the lower phase was removed and filtered into a 1.5 ml tube through a Pasteur pipette fitted with paper tissue and approximately 50 mg of anhydrous sodium sulphate.

11 The tubes, with their lids open, were placed in a dry bath set at 45 °C until they were dry. All the samples were resuspended in 500 µl of dichloromethane. 50 µl of sample, 50 µl of pyridine and 250 µl ofBSTA (N,0-bis(trimethylsilyl)acetamide) were added in a glass GC vial and mixed well. The vials were incubated for 2 hours at room temperature. The samples were then analyzed with a GC-FID system and the levels of syringyl (S) and guaiacyl (G) lignin (Figure 4) were determined as per Robinson and

Mansfield (2009). 602.JVVV' sVocH3 o"'l. o..,. G s

Figure 4. Chemical structures of guaiacyl (G) and syringyl (S) lignin (Samuel et

al., 2014).

4. Results and Discussion

4.1. Plant Transformation Efficiency

To assess the transformation efficiency of A. thaliana of varying bolt heights, the number of seeds germinating on hygromycin-containing media were compared to the total number of seeds planted across all replicates. Figure 5 shows the number of seeds planted versus number of seeds germinated from each replicate and height category. The data indicate that the plates containing seeds from medium-height plants (5-10 cm bolts) had the highest germination rates, although no statistical difference was found. The total

12 number of seeds germinating from the transformed plants grown in hygromycin­ containing media ranged from 0 to 9. Figure 6 shows the germination numbers from the seeds of the second generation of A. thaliana (those from self-crossing of the initial transformants). The germination rate from these plants were high, ranging from 75% to

90%, indicating transgene stability into the next generation.

4.2. Lignocellulose Analysis

The carbohydrates in lignocellulose of fungal-treated and untreated biomass were examined using acid hydrolysis. There are primarily five different carbohydrates of interest: glucose, xylose, galactose, arabinose and mannose. The final carbohydrate mannose was found only in trace amount and so was excluded from further evaluation.

HPLC analysis after acid hydrolysis shows that there was significant increases in the glucose content in miscanthus after fungal treatment, suggesting that both fungi (P. chrysosporium and T. versicolor) play a role in enhancing glucose extractability through lignocellulose deconstruction (Figure 7). This trend was not observed for sunflower as there was no significant change in the glucose content between the fungal-treated and untreated biomass. For maple, there was increase in the glucose in the biomass treated with P. chrysosporium but not in the biomass treated with T. versicolor (Figure 7).

Unlike the data from the glucose analysis, there was no significant change in xylose content between P. chrysosporium-treated miscanthus and untreated miscanthus. But it was seen that the xylose presence in T. versicolor-treated miscanthus was, in fact, lower than that in untreated biomass (Figure 8). In sunflower, the xylose content was significantly lower in P. chrysosporium-treated biomass than untreated, but remained unchanged between T. versicolor-treated and control biomass. For xylose content in

13 maple, there was a significant difference in the fungal-treated biomass, but there was not a significant change when comparing treated maple to the untreated maple (Figure 8).

Figure 9 shows the percentage of galactose in biomass. Though the sugar was found to be in relatively lower amount, some differences were observed with the miscanthus biomass. There was a significant increase in galactose content between P. chrysosporium-treated miscanthus and untreated biomass. But no any changes were observed in all other comparisons including the fungal-treated and untreated sunflower and maple biomass. Arabinose (Figure 10) was also present in lower quantity

(approximately 1.5-3% biomass). But a few changes were observed. There was a

significant difference in arabinose content between the fungal-treated miscanthus.

However, no significant differences were observed among the sunflower samples. Maple

biomass showed a significant reduction in this sugar after treatment with T. versicolor compared to the untreated sample (Figure 10).

Figure 11 shows the S /G lignin ratio in all three biomass types among the fungal­ treated and untreated samples after analysis with thioacidolysis. The data show that there were not significant differences in the S/G ratio after the fungal treatment in any biomass

sample. That is, the results were same for the fungal treated and untreated samples for all three types of biomass; however, these results should be viewed cautiously as thioacidolysis was not a reliable technique with respect to direct fungal pretreatment of

biomass for this set of experiments.

14 5. Conclusions

In the current research, an efficient cloning system was developed using In-Fusion technology. In addition, there appeared to be a trend towards higher transformation efficiency in medium-sized plants. The data also indicate that the techniques used during this research led to transgene stability into the T1 generation. Modified acid hydrolysis and thioacidolysis procedures were examined for their suitability with fungal-treated (i.e. unconventional) biomass. While acid hydrolysis was demonstrated to be a reliable technique for capturing carbohydrate differences between treatments and biomass types, the modified thioacidolysis procedure will require further optimization. Overall, this research generated a pipeline for efficient Agrobacterium-mediated plant transformation using cloning technology that allows for rapid insertion of genes of interest, followed by rapid and reliable carbohydrate biomass analysis using acid hydrolysis. This transformation and sugar analysis system is expected to be used in future experiments that will examine the effects of in planta expression of novel lignocellulose-modifying enzymes from P. chrysosporium and T versicolor.

15 soo ~ Seeds (TO)

4SO • No. of sprouted seeds in TO

400

3SO

300 Vl "C QJ QJ Vl 0 2SO -...... QJ .0 E ::I z 200

lSO

100

so

0 _ !!! II I Sl S2 S3 S4 SS S6 57 Ml M2 M3 M4 MS M6 M7 Tl T2 T3 T4 TS T6 T7 Transformed Plant

Figure 5. Total number of seeds sprouted in Arabidopsis thaliana after transformation with Agrobacterium tumefaciens. S, M and T are plants with corresponding heights: small (<5 cm), medium (5-10 cm) and tall (>10 cm).

16 4SO

400

3SO

300

VI "O Q) Q) 2SO ...... VI ....0 Q) .0 200 E z::::l lSO

100

so

0 51 52 SS 57 Ml M3 M4 MS M6 M7 T2 T3 TS T7 ES Seeds (Tl) • No. of Sprouted seeds in Tl

Figure 6. Second generation (T1) of transformed Arabidopsis thaliana plants. S, Mand T are plants with corresponding heights: small ( <5 cm), medium (5-10 cm) and tall (> 10 cm).

17 50.0 a a 45.0

40.0 a a ab e 35.0 Vl Vl ro 30.0 E >'- -0 25.0 ...... 0 c:: 20.0 0 ·.;:::; '- 0 0.. 15.0 0 '- Cl.. 10.0

5.0

0.0 Miscanthus Sunflower Maple

O Phanerochaete chrysosporium lirl Trametes versicolor Ill Control

Figure 7. Glucose content after acid hydrolysis of miscanthus, sunflower and maple after treatment with P. chrysosporium (dotted) and T versicolor (light-checkered). The dark- checkered bar is untreated biomass. Letter differences within each biomass indicate significant differences at the 95% level (ANOV A followed by Tukey's pairwise comparisons).

18 20.0

18.0 b b b 16.0

~ 14.0 V> V> ro E 12.0 >'- "O ..... 10.0 0 c .Q 8.0 .....I... 0 o_ 0 6.0 '- 0.. 4.0

2.0

0.0 Miscanthus Sunflower Maple

D Phanerochaete chrysosporium rnl Trametes versicolor 1111 Cont rol

Figure 8. Xylose content after acid hydrolysis of miscanthus, sunflower and maple after treatment with P. chrysosporium (dotted) and T versicolor (light-checkered). The dark- checkered bar is untreated biomass. Letter differences within each biomass indicate significant differences at the 95% level (ANOVA followed by Tukey's pairwise comparisons).

19 4.0 a 3.5 a * 3.0 V1 V1 a ro ab E 2.5 .._>- -0 2.0 0 -c: 0 ...... 1.5 '- 0 Q. 0 '- 1.0 CL

0.5

0.0 Miscanthus Sunflower Maple

D Phonerochaete chrysosporium Ill! Trometes versicolor Ill Control

Figure 9. Galactose content after acid hydrolysis of miscanthus, sunflower and maple after treatment with P. chrysosporiurn (dotted) and T. versicolor (light-checkered). The dark-checkered bar is untreated biomass. Letter differences within each biomass indicate significant differences at the 95% level (ANOVA followed by Tukey' s pairwise comparisons).

20 4.0

3.5 b

* 3.0 VI VI ro E 2.5 > I- a CJ b '+- 2.0 0 c .Q....., 1.5 I- 0 Q.. 0 I- 1.0 0...

0. 5

0.0 Miscanthus Sunflower Maple

O Phanerochoete chrysosporium Im Trometes versicolor Ill Control

Figure 10. Arabinose content after acid hydrolysis of miscanthus, sunflower and maple after treatment with P. chrysosporium (dotted) and T versicolor (light-checkered). The dark-checkered bar is untreated biomass. Letter differences within each biomass indicate significant differences at the 95% level (ANOVA followed by Tukey's pairwise comparisons).

21 1.6 a 1.4 a a a 1.2

0 1.0 :;::; ro a: ~ 0.8 Vl a 0.6 a

0.4

0.2

0.0 Miscanthus Sunflower Maple

D Phonerochoete chrysosporium ~ Trometes versicolor II Control

Figure 11. S and G lignin ratios (S/G) from thioacidolysis of miscanthus, sunflower and maple after treatment with P. chrysosporium (dotted) and T versicolor (light-checkered).

The dark-checkered bar is untreated biomass. Letter differences within each biomass indicate significant differences at the 95% level (ANOV A followed by Tukey's pairwise comparisons).

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