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Development of a Tissue Culture and Transformation Protocol for Seashore Paspalum (Paspalum vaginatum Swartz)

Permanent Link: http://ufdc.ufl.edu/UFE0021316/00001

Material Information

Title: Development of a Tissue Culture and Transformation Protocol for Seashore Paspalum (Paspalum vaginatum Swartz)
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Neibaur, Isaac E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 2, armyworm, auxin, bap, biolistic, bt, cry, cytokinin, dicamba, fall, gus, icp, paspalum, pest, resistance, salt, seashore, transformation, turfgrass, vaginatum
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Seashore paspalum (Paspalum vaginatum Swartz) is a salt-tolerant, fine textured turfgrass used on golf courses in coastal, tropical and subtropical regions. Targets for genetic engineering of seashore paspalum include improved disease and insect resistance. However, a genetic transformation protocol for seashore paspalum is lacking. In this work, a callus induction, plant regeneration, and transformation protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg L 1 3,6 dichloro 2 mehthoxybenzoic acid (dicamba) and 0.1 or 1.0 mg L 1 benzylaminopurine (BAP). A multifactorial experiment showed that callus induction medium containing 3 mg L 1dicamba and 1.0 mg L 1 BAP had a plant regeneration frequency that was 12 times higher than medium with 3 mg L 1 2,4 dichlorophenoxyacetic acid (2,4 D) alone, and 10 times higher than the combination of 3 mg L 1 2,4 D and 1.0 mg L 1 BAP. The correlation between transient and stable transformation frequency in seashore paspalum was evaluated using callus derived from six different callus induction media for biolistic gene transfer of a constitutive uidA (GUS) reporter gene expression cassette or an hph selectable marker expression cassette. Callus induction media using immature inflorescence segments as explants differed in auxin type (2,4 D or dicamba) and cytokinin concentration (0.0, 0.1 or 1.0 mg L 1 BAP). Media with 2,4 D supported significantly higher transient GUS expression. However, media containing dicamba showed higher plant regeneration frequencies. Transgenic plants regenerated on both 2,4 D and dicamba containing media. Transgenic plants grew vigorously and did not show phenotypic differences compared to nontransformed controls. This is the first report of the production of transgenic seashore paspalum plants.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Isaac E Neibaur.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Altpeter, Fredy.
Local: Co-adviser: Gallo, Maria.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021316:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021316/00001

Material Information

Title: Development of a Tissue Culture and Transformation Protocol for Seashore Paspalum (Paspalum vaginatum Swartz)
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Neibaur, Isaac E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 2, armyworm, auxin, bap, biolistic, bt, cry, cytokinin, dicamba, fall, gus, icp, paspalum, pest, resistance, salt, seashore, transformation, turfgrass, vaginatum
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Seashore paspalum (Paspalum vaginatum Swartz) is a salt-tolerant, fine textured turfgrass used on golf courses in coastal, tropical and subtropical regions. Targets for genetic engineering of seashore paspalum include improved disease and insect resistance. However, a genetic transformation protocol for seashore paspalum is lacking. In this work, a callus induction, plant regeneration, and transformation protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg L 1 3,6 dichloro 2 mehthoxybenzoic acid (dicamba) and 0.1 or 1.0 mg L 1 benzylaminopurine (BAP). A multifactorial experiment showed that callus induction medium containing 3 mg L 1dicamba and 1.0 mg L 1 BAP had a plant regeneration frequency that was 12 times higher than medium with 3 mg L 1 2,4 dichlorophenoxyacetic acid (2,4 D) alone, and 10 times higher than the combination of 3 mg L 1 2,4 D and 1.0 mg L 1 BAP. The correlation between transient and stable transformation frequency in seashore paspalum was evaluated using callus derived from six different callus induction media for biolistic gene transfer of a constitutive uidA (GUS) reporter gene expression cassette or an hph selectable marker expression cassette. Callus induction media using immature inflorescence segments as explants differed in auxin type (2,4 D or dicamba) and cytokinin concentration (0.0, 0.1 or 1.0 mg L 1 BAP). Media with 2,4 D supported significantly higher transient GUS expression. However, media containing dicamba showed higher plant regeneration frequencies. Transgenic plants regenerated on both 2,4 D and dicamba containing media. Transgenic plants grew vigorously and did not show phenotypic differences compared to nontransformed controls. This is the first report of the production of transgenic seashore paspalum plants.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Isaac E Neibaur.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Altpeter, Fredy.
Local: Co-adviser: Gallo, Maria.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021316:00001


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1 DEVELOPMENT OF A TISSUE CULTURE AND TRANSFORMATION PROTOCOL FOR SEASHORE PASPALUM ( P aspalum vaginatum SW ARTZ ) By ISAAC ESPI NEIBAUR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILL MENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 Isaac Espi Neibaur

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3 T o friends and family I relied on to keep me going.

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4 ACKNOWLEDGMENTS I thank Dr Fredy Altpeter for his continual guida nce, scientific resources, and monetary unending patience and continual personal pursuit for progress will continue to inspire me long after my time as his student I am also deeply grateful to the time and effort my committee members dedicated in helping me to complete this work. In particular I would like to thank Dr Altpeter and Dr Gallo for their assistance on this work and their efforts in my contributions to the scientific literature. Thanks to Dr Kenworthy for use of his facilities, and special thanks to Dr Trenholm for joining my committee after my proposal seminar. I thank Springer Verlag for their permission to use material submitted for publication in In Vitro Cellular Developmental Biol ogy Plant under the working title of "The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum ( Paspalum vaginatum Swartz)". I would especially lik e to thank my parents and sisters for their encouragement and unfaltering faith in me. My parents taught me to always be mindful curious, and tolerant of differing thoughts, opinions, and beliefs. This instilled a solid base for me to further develop my scientific reasoning and curiosity. My sisters taught me that it i s all right to just enjoy living in the moment Finally I would like to deeply thank my wife for her continual love and support. She is truly the yin to my yang, very direct and realisti c. As I grow out of my childish naivety, she helps bring me out of the clouds and helps me focus on making progress in work, life, and spirituality. She is a rare gem and I look forward to facin g l

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5 TABLE OF CONTENT S page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 9 LIST OF FIGURES ................................ ................................ ................................ ....................... 10 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................................ ... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 14 Introduction ................................ ................................ ................................ ............................. 14 Seashore Paspalum ................................ ................................ ................................ ................. 15 Objectives of the Research ................................ ................................ ................................ ..... 18 2 LITERATURE REVIEW ................................ ................................ ................................ ....... 20 Turfgrass Tissue Culture ................................ ................................ ................................ ......... 20 Gene Transfer to Grasses ................................ ................................ ................................ ........ 24 Selectable and Scorable Markers Used for Grass Transformation ................................ ......... 26 Fall Armyworm [ Spodoptera frugiperda (J. E. Smith)] ................................ ......................... 27 Bacillus thuringiensis ................................ ................................ ................................ ............. 27 3 MATERIALS AND METHODS ................................ ................................ ........................... 32 Development of a Tissue Culture Pr otocol for Seashore Paspalum ................................ ....... 32 Plant Material ................................ ................................ ................................ .................. 32 Preparation of Explants ................................ ................................ ................................ ... 32 Experimental Design ................................ ................................ ................................ ....... 32 Callus Induction ................................ ................................ ................................ ............... 33 Plant Regeneration ................................ ................................ ................................ ........... 33 Statistical Analysis ................................ ................................ ................................ .......... 33 Transient Transformation of Sea Isle 1 Callus by Biolistic Gene Transfer ............................ 34 Plant Propagat ion and Preparation of Explants ................................ ............................... 34 Tissue Culture Composition ................................ ................................ ............................ 34 Transient Reporter Gene Expression ................................ ................................ ............... 34 Stable Genetic Transformation of Seashore Paspalum by Biolistic Gene Transfer ............... 35 Tissue Culture Composition ................................ ................................ ............................ 35 Stable Genetic Transformation ................................ ................................ ........................ 36 Evaluation of cry1Fa Integration and Expression ................................ ........................... 37

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6 Immunochromatography ................................ ................................ ................................ 37 Statistical Analysis ................................ ................................ ................................ .......... 38 4 RESULTS ................................ ................................ ................................ ............................... 39 Development of a Tissue Culture Protocol for Seashore Paspalum ................................ ....... 39 Callus Induction ................................ ................................ ................................ ............... 39 Callus Fresh Weight ................................ ................................ ................................ ........ 39 Shoot Regeneration ................................ ................................ ................................ ......... 40 Transient Expression of uidA in Seashore Paspalum Callus ................................ .................. 41 Stable Transformation ................................ ................................ ................................ ............ 41 5 DISCUSSION AND CONCLUSIONS ................................ ................................ .................. 53 Developing a Tissue Culture Protocol ................................ ................................ .................... 53 Transient Transformation of Seashore Paspalum ................................ ................................ ... 54 Stable Genetic Transformation of Seashore Paspalum ................................ ........................... 55 6 CONCLUSION ................................ ................................ ................................ ....................... 58 APPENDIX. LABORATORY PROTOCOLS USED FOR VECTOR CONSTRUCTION, TISSUE CULTURE AND MOLECULAR ANALYSIS OF TRANSFORMED SEASHORE PASPALUM PLANTS ................................ ................................ ..................... 60 Protocols For Molecular Cloning ................................ ................................ ........................... 60 Preparation of Electrocompetent E. coli ................................ ................................ .......... 60 Electroporation of E. coli ................................ ................................ ................................ 60 Glycerol Stocks ................................ ................................ ................................ ............... 61 Amplification and Purification of Plasmid DNA Using QIAprep Miniprep Kit ......... 61 Amplification and Purification of Plasmid DNA Using the QIAGEN Plasmid Midi Kit ................................ ................................ ................................ ........................ 62 Gel Extraction Using QIAquick Gel Extraction Kit ................................ ..................... 63 Vector Construction ................................ ................................ ................................ ......... 64 Digestion of pUbiGUS ................................ ................................ ............................. 64 Ligation of backbone fragment from HindIII dig ested pUbiGUS ........................... 64 Linearization of HindIII digested pUbiGUS and self ligation prevention ............... 64 Restriction Digestions ................................ ................................ ................................ ..... 65 The pHZubicry1Fa digestion ................................ ................................ ................... 65 Ligation of the ubiquitin promoter and cry1Fa coding sequence from pHZubicry1Fa into the pUbiGUS linearized b ackbone ................................ ........ 65 Protocols For Seashore Paspalum Callus Induction, Transformation, And Regeneration ..... 66 Protocol for Seashore Paspalum Immature Inflorescence In Vitro Cultivation .............. 66 Tissue Culture Conditions ................................ ................................ ............................... 66 Protocol for Particle Bombardment ................................ ................................ ................. 66 Gold stock (60 mg mL 1 ) preparation ................................ ................................ ....... 66 Sterilization of Biolistic Gene Delivery Device (PDS 1000, BioRad) Components ................................ ................................ ................................ .......... 67

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7 Preparation of DNA Coated Microparticles ................................ ................................ .... 67 Biolistic Bombardment ................................ ................................ ................................ .... 67 The GUS Assay ................................ ................................ ................................ ............... 68 Solution 1 ................................ ................................ ................................ ................. 68 Solution 2 ................................ ................................ ................................ ................. 68 The GUS stock solutions ................................ ................................ .......................... 68 The GUS Assay Procedure ................................ ................................ .............................. 68 Buffers And Medium ................................ ................................ ................................ .............. 69 Bacterial Growth ................................ ................................ ................................ ............. 69 The SOC medium ................................ ................................ ................................ ..... 69 Antibiotics ................................ ................................ ................................ ................ 69 Seashore Paspalum Tissue Culture Med ium ................................ ................................ ... 69 The IF medium ................................ ................................ ................................ ......... 69 Treatment medium ................................ ................................ ................................ ... 69 Sorbitol medium ................................ ................................ ................................ ....... 70 Selection medium ................................ ................................ ................................ ..... 70 Regeneration medium ................................ ................................ .............................. 70 Stock Solutions for Ti ssue Culture ................................ ................................ .................. 70 The 2,4 dichlorophenoxyacetic acid (2,4 D) ................................ ........................... 70 The 3,6 dichloro 2 methoxybenzoic acid (dicamba) ................................ ............... 70 The 6 benzylaminopurine (BAP) ................................ ................................ ............. 70 Hygromycin ................................ ................................ ................................ .............. 70 Paromomycin ................................ ................................ ................................ ........... 70 Molecular Techniques used in the Confirmation of Stable Transformants ............................ 71 Immunochromatographic Cry1Fa QuickStix Test ................................ .......................... 71 The ELISA for Presence of Cry1Fa Protein ................................ ................................ .... 71 Interpretion of results (Spectrophotometric measurements and general test criteria) ................................ ................................ ................................ .................. 72 Preparation of Solutions for ELISA ................................ ................................ ......... 72 Wash buffer ................................ ................................ ................................ .............. 72 Extraction buffer ................................ ................................ ................................ ...... 72 Sample preparation ................................ ................................ ................................ ... 72 Leaf testing ................................ ................................ ................................ ............... 73 The DNA Extraction Using the CTAB Met hod ................................ .............................. 73 Procedure ................................ ................................ ................................ .................. 73 The CTAB buffer (500 mL) ................................ ................................ ..................... 74 The PCR Reaction s ................................ ................................ ................................ .......... 74 Basic PCR set up using HotStarTaq DNA Polymerase (Qiagen) .......................... 74 Amplification of hph in modified phphcry1Fa ................................ ........................ 75 Amplification of cry1F in modified phphcry1Fa ................................ ..................... 75 Agarose Gel Electrophoresis ................................ ................................ ........................... 75 Procedure ................................ ................................ ................................ .................. 75 The TBE stock ................................ ................................ ................................ .......... 76 Using the Nanodrop ND 1000 Spectrophotometer ................................ ......................... 76

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8 LIST OF REFERENCES ................................ ................................ ................................ ............... 77 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 95

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9 LIST OF TABLES Table page 4 1 Callus induction and regeneration in response to the original position of immature inflorescence segments ................................ ................................ ................................ ...... 43 4 2 Callus induction and regeneration as influenced by the immature inflores cence segments, auxin type ................................ ................................ ................................ .......... 44 4 3 Callus induction and regeneration as influenced by auxin type BAP concentration and interaction of auxin type and BAP concentration ................................ ....................... 45 4 4 Transformation efficiencies ................................ ................................ ............................... 46

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10 LIST OF FIGURES Figure page 3 1 The pUbiGUS expression cassette ................................ ................................ .................... 38 3 2 The p HZubicry1Fa expression cassette ................................ ................................ ............. 38 4 5 Seashore paspalum response to comp osition of tissue culture media ............................... 47 4 6 Transient GUS gene expression in callus induced on different culture media and following biol istic transfer of the uidA gene ................................ ................................ ..... 48 4 7 Tran sient GUS express ion in seashore paspalum callus ................................ ................... 49 4 8 Transgenic seashore paspalum plants co transformed with cy1Fa and hph, selected on callus induction media with 30 mg L 1 ................................ ................................ ......... 50 4 9 The Cry1Fa protein expression detected by the immunochromatographic Cry1Fa QuickStix Test ................................ ................................ ................................ ................... 51 4 10 The PCR of genomic DNA from seashore paspalum lines suggesting the presence of Ubicry1Fa ................................ ................................ ................................ ......................... 52

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11 LIST OF ABBREVIATION S 2,4 D 2,4 Dichlorophenoxyacetic acid 2,4,5 T 2,4,5 Trichlorophenoxyacetic acid BAP Benzylaminopurine Bt Bacillus thuringiensis d icamba 3,6 Dic hloro 2 mehthoxybenzoic acid GFP Green fluorescent protein GUS glucuronidase ICP Insecticidal crystal protein NAA Naphthaleneacetic acid PCR Polymerase chain reaction PMI Phosphomannose isomerase SI1 Sea Isle 1

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12 Abstract of Thesis Presented to the Gra duate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF A TISSUE CULTURE AND TRANSFORMATION PROTOCOL FOR SEASHORE PASPALUM ( P aspalum vaginatum SW ARTZ ) By I s aac E spi N eibaur August 2007 Chair:Fredy Altpeter Cochair:Maria Gallo Major: Agronomy Seashore paspalum ( Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical and subtropic al regions. Targets f or genetic engineering of seashore paspalum include improved disease and insect resistance. However, a genetic transformation protocol for seashore paspalum is lacking. In this work, a callus induction, plant regeneration and transformation protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 40 0 shoots per cultured immature inflorescence ( 1 cm in length) was achieved by culturing 0. 2 cm segments on media with 3 mg L 1 3 ,6 d ichloro 2 mehthoxybenzoic acid (dicamba) and 0.1 or 1. 0 mg L 1 benzylaminopurine (BAP). A multifactorial experiment showed that callus induction medium containing 3 mg L 1 dicamba and 1. 0 mg L 1 BAP had a plant regeneration frequency that was 12 times higher than medium with 3 mg L 1 2,4 d ichlorophenoxyacetic acid (2,4 D) alone and 10 times higher than the combination of 3 mg L 1 2,4 D and 1. 0 mg L 1 BAP. The correlation between transient and stable transformation frequency in seashore paspalum was ev aluated using callus derived from six different callus induction media for biolistic gene transfer of a constitutive uidA (GUS) reporter gene expression cassette or an hph selectable marker expression

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13 cassette. Callus induction m edia using immature inflor escence segments as explants differed in auxin type (2,4 D or dicamba) and cytokinin conc entration (0.0 0.1 or 1. 0 mg L 1 BAP). Media with 2,4 D supported significant ly higher transient GUS expression. However, media containing dicamba showed higher pla nt regeneration frequencies T ransgenic plants regenerated on both 2,4 D and dicamba containing media T ransgenic plants grew vigorously and did not show phenotypic differences compared to nontransformed controls This is the first report o f the product ion of transgenic seashore paspalum plants.

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14 CHAPTER 1 INTRODUCTION Introduction Fresh water availability is one of the many challenges facing society on a global scale. According to the UN in 2025 nearly 3 billion people will face severe water shortag es if water consumption continues at the current rate ( Montaigne 2002 ). Thus for issues like turf management it will become impractical to continue using fresh water for irrigation. Indeed when water shortages do occur, the use of fresh water for turfg rass maintenance will be a low priority (Kjelgren et al. 2000). Besides fresh water shortages, currently many land areas face salinity issues. As of a decade ago, 10 to 2 0 Mha of irrigated land lost productivity from salinity related toxicity annually ( H amdy 1996 ; Choukr Allah 1996 ). To mitigate shortages of fresh water states such as California and Arizona have passed laws requiring the use of saline sources for turfgrass irrigation (California State Wa ter Resource Control Board 1993; Arizona Department of Water Resources 1995). These sources include sewage effluent or brackish water. Emphasis on better management of fresh water use is particularly important in coastal areas. These regions face unique challenges because overused aquifers fall victim t o salt intrusion ( Parker 1975 ; Murdock 1987 ). Therefore it is not surprising that using recycled water as the primary source for irrigating golf courses in Florida has increased from only 8% in 1974 to nearly 50% in 2000 ( Haydu and Hodges 2002 ). Althoug h these practices may address issues pertaining to fresh water conservation they do not alleviate saline stresses that would result from irrigating with these recycled sources. As fresh water becomes limiting, alternative flora with adaptation to saline conditions will be needed. U sing a salt tolerant turf would reduce poor performance of turf in saline conditions. Because plants are more susceptible to salt stress under hot dry conditions than cool humid ones

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15 ( Hoffman and Rawlins 1971 ), a turfgrass show ing high levels of salt tolerance grown in tropical conditions would be economically advantageous to incorporate in areas w h ere fresh water irrigation is no longer a viable option. Seashore paspalum ( Paspalum vaginatum Swartz) is a turfgrass that has show n much promise for its excellent turf quality under saline conditions ( Duncan and Carrow 2000a ). Seashore Paspalum Seashore paspalum, Paspalum vaginatum Swartz is also known as saltwater couch, sand knotgrass, or siltgrass It is a h a lophytic warm season perennial grass highly adapted to saline, tropical environments and sandy soils ( Malcom and Laing 1969 ; Morton 1973 ; review Duncan and Carrow 2000b ). Seashore paspalum is native to coastal environments where it is able to grow directly on sand dunes adja cent to breaking waves ( Duncan 1996 ). Seashore paspalum has good potential for use in soil bioremediation in part due to its great adaptability to soil pH s ranging from 4.0 to 9.8, and its tolerance to a wide range of soil types ( Duncan 1996 ) as well as to flooding ( Malcom 1969 ; Morton 1973 ; Peacock and Dudeck 1985) and drought ( Beard et al. 1991b ). Being a warm season perennial suitable for golf, seashore paspalum is often compared to bermudagrass ( Cynodon dactylon L. ) A study comparing wear toleranc e between bermudagrass and seashore paspalum found that wear tolerance of both species can be attributed to high shoot density and leaf moisture, suggesting that turfgrass managers can maintain high levels of wear tolerance in seashore paspalum like bermud agrass by avoiding drought stress and adjusting their cultural practice to maintain a high shoot density ( Trenholm et al. 2000 ). Another study on the wear tolerance of seashore paspalum also found cultivars with high shoot densities to be equal to or bett er than tested bermudagrasses while cultivars of seashore paspalum with low shoot densities had poor wear tolerance ( Trenholm et al. 1999 ).

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16 Much work has been done on screening for salinity tolerance in seashore paspalum ( Dudeck and Peacock 1985 ; Lee et a l. 2002 ; 2004a ; 2004 b). Seashore paspalum show ed a greater salt tolerance than cultivars of bermudagrass (Lee et al. 2002). One cultivar of seashore Sea Isle 1 ( SI1 ) salinity, dark Duncan 2002 ). SI1 was selected with criteria important to golf managers looking for aggressive growth and tolerance to close mowing. It has unknown parentage from Argentina (Duncan 2 002). It is also useful for erosion control because of its ability to tolerate waterlogged conditions and occasional mesosaline flooding (Duncan 2002). A botanical description of seashore paspalum subfamily Panicoideae, includes unmowed cu lm s ranging in height from 8 to 1 5 cm with glabrous nodes. It has fine textured, distichouss leaves up to 5 0 mm in length and 2 mm in width and lacking auricles. The lamina shape is linear triangular and taper s to a narrow apex. Other features of the leaf include a pr ophyllum of 2 0 mm in length, a 1 mm ligule with a distinguishable pubescent collar, obscure venation, with smooth edges on the leaf blades. Internode lengths range from 7 to 9 mm and nodes are pubescent. The inflorescence has a digitate shape with two un ilateral short spreading racemes 20 to 2 5 mm in length including 16 to 25 twin rowed spikelets on each primary raceme The spikelets are solitary, plano convex, subsessile, elliptic, approximately 2. 5 mm in length, and 0.9 to 1. 5 mm wide. Anthers are 1.2 to 1. 4 mm in length. Glumes are glabrous. It is a creeping, mat forming, short, species having both stolons and rhizomes. Vegetative cuttings are the primary method of propagation (Duncan 2002 ; Loch and Roche 2003)

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17 Seashore paspalum is diploid (2n=2x=2 0) and highly self incompatible ( Burson 1981 ). Seashore paspalum can be found world wide, but is mostly found in the tropical and subtropic al east coast of the Americas. Initially seashore paspalum was not popular as turf due to incorrect management from high fertilizer rates which caused significant problems with thatch ( Duncan 1996 ). The best way to maintain high quality seashore paspalum plants is to avoid excess nitrogen application, irrigation in the latter part of the day, scalping, shade and area s with poor air circulation ( Trenholm and Unruh 2002 ). Some seashore paspalum cultivars have shown high quality without any additional N fertilizer a key factor in becoming a practical turfgrass for golf courses in subtropical areas (Beard et al. 1982; 1 991b; Duble 1989) Also, some cultivars of seashore paspalum tolerate low mowing heights of less than 1 3 mm ( Beard et al. 1991a ; 1991b ). There are potential advantages to using seashore paspalum in home lawns including good wear tolerance, low fertilit y requirements and the production of extensive root systems ( Trenholm and Unruh 2002 ). However this turfgrass has significant hurdles to overcome to be a success in the home landscape Shaded turf area is estimated to be at 25% in the US ( Beard 1973 ) an d seashore paspalum has poor shade tolerance ( Trenholm and Unruh 2002 ). Weed control is limited because seashore paspalum has poor tolerance to most conventional herbicides ( Trenholm and Unruh 2002 ). It also performs best at consistent mowing heights of one to two inches. It is also important to not remove more than 1/3 of the leaf blade when mowing as this can lead to scalping and leave the grass susceptible to fungal and insect problem s ( Trenholm and Unruh 2002 ). As such current cultivars of seashore paspalum do perform be st as turf in a well maintained golf course.

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18 In a study with two paspalum cultivars, two zoysiagrass ( Zoysia japonica Steud. ) cultivars, and two bermudagrass cultivars resistance to fall armyworm was greatest in zoysiagrass and wors t in the paspalum cultivars with SI1 being the most susceptible ( Braman et al. 2004 ). Fall armyworm has been found to be one of the most serious pests in corn and grasses throughout the Americas ( Ashley et al. 1989 ). Increasing resistance to fall armywor m in seashore paspalum and particularly in SI1 may make it more popular as a turf. Other targets for genetic improvement of seashore paspalum include shade ( Duncan 1996 ), cold (Ibitayo and Butler 1981) and enhanced drought tolerance as well as disease resistance. While genetic improvement by traditional plant breeding is complicated by the high self incompatibility and poor seed set of the species, g enetic diversity for both cold tolerance and a certain level of tolerance to mole crickets and fall army worm have been described ( Duncan 1996 ). Other warm season turfgrasses have also shown genetic variability in pest tolerance ( Quisenberry 1990 ; Braman et al. 1994 ; Shortman et al. 2002 ). Furthermore, in one study, a high level of variation was found in to lerance to drought for seashore paspalum suggesting that breeders can use these variations to improve cultivars ( Carrow 2005 ). G enetic transformation allows for the introduction and expression of heterologous genes from genetically distant sources with th e potential to significantly improve seashore paspalum. However, a genetic transformation protocol is not yet available for seashore paspalum. For genetic transformation to be successful protocols for in vitro tissue culture, gene delivery, selection and regeneration of transgenic plants must be developed Objectives of t he Research Given the above background, t he objectives of the research were to: 1) E nhance the regeneration response of seashore paspalum tissue culture through optimization of explant ty pe and culture media composition ;

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19 2) Use highly regenerable tissues as target s for the co transfer of a selectable marker gene and an insect resistance gene cry1Fa from Bacillus thuringiensis ; 3) Optimize a protocol for the selection of transgenic events ; and 4) Perform m olecular analysis for evaluation of successful transgene integration and expression in seashore paspalum plants.

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20 CHAPTER 2 LITERATURE REVIEW Turfgrass Tissue Culture There are 14,000 golf courses in the US and an additional 300 golf cou rses are being added annually ( Lee 1996 ). Golf managers would prefer turf that requires less management and fewer pesticide applications ( Lee 1996 ). Traditional plant breeding has made impressive progress in the development of advanced cultivars but face s the limitations given by natural hybridization barriers Transgenic technology can overcome these limitations and contribute to accelerated development of genetically improved germplasm ( Vasil 1995 ). This certainly would apply to seashore paspalum wher e progress by traditional plant breeding is limited by poor fertility and genetic improvement for insect and disease resistance is highly desirable. Since a genetic transformation protocol for seashore paspalum is not available its development was the f ocus of this research As a prerequisite for genetic transformation of grasses a protocol for efficient plant regeneration from undifferentiated tissues must be developed ( Ritala et al. 1995 ). Plant tissue culture and regeneration systems have been extend ed from major crop species to other species like turf and forage type grasses (Chai and Sticklen 1998). Progress with grasses, once considered extremely recalcitrant to tissue culture, can be attributed to important factors like genotype ( Maddock et al. 19 83 ; Krumbiegel Schroeren et al. 1984 ), donor plant quality ( Lu et al. 1984 ; Zimmy and Lrz 1989), explant type and media composition ( Eapen and Rao 1982 ; 1985 ; Lu et al. 1984 ). Regenerable tissue cultures have been developed for forages ( Chen et al. 1977 ; Lo et al. 1980 ; Baja et al. 1981 ; Songstad e t al 1983 ; Johnson and Worthington 1987 ; Metzinger et al. 1987 ; Straub et al. 1989 ; Franklin et al. 1990 ; Akashi and Adachi 1992 a ;1992 b ). However, progress in tissue culture of turfgrass has been slow because of the initial use of mature and

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21 differentiated tissues as explant s ( Vasil and Vasil 1994 ). According to Vasil (1995) there are some strategies to successfully establishing regenerable callus cultures with turfgrass that involve choosing explant tissues made of meristematic tissues and undifferentiated cells and using culture media supplemented with strong auxins. For the tissue culture phase to be successful callus induction from the explant induction of embryoids, and efficient regenerati on of normal plants must be optimal (Street 1979 ). Genotypic diversity in tissue culture response has been reported in varying cultivars of selfing cereals ( Lazer et al. 1983 ) like barley ( Hordeum v ulgare ; Bregitzer 1992 ) and wheat ( Triticum spp.; Fennell et al. 1996 ) and grasses ( Altpeter and Posselt 2000 ) For different cereals genotypic differences can affect callus initiation and plant regeneration in tissue culture ( Dornelles et al. 1997 ). In one study 25 tall fescue ( Festuca arundinacea Schreb ) cultivars we re tested and significant differences were found for callus formation ranging from 4.4% to 40.3% and for shoot regeneration ranging from 16.7% to 54.5% ( Bai and Qu 2000 ). One of the first observations of somatic embryogenesis in grass species was made usin g immature embryos of Italian ryegrass, Lolium multiflorum Lam. ( Dale 1980 ). Many subsequent studies have successfully regenerated whole plants from callus initiated with immature inflorescences (Ahn et al. 1985 ; 1987; Artunduaga et al. 1988 ; 1989 ; Chaudh ury and Qu 2000 ; Dale et al. 1981 ; Dale and Dalton 1983 ; Baja et al. 1981; Xu et al. 1984 ; Songstad et al. 1986 ; Van der Valk et al. 1989 ; George and Eapen 1990 ; Straub et al. 1992 ). Immature inflorescences have been shown repeatedly to be one of the best explant sources in grasses to generate callus. The use of immature inflorescences for callus induction has been reported for common bermudagrass (Ahn et al. 1985 ; 1987), tall fescue ( Eizenga and Dahleen 1990 ), pentaploid bermudagrass ( 2n=5X=45; Jain et al. 2005 ), perennial ryegrass ( Lolium

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22 perenne L. ; Can et al. 2004 ), Kentucky bluegrass ( Poa pra tensis L. ; Van der Valk et al. 1989 ), Chinese leymus ( Leymus chinensis ) ( Caswell et al. 2000 ), species of Paspalum (Bovo and Mroginski 1985), and in seashore paspal um genotypes ( Cardona and Duncan 1997 ). The maturity of the cultivated immature inflorescence is critical in obtaining regenerable callus with only inflorescences of specific length s and a yellowish to pale green color being effective ( Lu and Vasil 1982 ). In many instances 2,4 D ( 2,4 Dichlorophenoxyacetic acid ) is the preferred auxin for callus induction in grasses ( Bhaskaran and Smith 1990 ; Chaudhury and Qu 2000 ). Various concentrations of 2,4 D have shown positive response in embryogenic callus induc tion. Embryogenic callus was induced in buffelgrass ( Cenchrus ciliaris ) with 2,4 D ( 3 and 9 mg L 1 ; Colomba et al. 2006 ), tall fescue ( 9 mg L 1 2 ,4 D ; Bai and Qu 2000 ), and pentaploid bermudagrass ( 3 to 6 mg L 1 2, 4 D ; Jain et al. 2005 ). Therefore au xin concentration is an important factor to consider when optimizing a tissue culture protocol, but tissue can also have a different callus response based on the auxin source. There are alternative auxin sources that have been shown to be successful in th e induction of somatic embryos in turfgrass species. In a comparative study of auxin types for induction of embryogenic callus of s orghum ( Sorghum bicolor ) 2,4 D was superior followed by Naphthalene acetic acid (NAA) dicamba, picloram, and 2,4,5 T richlorophenoxyacetic acid (2,4,5 T) (Joges w ar et al. 2007). Dicamba is an alternative auxin that has been used to induce embryogenic and somatic callus in monocot tissue culture. The use of d icamba instead of 2,4 D led to much higher quality callus across four genotypes of barley ( Tiidema and Truve 2004 ). In one study on wheat, dicamba was more effective than 2,4 D in inducing embryogenic callus from cultivated mature embryos ( Filippov et al. 2006 ). Varyin g l evels of dicamba were tested with immature

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23 inflorescences f ro m perennial ryegrass and the highest generation of fresh weight was with dicamba at 7. 5 mg L 1 while the highest shoot regeneration was with 5. 0 mg L 1 dicamba ( Can et al. 2004 ) Dicamba was also better at maintaining prolonged cell cultures in barley and bahiagrass ( Paspalum notatum Flugge ) than 2,4 D ( Castillo et al. 1998 ; Altpeter and Positano 2005 ). Supplementation of callus regeneration media with cytokinins like benzylamin opurine (BAP) also typically enhances formation of shoot primordia and plant regeneration. In a study with triploid bermudagrass regeneration media lacking cytokinins had a significantly lower plant regeneration frequency than media containing cytokinins ( Lu et al. 2006 ). Similarly, i nclusion of the cytokinin BAP in the regeneration medium increased plant regeneration rates of St Augustinegrass ( Stenotaphrum secundatum ; Li et al. 2006 ). Low levels of a cytokinin in the callus induction medi a in combinat ion with an auxin enhanced the embryogenic nature and plant regeneration of callus in several grasses including ryegrass ( Altpeter and Posselt 2000 ; Bradle y et al. 2001), bermudagrass (Chaudhury and Qu 2000), barley ( Cho et al. 2000 ), tall fescue ( Bai and Qu 2001 ), and bahiagrass ( Altpeter and Positano 2005 ). The correct combination of a uxin type to cytokinin type and the appropriate auxin and cytokinin concentration must be determined to optimize a tissue culture medium (Hagio et al. 1995 ; Altpeter and Po sitano 2005 ; Jain et al. 2005 ). Species specific differences however, cannot be ignored. A recent study on buffelgrass, a warm season perennial forage grass, found that the inclusion of BAP with 2,4 D inhibited embryogenic callus growth and did not perm it plant regeneration ( Colomba et al. 2006 ). Similar findings have been found in tissue culture response of Kentucky bluegrass where 2,4 D in association with BAP led to lower frequencies of callus induction than 2,4 D alone (Ha et al. 2001 ).

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24 Other medium components also affect tissue culture response. For example, h igher levels of CuSO 4 have been shown to enhance tissue culture response in wheat ( Purnhauser 1991 ), barley ( Cho et al. 1998 ) and rye ( Popelka and Altpeter 2001 ) Long tissue culture periods generally increase the risk of producing somaclonal variants and abnormal plants ( Zaghmout and Torello 1992 ). Dicamba is thought to lead to higher rates of somaclonal variation than 2,4 D ( Bregitzer et al. 1998 ). However, s omaclonal variation can be an e ffective tool in contributing useful variations to populations that breeders could exploit Breeders have used somaclonal variation to increase disease resistance in sugarcane ( Saccharum spp.; Ramos Leal et al. 1996), and enhance drought tolerance in berm udagrass ( Lu et al. 2006 ). Somaclonal variation has also been used in an attempt to improve resistance to fall armyworm in common bermudagrass ( Croughan and Quisenberry 1989 ). However, the large number of undesirable somaclonal variation events typically outweighs the desirable events M inimizing the period in tissue culture typically also contributes to higher reproducibility of genetic transformation protocols in recalcitrant species and would lessen the frequency of random somaclonal variations ( Altpet er et al. 1996 ). Gene Transfer to Grasses The first report of a perennial transgenic grass was in 1988 using protoplast mediated gene transfer into orchardgrass Dactylis glomerata L. (Horn et al. 1988). Protoplast derived generation of transgenic plants was also successful for red fescue ( Festuca rubra L.) (Spangenberg et al. 1994) and zoysia ( Zoysia japonica Steud.) (Inokuma et al. 1998). Biolistic gene transfer (Sanford et al. 1993) is less genotype dependent than protoplast derived genetic transformati on and allows for a shorter tissue culture period which reduces the chance of somaclonal variation and increases reproducibility of the genetic transformation protocol. Consequently m any turf and forage grasses have been genetically

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25 transformed using biol istic gene transfer such as tall fescue ( Wang et al. 1992 ; Ha et al. 1992 ), creeping bentgrass ( Agrostis stolinifera L.) ( Zhong et al. 1993 ; Hartman et al. 1994 ), perennial ryegrass (Spangenberg et al. 1995 a ; Altpeter et al. 2000), red fescue ( Altpeter and Xu 2000 ), Kentucky blue grass ( Ha et al. 2001 ), bahiagrass, ( Smith et al. 2002 ; James et al. 2004 ; Gondo et al. 2005 ), bermudagrass ( Zhang et al. 2003 ; Goldman et al. 2004 ; Li and Qu 2004 ), and zoysiagrass (Qi et al. 2006). Agrobacterium mediated transfor mation is the method of choice for obtaining transgenic plants with lower copy number and stable gene expression. However, c ompared to Agrobacterium mediated gene transfer, biolistic gene transfer is more versatile in that it creates transgenic events wit h a wider range of transgene expression Biolistic gene transfer is also the preferred method for the introduction of multiple transgene expression cassettes typically required for pathway engineering or gene stacking (reviewed in Altpeter et al. 2005 ). I n the past Agrobacterium mediated gene transfer was considered not applicable for monocot transformation because monocots are not natural hosts for Agrobacterium tumefaciens ( De Cleene and Deley 1976 ). However, it was subsequently discovered that success ful Agrobacterium m ediated transformation of grasses can be achieved and the transformation frequency can be enhanced by using hyper virulent strains, virul ence gene inducing agents ( i.e. a cetosyringone ) effective means for elimination of Agrobacterium a fter cocultivation, and antioxidant s and cysteine in the co culture medium ( Hiei et al. 1994 ; Ishida et al. 1996 ; Tingay et al. 1997 ; Frame et al. 2002). Agrobacterium tumefaciens has been used successfully to transform perennial grasses like creeping ben tgrass (Yu et al. 2000), switchgrass ( Panicum virgatum L. Somleva et al. 2002), zoysiagrass (Toyama et al. 2003), tall fescue (Bettany et al.

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26 2003), perennial ryegrass ( Altpeter 2006 ), bermudagrass (Ge and Wang 2006) and orchardgrass (Lee et al. 2006). S el ectable and Scorable Markers U sed for Grass Transformation Herbicide and antibiotic selectable marker genes have been frequently used for the generation of transgenic grasses T he bar gene conferring resistance to the herbicide phosphinothricin was intr oduced into creeping bentgrass ( Hartman et al. 1994 ), tall fescue ( Dalton et al. 1995 ), zoysia (Toyama et al. 2003) and bahiagrass ( Smith et al. 2002 ). T he nptII gene was introduced into perennial ryegrass ( Altpeter et al. 2000 ), red fescue ( Altpeter and Xu 2000 ), and bahiagrass ( James et al. 2004 ) for selection of transgenic plants with the antibiotic paromomycin. The hph gene in combination with h ygromycin selection has been used widely for grass transformation and hygromycin was effective to suppress n on transgenic tissues and shoots at concentrations of 2 0 mg L 1 in orchardgrass (Horn et al. 1988) to concentrations as high as 25 0 mg L 1 f or selection in transformed tall fescue ( Wang et al. 1992 ; Ha et al. 1992 ; Dalton et al. 1995 ; Spangenberg et al. 19 95 b ). As an alternative to herbicide or antibiotic selectable marker genes an Escherichia coli phosphomannose isomerase (PMI) encoding gene was successfully used for the generation of transgenic creeping bentgrass (Fu et al. 2005) and sugarcane (Jain et al. 2007). The E. coli uidA gene encoding glucuronidase (GUS) ( Jefferson et al. 1987 ) has been used as a reporter gene for optimization of stable or transient expression in grasses and also to study expression patterns generated by different promoters ( e.g. Toyama et al. 2003 ; review: Basu et al. 2004 ; Gondo et al. 2005 ; Lee et al. 2006). The gene encoding the green fluorescent protein (GFP) is an alternative reporter which in contras t to the GUS reporter system has the advantage of allowing non destr uctive monitoring of reporter gene expression over time. It was

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27 successfully used to establish genetic transformation protocols in bentgrass (Yu et al. 2000) and perennial ryegrass ( Altpeter 2006 ). Fall A rmyworm [ Spodoptera frugiperda (J. E. Smith)] F all a rmyworm is one of the most severe pests in the southeastern US, causing significant seasonal economic losses in forage and turf grasses and other crops (Sparks 1979 ; Meagher and Nagoshi 2004). This lepidopteran pest has two morphologically identical host s trains differing in host preference and pesticide susceptibility ( Nagoshi and Meagher 2004 a ; 2004b ). The corn strain is specialized on maize and sorghum, while the rice strain is found predominately in turf grasses, forage grasses and rice ( Oryza spp.) To distinguish between the two strains, molecular methods are required ( Meagher and Gallo Meagher 2003; Nagoshi an d Meagher 2004 a ; 2004b ; Nagoshi et al. 2006). Bacillus thuringiensis Bacillus thuringiensis (Bt) a gram positive spore forming bacterium, is known to produce safe and effective insecticidal crystal proteins (ICP s ) endotoxins ( Dulmage 1981 ). Bt is a ubiquitous soil bacterium present in considerable variety at low levels world wide ( Bernhard et al. 1997 ). Bt ICP has been used for over 4 0 years in a spray form but through genetic engineering has be come more popular in modern day agriculture ( Tabashnik et al. 1998 ). The re are a number of ICPs encoded by various genes. At least 180 cry and cryt genes have been discovered ( Crickmore et al. 2000 ). The cry gene products (ICPs) are harmless to humans, f ish, wildlife, and agriculturally important insects ( McClintock et al. 1995 ). The first step to activating the protoxin (encoded by the cry gene) is ingestion by the target, a phytophagous insect with an alkaline midgut The ICP is then solubilized in th e midgut of the pest and the protoxin is cleaved by proteases into active toxin ( Gill et al. 1992 ). Midgut proteases process the protoxin into a protease resistant core fragment making this compound toxic. This toxin then

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28 passes through the peritrophic m embrane and bind s to the midgut epithelium receptors in the insect where it is effective (Hof fma n et al. 1988a ; Van Rie et al. 1990a ; Van Rie et al. 1990b ). Partial insertion of the toxin into the midgut cell occurs and this in connection with the protein bindin g l eads to pore formation, ultimately leading to cell lysis and death of the insect ( Gill et al. 1992 ; Schnepf et al. 1998 ). Although Bt ICP as a biopesticide can be a useful alternative to commercial insecticides it is limited in its field stabilit y, its ability to reach insects parasitizing cryptic parts of the plant, and its narrow spectrum of activity (Ferr and Van Rie 2002). Bt ICP s produced in transgenic plants are more effective than a topical application of Bt ICP biopesticides. B iopestici des contain more toxins on a gram per hectare basis however they are not ensured to be effective to control larvae which may occur several weeks after application or may feed inside the plant or on the roots of the plant. However, t hese larvae would be af fected immediately when consuming transgenic Bt crops ( Cannon 2000 ). Bt endotoxins genes have been introduced into several crops such as cotton, potato, soybean and corn and extensively released in the US and other countries ( James 200 6 ). Currently, Bt transgenic crops are grown on more than 1 4 million hectares worldwide (J ames 2005). In the US, Bt crops are grown on approximately 20% of the crop acreage. The use of Bt genes has reportedly led to a reduction in the use of synthet ic insecticides (Constable 1998 ; Roush 1997). Their use has been directly linked to higher yiel ds and profits (Cannon 2000). To remain effective against pests, crops need to express sufficient levels of the endotoxin to significantly reduce the fitness of the phytophagous pest. A high dose is defined as being capable of causing mortality of all het erozygous individuals that feed on the transgenic tissue (Alstad and Andow 1999). The EPA has endorsed a toxic concentration of Bt to be 25 times

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29 more concentrated than the level required to kill 99 % of the pests ( Renner 1999 ). The expression of the cry gene can be affected by where in the genome the gene construct is inserted, the environment where the plant is grown tissue age and tissue type ( Sachs et al. 1998 ; Greenplate 1999 ). Bt expression levels in transgenic plants were successfully increased b y codon optimization of cry sequences, the reduction of AT sequences and the truncation of the native cry sequence (Schnepf et al. 1998 ; Bohorova et al. 2001 ; Kaur 2006). Stacking of different cry genes (Ka ur 2006), expression of C ry fus ion constructs (B ohorova et al. 2001), and pyramiding genes including cry genes with genes encoding proteins having alternative insect control mechanisms like vegetative insecticidal proteins or proteinase inhibitors is hypothesized to reduce the risk of i nsects developin g resistance to Bt toxins (Ferry et al. 2006). In 1985 the first development of resistance to Bt ICP was reported ( McGaughey 1985 ) followed by additional reports ( Bauer 1995 ; Ferr et al. 1995 ; Frutos et al. 1999 ; Schnepf et al. 1998 ; Tabashnik 1994 ; Van Rie and Ferr 2000). Interestingly d iamondback moth ( Plutella xylostella L.) has developed resistance to four Bt toxins ( Tabashnik et al. 1997b ). Therefore there is a possibility that pests can become resistant to a range of Bt endotoxins and thus measu res must be taken to reduce resistance acquisition in these pests. Resistance management should rely on four key strategies, the diversification of lethal sources, the use of refuges to reduce selection pressure, prediction and monitoring of resistance, a nd policy implementation ( Whalon and Norris 1996 ). Maintaining non Bt ICP crops in the retaining zone ensures a population of non resistant pests and should slow down the evolution of resistance to Bt crops ( Roush 1996 ). To date, three biochemical mechanis ms for resistance have been found in resistant populations ranging from proteolytic processing of protoxins to repair of damaged midgut cells

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30 and modification of affected binding sites (Ferr and Van Rie 2002). Unfortunately there is some overlap and occa sionally resistance to one Cry protein will lead to resistance to several other cry gene products ( Tabashnik et al. 1997a ; 1997b ). Still, there are many cry genes coding for different Cry proteins and although some insects have shown a buildup of resistan ce to certain cry genes other cry genes oftentimes remain effective ( Mller Cohn et al. 1996 ; Tabashnik et al. 1997b ). In most reported cases resistance has been unstable (Ferr and Van Rie 2002). In several studies selection has been removed a fter re sistance was detected in a population and in most instances resistance was lost likely due to fitness costs (Tabashnik et al. 1994, Ferr and Van Rie 2002). Extensive high dose feeding studies and field studies have shown no adverse affect to non targets such as adult and larval honeybees, ladybird beetles, collembolan, parasitic wasps, and lacewigs ( Armstrong et al. 2000 ; Sims and Martin 1997 ). The EPA has also concluded that tested Cry proteins are nontoxic to mammals ( EPA 1998a ; EPA 1998b ). Receptors l ike those found in insects affected by Cry proteins have not been found in mammalian species (Hof fman et al. 1988b ; Sacchi et al. 1986 ). Furthermore, Cry proteins are also known to degrade rapidly under natural conditions ( Palm et al. 1993 ; Palm et al. 199 4 ; Palm et al. 1996 ). Therefore, these Cry proteins do not adversely affect non target pests, beneficial insects addressed or other animals. Currently, marketed Bt ICP products include Bt corn containing the cry1Ab cry1Fa cry3Bb1 and stacked cry1Ab and c ry3Bb1 genes for controlling European corn borer [ Ostrinia nubilalis Hbner], southwestern corn borer [ Diatraea grandiosella Dyar] and corn rootworm [Diabrotica barberi Smith and Lawrence], and Bt cotton containing cry1Ac stacked cry1Ac and cry2Ab2, stack ed cry1Ac and cry1Fa for controlling tobacco budworm [ Heliothis virescens

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31 Fabricius], cotton bollworm [ Helicoverpa zea Boddie], and pink bollworm [ Pectinophora gossypiella Saunders] ( Castle et al. 200 6 ). Cry1Fa has been reported to control fall armyworm in cotton ( Adamczyk and Gore 2004) and bahiagrass (Luciani et al. 2007). Hence, t he overall goal of this work was to develop a genetic transformation protocol for seashore paspalum to evaluate the expression of a synthetic Cry 1Fa gene in transgenic seashore paspalum and its effect on resistance to fall armyworm.

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32 CHAPTER 3 MATERIALS AND METHOD S Development of a Tissue Culture Protocol for Se a shore Paspalum Plant M aterial Seashore paspalum cultivar SI1 was obtained from Turfgr ass America, Houston, TX. Plant material was propagated in Fafard #2 soil mix (Fafard, Inc., Apopka, FL) in an airconditioned greenhouse at 30 C day / 25 C night temperature. The photoperiod was extended to a 1 6 h day length using 100 0 w att sodium vapor lights. Miracle Gro Bloom Booste r (Scotts Miracle Gro Products Inc, Marysville, OH) was applied biweekly at the recommended rate to enhance flowering. Automatic, daily irrigation was achieved with an ebb and flow irrigation system. Preparation of E xplants Shoots with i mmature infloresce nce s were collected at the end of June 2006. Excess leaf material was removed before surface sterilization with 70% ethanol for 4 min followed by a 10% sodium hypochlorite solution with 0.01% T ween 20 for 2 0 min. After sterilization, tissue was rinsed five times with sterile distilled water with the last two rinses including a 1 0 min soaking period. I mmature inflorescences were then excised with a sterile scalpel in aseptic conditions and inflorescences of 1 cm in length were cut into five 0. 2 cm segme nts and placed onto the culture medi um I nflorescence segments were numbered 1 to 5, with 1 being the apex of the inflorescence and 5 being the base just above the immature peduncle Experimental D esign The experimental design consisted of four randomized blocks, each represented by six media treatments. Each replication was represented by 10 inflorescences cut into 50 segments and cultured on five plates with the same medium. In total 240 immature inflorescences were

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33 cut into 1200 segments before cultu re initiation. Callus was maintained at 25C in a n incubator ( Percival I36LLVL, Percival Scientific Inc, Perry IA ) with 3 0 E m 2 s 1 light and 1 6 h light / 8 h dark photoperiod. The six treatments included one of the two auxin types (2,4 D or d icamba) at 3 mg L 1 with the cytokinin BAP at 0, 0.1 or 1. 0 mg L 1 Callus I nduction Callus induction media components (PhytoTechnology Laboratories, Shawnee Mission, KS,) consisted of 4. 3 g L 1 MS salts ( Murashige and Skoog 1962 ) supplemented with 12.4 5 mg L 1 C uSO4 and 2 0 g L 1 sucrose. Before the addition of 3 g L 1 phytagel, the pH was adjusted to 5.8. Media was autoclaved at 121C and 12 0 kPa for 2 0 min. MS vitamins ( Murashige and Skoog 1962 ), auxins and BAP were added from concentrated and filter steriliz ed stock solution s after autoclaving. Callus was sub cultured weekly and callus induction was recorded weekly for 5 6 d. The time of c allus induction was determined to be when undifferentiated cells were observed on the inflorescence segment. Callus f res h weight was measured 1 0 wk after culture initiation. Plant R egeneration Two wk after assessing callus fresh weight, callus was transferred to plant regeneration media under 15 0 Em 2 s 1 light at 25C with a 1 6 h light / 8 h dark photoperiod. Composition of regeneration media was the same as the callus induction media, except that neither auxin nor cytokinin were included. N umber of shoots was determined at 0, 48, and 9 4 d after transfer to regeneration medium. Statistical A nalysis Statistical analysis wa s performed according to the randomization structure using the GLM procedure of SAS version 9.1 ( SAS Institute Inc. 2005). Means were compared by the t test (LSD, p< 0.05).

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34 Transient Transformation of Sea Isle 1 C allus by Biolistic Gene Transfer Plant P ro pagation and Preparation of Explants Seashore paspalum cultivar SI1 plant propagation and preparation of explant s were all done as described earlier in the chapter : The Development of a Tissue Culture Protocol for Seashore Paspalum Tissue Culture C omposit ion Callus induction media w ere made as described earlier A uxins and BAP were added as concentrated and filter sterilized stock solution after autoclaving. Sorbitol media for osmotic pre bombardment treatment was made by the addition to the respective ex perimental media of 72.8 5 mg L 1 of sorbitol before sterilization. Transient Reporter Gene Expression Callus initiated from four randomized blocks with all six treatments of media (2,4 D or dicamba with 0, 0.1 or 1. 0 mg L 1 BAP) was used for bombardment 1 5 wk after callus initiation to determine biolistic bombardment efficiency with a GUS reporter gene assay (Jefferson 1987). Non embryogenic or necrotic callus was not included in the experiment. The vector pUbiGUS (Fig. 3 1 ) containing the maize u biquitin promoter with first intron (Christensen et al. 1992), the uidA gene (Novel and Novel 1973), and the n os terminator ( Bevan 1984 ; Fraley et al. 1983 ) was u sed for bombardment. DNA gold solutions were made with 1m gold (BioRad Laboratories, Hercules, CA) co ated with 1 g of p UbiGUS following the protocol described by Somers et al. ( 1992). To generate four independent replications of each treatment, plasmid was precipitated on gold particles in four independent reactions. Each reaction was used for six shot s, one shot for callus on each media treatment resulting in a total of 24 shots for all four replications. Four h before bombardment callus was placed within a circle of 1 8 mm in diameter on its respective culture

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35 media supplemented with the addition of sorbitol evenly and without gaps between callus pieces. Using a PDS 1000/He Particle Delivery System (Biorad) callus was placed 6 cm below a 110 0 once 110 0 psi was r eached After bombardment callus was transferred to callus induction medium and propagated an additional 4 d under the same conditions it was cultivated on before exposure to GUS substrate (Jefferson et al 1987). Immediately after immersing callus in the GUS assay substrate the callus 0 min Callus exposed to substrate was then incubated for 1 6 h at 37 C in the dark. After incubation GUS transient expression was determined by counting the number of blue foci obser ved using a S4E (Leica Microsystems) dissecting scope. Each blue focus was interpreted as an individual transformation event ( Fig. 4 6 4 7 ). Stable Genetic Transformation of Seashore Paspalum by Biolistic Gene Transfer Tissue Culture Composition Callus i nduction media w ere made as described earlier Sorbitol media for osmotic pre bombardment treatment w ere made with the addition to the respective experimental media of 72.8 5 mg L 1 of sorbitol before sterilization in an autoclave. Callus selection media w ere identical to callus induction media except for being supplemented with 3 0 mg L 1 hygromycin added as concentrated and filter sterilized stock solution after autoclaving and 6 mg L 1 agarose. Regeneration selection media were identical to callus induct ion media except that they lacked auxins and cytokinins and were supplemented with 6 mg L 1 agarose and hygromycin at either 10 or 2 0 mg L 1 For the first two months on regeneration media hygromycin was added at concentrations of 2 0 mg L 1 followed by a reduction of the hygromycin concentration to 1 0 mg L 1

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36 Stable Genetic T ransformation Callus was induced on six different media (2,4 D or dicamba with 0, 0.1 or 1. 0 mg L 1 BAP) and bombarded 1 5 wk after callus initiation to evaluate stable transformation using the hph selectable marker gene. Non embryogenic or necrotic callus was not included in the experiment. SI1 callus was arranged within a circle of 2 5 mm in diameter on callus induction medium containing sorbitol and bombarded with microprojectiles coated with linearized phph c ry1Fa plasmid, containing a constitutive c ry1Fa cassette linked to a constitutive hph selectable marker expression cassette (Fig. 3 2 ). A total of 114 P etri dishes of SI1 callus were bombarded From media containing dicamba an d BAP (0, 0.1 or 1. 0 mg L 1 ) 9, 23, and 25 P etri dishes were bombarded respectively, while from media containing 2,4 D and BAP (0, 0.1 or 1. 0 mg L 1 ) 16, 22, and 19 petri dishes were bombarded respectively. Selection schemes were consistent for the dif ferent media treatments. Callus bombarded with hph was allowed to recover after bombardment on its respective media without any selection pressure for seven d and then transferred to selection media containing 3 0 mg L 1 hygromycin and the same composition as the callus induction medium. Calli were subcultured on selection media every two wk for a period of three months with 3 0 E m 2 s 1 light and 1 6 h light / 8 h dark photoperiod and 25 C. Then callus was transferred every three wk to media without growt h regulators and 2 0 mg L 1 hygromycin with 15 0 Em 2 s 1 illumination and a 1 6 h light / 8 h dark photoperiod at 25 C. Two months later the hygromycin concentration was reduced to 1 0 mg L 1 hygromycin. After two to three subcultures on this selection medi um regenerated plants were transferred to soil and grown under the same greenhouse conditions as the original explant material described.

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37 Evaluation of c ry1Fa I ntegration and E xpression Genomic DNA was extracted using the CTAB method (Doyle and Doyle 1987 ; as modified by Cullings 1992). After extraction DNA concentration was measured with a ND 1000 spectrometer (NanoDrop technologies, Wilmington, DE) and concentrations were adjusted to 5 0 ng l 1 for the polymerase chain reaction ( PCR ) PCR was carried out using the PCR core system II (Promega; Madison, WI) in an iCycler thermocyler (BioRad) Using HotStarTaq DNA Polymerase (Qiagen) the putative transgenic DNA samples (5 0 ng) non transformed SI1 negative control DNA (5 0 ng) negative PCR control (no DNA ) and a positive con trol ( 5 0 pg, phph c ry1Fa) were used for PCR All samples were mixed well by tapping, and then spun briefly ( 5 s) in a table top centrifuge before undergoing PCR The PCR program was first set for 1 5 min at 95 C to activate the HotStar Taq DNA polymerase. To amplify hph CCC GAT ATG AAA AAG CCT GA GAT GTT GGC GAC CTC GTA TT were used resulting in a PCR fragment of 889 bases Thirty cycles were used with 1 min at 95 C, 1 min at 48 C, and 1 m in at 72 C. After the 30 th cycle the samples were held at 72 C for 5 min and then held at 4C until use To amplify c ry1Fa in phph c ry1Fa the same procedure was done with a different forward primer of CCG GGA CCA TTG ACT CTC TA CAC TTC GTT GCC TGA ACT GA 3 resulting in a PCR fragment of 581 bases. PCR amplification was verified by electrophoresis with a 0.8% agarose gel. Immunochromatography Testing for Cry1Fa was done with the commercially available immunochromatographic Cry1F a QuickStix test ( EnviroLogix; 500 Riverside industrial parkway, Portland, ME 04103) To conduct the immunochromatographic Cry1F a QuickStix test, four 3 cm segments of leaf tissue were ground with a sterile micro pestle in a 1. 5 mL eppendorf tube with 12 5 L of protein

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3 8 extraction buffer, provided by the manufacturer. After centrifugation for 5 s with a table top centrifuge the supernatant was transferred to a fresh eppendorf tube and the QuickStix strip was then placed into the protein extract solution. Approximately 1 0 min later results were visible (Figure 4 9 ) Statistical A nalysis Statistical analysis for the GUS assay was performed according to the randomization structure using the GLM procedure of SAS version 9.1 (SAS I nstitute Inc. 2005). Means were compared by the t test (LSD, p < 0.05). Figure 3 1. The pUbiGUS expression cassette Plasmid map showing the uidA (GUS) gene, the ubiquitin promoter, the nos poly(A) region Figure 3 2. The pHZ ubicry1Fa expression c assette Plasmid map showing the synthetic cry1Fa gene, the constitutive promoter (CaMV35S), the ubiquitin promoter, the nos poly (A) region the hygromycin phosphotransferase gene (hph) for hygromycin resistance

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39 CHAPTER 4 RESULTS Development of a Tiss ue Culture Protocol for Seashore Paspalum Callus Induction P reliminary experiments confirmed that immature inflorescences of a size of 1 cm were best suited for induction of embryogenic, regenerable callus. While all segments of these immature inflorescen ces were able to induce callus, the callus induction frequency differed significantly by segment position in the inflorescence ( Table 4 1 ). Twenty one d after culture initiation 60% of the cultured basal inflorescence segments and 35% of the cultured api cal inflorescence segments had formed embryogenic callus ( Fig. 4 2 ). The auxin type showed significant effects for all evaluated parameters. Callus induction percentages were calculated by averaging the rate of callus induction per inflorescence across al l five segments of the original immature inflorescence. Significant auxin effects on callus induction were recorded after 1 4 d of culture initiation ( Table 4 2 ) with callus induction being highest on 2,4 D containing media ( Table 4 2, 4 3 ). At 2 1 d after culture initiation 2,4 D induced callus from 54% of the cultured inflorescences, while dicamba supported callus induction from 42% of the cultured inflorescences ( Table 4 3 ). The different BAP concentrations had no significant effect on callus induction ( Fig. 4 1, 4 2 ). Callus Fresh Weight C allus fresh weight gain was significantly higher only for segment 2 (31.7 7. 3 mg) which was not significantly different than the fresh weight gained for segment 1 (27. 3 7. 3 mg) despite the lower callus induction fr equency. Average fresh weight was lowest for segment 4 at an average of 22. 3 7. 3 mg of callus generated ( Table 4 1 ).

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40 Auxins tested had a clear significant effect on callus fresh weight gain Dicamba was significantly higher with 92. 5 mg of average we ight gained per inflorescence compared to 40. 4 mg of average weight gained per inflorescence on 2,4 D ( Fig. 4 2 ). Auxin and cytokinin interaction also led to significant differences in the accumulated callus fresh weight. Callus generated on dicamba with 0 and 0. 1 mg L 1 BAP had significantly higher average callus fresh weight per inflorescence than all callus induced on media with 2,4 D regardless of BAP concentration. However callus induced on dicamba with 1. 0 mg L 1 BAP had a fresh weight average that was not significantly different than callus generated on 2,4 D with 0. 1 mg L 1 BAP. Callus generated on dicamba with 0. 1 mg L 1 BAP average fresh weight of induced callus per inflorescence was 102. 0 mg while that of callus generated on 2,4 D with no BAP was only 23. 6 mg ( Fig. 4 2 ). Shoot Regeneration After callus induction on dicamba containing media, call us rapidly produced a large number of shoot primordia. In contrast, calli initiated on 2,4 D containing media remained mostly undifferentiated at 4 8 d on regeneration media (Fig. 4 5 ) The formation of shoot primordia was more pronounced on media supplemented with BAP ( Fig. 4 2 ). After a cultivation period of 4 8 d on regeneration media, callus generated on dicamba had an average of 336.6 shoots per in florescence while callus generated on 2,4 D had an average of only 35.3 shoots per inflorescence, a highly significant difference ( Pr > F <0.0001 ; Fig. 4 1, 4 2 ). The effect of cytokinins on shoot formation was less drastic but still significant ( ; Fig. 4 1, 4 2 ). While calli induced on BAP free media produced on average 133.7 shoots per inflorescence, those induced on 0.1or 1. 0 mg L 1 BAP produced 207.1 or 223.2 shoots per inflorescence respectively ( Fig. 4 2 ). A significant auxin type BAP concentration interaction was also observed (Pr>F <0.0659; Fig. 4 1) Addition of BAP to 2,4 D containing media had no

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41 significant effect on shoot regeneration compared to 2,4 D containing media without BAP. However, shoot regeneration almost doubled when 0.1or 1. 0 mg L 1 BAP was added to dicamba containing media, compared to dicamba containing media without BAP ( Fig. 4 2 ). Auxin type and BAP concentration did not significantly affect the proportion of cultured inflorescences that had the ability to r egenerate at least one shoot ( Fig. 4 1, 4 2 ). Transient Expression of uidA in S eashore Paspalum Callus Transient expression was found to be significantly higher when using 2,4 D with average expression rates of 334.9 2 155.9 blue foci, while dicamba had an average rate of 49.1 7 22.5 blue foci BAP levels also had a significant effect on transient GUS expression. Average transient expression was highest in callus originating from media with 2,4 D and 1. 0 mg L 1 BAP ( Fig. 4 6 4 7 ). The next transient expression level was observed in calli derived from media with 0.1 or 0. 0 mg L 1 BAP and 2,4 D While all callus derived from dicamba with (0, 0.1, 1. 0 mg L 1 ) show ed significa ntly lower transient expression ( Fig. 4 6 ). Stable T ransformation S tabl e trans formed lines had vigorous shoot and root growth while on selection medi um (Fig. 4 8 ). T ransgenics were recovered showing a strong Cry1F a specific signal in the immunochromatographic analysis suggesting high level expression of c ry1F a (Fig. 4 9 ) Four Cr y1F a expressin g l ine s w ere generated one from callus induction media originally containing 2,4 D with 0 mg L 1 BAP and one from callus induction media originally containing dicamba with 0. 1 mg L 1 BAP. Two transgenic lines were generated from media origi nally containing 2,4 D with 0.1 mg L 1 There were 214 inflorescences used to induce callus for this experiment with callus generated on dicamba BAP (0, 0.1, 1. 0 mg L 1 ) originating from 19, 40, and 41 inflorescences respectively, while callus originating on media with 2,4 D BAP (0, 0.1, 1. 0 mg L 1 ) originating from 39, 38, and 37 inflorescences respectively. Callus initiated on

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42 dicamba media had a 1.75% and 1% transformation efficiency from bombarded P etri dish and cultured immature inflorescences respectively. Callus initiated on 2,4 D media had a 5.26% and 2.63% transformation efficiency from bombarded P etri dish and cultured immature inflo rescences respectively (Table 4 4) Transgenic plants did not show any phenotypic difference from wildtype (Fig 4 5 ) PCR confirmed the presence of the c ry1Fa gene in genomic DNA extract s of transgenic lines (Fig. 4 10 ).

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43 Table 4 1. Callus induction an d regeneration in response to the original position of immature inflorescence segments Induction of callus (%) a Callus fresh weight (mg) b Number of shoots per segment c Segment 1 34.9 C 27.3 AB 33.3 C Segment 2 46.6 B 31.7 A 101.2 A Segment 3 52.9 AB 24.5 B 7 5.9 AB Segment 4 48.9 B 22.3 B 60.4 BC Segment 5 60.2 A 22.9 B 47.5 BC a = 2 1 d after initial platting on callus induction media. b = 1 0 wk after initial platting on callus induction media. c = 4 8 d after platting of callus onto regeneration media. Same lette rs in a column Immature inflorescences 1c m in size were cut into 5 mm segments. The segments are numbered in order with the apical segment is segment 1 the basal segment is segment 5.

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44 Table 4 2 C allus ind uction and regeneration as influenced by the immature inflorescence segments, auxin type, BAP concentration, and interaction of auxin type BAP concentration. Induction of callus in % of cultured inflorescences after culture initiation Callus fresh weig ht (mg) per inflorescence a Number o f shoots per inflorescence b % of cultured inflorescences regenerating shoots c 7 d 1 4 d 2 1 d Segment (df = 4) MS 0.9 2.3 1.9 1777.7 23595.6 F Value 6.3 10.8 *** 9.4 *** 2.2 ns 3.57 ** P Value <0.0001 <0.00 01 <0.0001 0.0664 0.0070 Treatment (df=5) MS 2.5 5.4 5.1 37748.3 54112.9 F Value 1.4 ns 2.0 ns 1.8 ns 7.1 *** 19.1 *** P Value 0.2179 0.0843 0.1059 <0.0001 <0.0001 [A] Auxin (df=1) MS 3.6 21.5 20.9 162921.9 236464.9 F Value 1.8 ns 6.7 ** 5.7 25.2 *** 80.2 ** P Value 0.1798 0.0103 0.0177 <0.0001 <0.0001 [C] Cytokinin (df=2) MS 1.7 1.8 1.7 8732.6 9732.6 F Value 0.9 ns 0.6 ns 0.5 ns 1.4 ns 3.3 P Value 0.4132 0.5728 0.6319 0.2609 0.0414 A C (df=2) MS 3.1 0.8 0.6 417 7.2 8269.7 0.8 F Value 1.6 ns 0.3 ns 0.2 ns 0.7 ns 2.8 ns 5.7 ** P Value 0.2124 0.7824 0.8530 0.5248 0.0659 0.0048 a = 1 0 wk after originally platting callus on callus induction media. b and c = 4 8 d after transferring callus to regeneration media. *,**,*** indicate sig were cut into 5 mm segments. The most apical segment is segment 1 the most basal segment is segment 5. ns = non significant

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45 Table 4 3 Callus ind uction and regeneration as influenced by auxin type BAP concentration and interaction of auxin type and BAP concentration Induction of callus in % of cultured inflorescences after culture initiation Callus fresh weight (mg) per inflorescence a Number of shoots per inflorescence b % of cultured inflorescences regenerating shoots c 7 d 1 4 d 21 d Dicamba 20.0 A 34.0 B 42.0 B 92.5 A 336.6 A 87.5 A 2,4 D 16.0 A 46.0 A 54.0 A 40.4 B 35.3 B 73.9 A BAP 0 mg L 1 16.0 A 40.0 A 50.0 A 57.7 A 133.7 B 73.3 A BAP 0. 1 mg L 1 2 0.0 A 44.0 A 50.0 A 78.0 A 207.1 AB 87.5 A BAP 1. 0 mg L 1 22.0 A 36.0 A 44.0 A 63.6 A 223.2 A 81.3 A Dicamba BAP 0 mg L 1 18.0 AB 34.5 B 45.0 AB 91.8 A 221.3 B 100.0 A Dicamba BAP 0. 1 mg L 1 26.7 A 35.0 B 43.5 AB 102.0 A 383.0 A 100.0 A Dicamba BAP 1. 0 mg L 1 20.5 AB 33. 0 B 38.5 B 83.7 AB 405.5 A 62.5 B 2,4 D BAP 0 mg L 1 14.0 B 46.5 AB 52.8 AB 23.6 C 33.7 C 85.7 AB 2,4 D BAP 0. 1 mg L 1 13.5 B 51.0 A 58.0 A 54.0 BC 31.2 C 75.0 B 2,4 D BAP 1. 0 mg L 1 23.0 AB 41.0 AB 51.6 AB 43.5 C 40.9 C 62.5 B a = 1 0 wk after originally platting callu s on callus induction media. b and c = 4 8 d after transferring callus to regeneration media. Same letters in a column

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46 Table 4 4 Transformation efficiencies Number of bombarded Petri Dishes Number of cultured immature inflorescences Transgenic events Efficiency of gene transfer. Transgenic lines in % of bombarded Petri Dishes cultured inflorescences Dicamba 57 100 1 1.75 1 2,4 D 57 114 3 5.26 2.63 BAP 0 mg L 1 25 58 1 4 1.72 BAP 0. 1 mg L 1 45 78 3 6.67 3.85 BAP 1. 0 mg L 1 44 78 0 0 0 Dicamba BAP 0 mg L 1 9 19 0 0 0 Dicamba BAP 0. 1 mg L 1 23 40 1 4.35 2.5 Dicamba BAP 1. 0 mg L 1 25 41 0 0 0 2,4 D BAP 0 mg L 1 16 39 1 6.25 2.56 2,4 D BAP 0. 1 mg L 1 22 38 2 9.09 5.26 2,4 D BAP 1. 0 mg L 1 19 37 0 0 0

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47 Figures 4 5 Seashore paspalum response to composition of tissue culture media (a) Sterile immature inflorescence of SI1 ( 1 cm in length). ( b ) S egments from i mmature inflorescence ( 1 cm in size) 7 d after culture initiation on callus induction medium. (c) Callus 1 5 wk after culture of inflorescence segments on different media, from left to right 2,4 D (0; 0.1; 1. 0 mg L 1 BAP) and dicamba (0; 0.1; 1. 0 mg L 1 BAP). ( d ) Regeneration of callus following initiation on medium supplemented with 2,4 D ( 3 mg L 1 ) and BAP (0. 1 mg L 1 ) and after being cultivated on hormone free shoot regeneration media for 4 8 d. ( e ) Regeneration of callus following initiation on medium supplemented with dicamba ( 3 mg L 1 ) and BAP (0. 1 mg L 1 ) after being cultivated on hormone free shoot regeneration media for 4 8 d. (f) On the left, a SI1 transgenic plant regenerated from callus induction media with 2,4 D 3 mg L 1 with no BAP and on the right, a SI1 1 non transformed plant bar equal to 1 cm

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48 Figure 4 6 Transient GUS gene ex pression in callus induced on different culture media and following biolistic transfer of the uidA gene (a) Average number of blue foci resulting from callus induced on six different media F our petri dishes with callus placed in ring of 1 8 mm in diamete r were bombarded from each media (b) Average transient expression of blue foci from callus induced on 2,4D or dicamba containing media. Twelve petri dishes with callus placed in ring of 1 8 mm in diameter were bombarded from each media

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49 Figure 4 7 Tr ansient GUS expression in seashore paspalum callus Transient GUS expression on c allus induced on media supplemented with 2,4 D and BAP 1. 0 mg L 1

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50 Figure 4 8 T ransgenic seashore paspalum plants co transformed with cy1Fa an d hph selected on callus induction media with 3 0 mg L 1 hygromycin and regenerating on media containing 1 0 mg L 1 hygromycin. (a ) I nduced on callus induction media with dicamba ( 3 mg L 1 ) and BAP (0. 1 mg L 1 ). (b ) I nduced on callus induction media with 2 ,4 D ( 3 mg L 1 ) and no BAP.

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51 Figure 4 9 The Cry1Fa protein expression detected by the i mmunochromatographic Cry1Fa QuickStix Test Samples were taken from SI1 Wt and SI1 lines that had gone through a tissue culture phase involving the induction of cal lus, transformat i o n with the cry1Fa and hph gene, callus subculture under selection and shoot regeneration under selection with 3 0 mg L 1 hygromycin and 1 0 mg L 1 hygromycin used for selection respectively The lines represented above were induced under di f ferent tissue culture media describe as follows from left to right : 2,4 D and no BAP, 2,4 D and 0 1 mg L 1 BAP, dicamba and 0. 1 mg L 1 BAP ( d) 2,4 D and 0 .1 mg L 1 BAP The white arrow points to the line represent ing the control line indicat e the function al integrity of the strip W hile the black arrow points to the line indicat ing the presence of the Cry1Fa endotoxin in the sample.

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52 Figure 4 10 The PCR of genomic DNA from seashore paspalum lines suggesting the presence of Ubic ry1Fa 1 kb ladder with the lowest band being equal to 500 base pairs the positive control which is an amplification of 5 0 pg of phph c ry1Fa with fragment size of 581 base pairs Lanes 1, and 2 indicate the presence of the c ry1F gene in confirmed c ry1F a transgenic bahiagrass ( Paspalum notatum ) used as a positive control plant in this assay Lane 3 represents a PCR amplification product from genomic DNA of transgenic seashore paspalum SI1. WT and NC refer to SI1 wildtype, and buffer control respectivel y.

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53 CHAPTER 5 DISCUSSION AND CONCLUSION S Developing a Tissue Culture Protocol Type and concentration of plant growth regulators like auxins and cytokinins are critical components in callus induction and plant regeneration. For the generation of regenerabl e callus from seashore paspalum 2,4 D in combination with BAP was used earlier (Cardona and Duncan 1997) However, a comparison of different auxin types was not reported. Here it is shown that the use of dicamba drastically increases plant regeneration f requency from immature inflorescence derived callus of the commercially important seashore paspalum cultivar SI1. Compared to an equimolar application of 2,4 D, dicamba increased callus average fresh weight two times and the number of average regenerated plants per explant 10 times. T he use of dicamba was also identified as the most important factor in optimizing the tissue culture and stable genetic transformation response of barley (Trifonova et al. 2001; Castillo et al. 1998). Dicamba was also superio r to 2,4 D in generation of regenerable callus in rye ( Zimmy and Lrz 1989). However, 2,4 D was superior to dicamba for induction of embryogenic callus in pentaploid bermudagrass, sorghum and millets (Jain et al. 2005; Rao et al. 1995; Jogeswar et al. 200 7; Kavi Kishor et al. 1992). Seashore paspalum plants regenerated faster from dicamba containing media and grew more vigorously when transferred to soil, similar to nontissue cultured plants Different auxin types might have a different impact on the freq uency of undesirable somaclonal variation (Deambrogio and Dale 1980; Murata 1989). A reduction in the tissue culture period has been reported as an effective means to reduce somaclonal variation in other grasses (Altpeter et al. 1996; 2000).

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54 Preliminary e xperiments also show the size of the inflorescence as a critical factor in induction of highly regenerable callus. This has been shown in other grasses (Cai and Butler 1990; Lu and Vasil 1982; Qu and Chaudhury 2001; Poeaim et al. 2005). However, when seg menting immature inflorescences it was not clear which effect the original position of the segment had on callus quality. Although the segment below the ap ical segment produced three times more shoots than the apical segment and two times more shoots than the basal segment it seems reasonable to include all segments of the immature inflorescence for callus induction due to the limited seasonal availability of immature inflorescence and the cumbersome process involved in surface sterilizing, and isolating t hese explants While BAP did not have a significant effect on callus induction or callus fresh weight both 0.1or 1. 0 mg L 1 BAP increased the number of shoots per inflorescence 1.7 or 1.8 fold respectively. Surprisingly, this BAP effect was only signi ficant for callus induction media containing dicamba but not for media containing 2,4 D. Earlier reports described that the cytokinin BAP has a positive impact on the induction and maintenance of embryogenic callus from grasses (Altpeter and Posselt 2000 ; Chaudhury and Qu 2000; Cho et al. 2000; Bradley et al. 2001). These earlier reports did not describe a significant auxin cytokinin interaction. In tissue culture with pentaploid bermudagrass Jain et al. (2005) found that using 2,4 D and BAP to be th e only auxin / cytokinin combination effective for callus induction, growth, and establishment of regenerable callus; and not dicamba or IAA with any combination of BAP. This is suggestive of an important auxin cytokinin interaction depend e nt on the genot ype being induced to form callus. Transient Transformation of Seashore Paspalum The E. coli glucuronidase (GUS) encoding gene uidA (Jefferson et al. 1987) has been extensively used in earlier studies as a reporter gene for optimization of stable and tran sient

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55 expression in grasses and also to study expression patterns of different promoters (Toyama et al. 2003; review: Basu et al. 2004; Gondo et al. 2005; Lee et al. 2006). S eashore paspalum callus initiated and maintained on 2,4 D media produced six time s more transient GUS expression events than callus initiated on dicamba media. However transient reporter systems may not translate directly to stable transgene expression There is a positive correlation between the amount of particles used in biolistic gene transfer and transient expression rates (Chibbar et al 1991; Finer et al. 1992). However p lant regeneration potential may become limiting and can be partially compromised by tissue damage caused by particle mediated gene transfer ( Popelka et al. 20 03 ; Castillo et al. 1994) For example, a study in apple cultivars found very high transient transformation events but low stable transformation events, as transformed callus were not able to form leaf primordia (Maximova et al. 1998). Similar findings have been reported in wheat (Becker 1994) and barley (Hard wood et al. 2000), where higher gold concentrations led to greater tissue damage and less stable transformation events. Interestingly in the present study dicamba supported significantly higher sh oot regeneration while 2,4 D supported significantly higher transient reporter gene expression. Therefore, these results need to be followed up with stable transformation experiment s to clarify which factor has a greater influence on stable integration of transgenes in seashore paspalum Stable Genetic Transformation of Seashore Paspalum In order to maximize stable transformants several parameters need to be optimized. One critical aspect in generating transgenic plants is that a selection scheme suppre sses non transgenic events from callus propagation and plant regeneration (Wang and Ge 2006). Species, genotypic and tissue specific difference were observed regarding tolerance to antibiotics that are used in the selection of transgenic events (Lee 1996 ). Hygromycin has been used effectively at concentrations of 2 0 mg L 1 in protoplast derived cell cultures of orchardgrass

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56 (Horn et al. 1988) and has been used in concentrations as high as 25 0 mg L 1 for selection in transformed callus of tall fescue (Wan g et al. 1992; Ha et al. 1992; Spangenberg et al. 1995a; Spangenberg et al. 1995b; Dalton et al. 1995). The selection scheme used in the present study with 3 0 mg L 1 hygromycin created a relative low selection pressure and allowed the regeneration of nont ransgenic escape plants (94% escapes) Despite the low selection pressure one stable transgenic plant per 2 9 shots or one transgenic plant per 5 6 cultured inflorescences was obtained Although a more rigorous selection scheme using a higher concentratio n of hygromycin could have been implemented to reduce the number of escapes, in doing so there is a risk of reducing the number of transgenic plants. S everal parameters such as microcarrier concentrations, particle acceleration alternat ive promoters and selection protcols should be investigated to achieve higher transformation efficienc ies Alternative g e notypes of seashore paspalum are available and genotypic differences can contribute to significant improveme nt of transformation efficiency (Popelka an d Altpeter 2001 ). Biolistic gene transfer could be improved by optimized procedures for particle coating. For example Able et al. (2001) found that reporter gene expression levels were reduced 44% and 71% after using spermidine stored at 20C for 21 and 9 0 d, respectively. Since calli induced on dicamba containing media produce a large number of plants it is less likely that regeneration potential becomes a limiting factor in these cultures. Therefore something like the ratio and final concentration of DNA and microcarrier particle or other biolistic gene transfer parameters affecting particle penetration into tissues should be further explored While further optimization of the described protocols are desirable this research achieved for the first ti me stable genetic transformation of seashore paspalum and the stable expression of a promising insect resistance gene shown in expressing lines planted to soil from tissue culture

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57 This is expected to exemplify the potential of genetic transformation for the improvement of seashore paspalum

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58 CHAPTER 6 CONCLUSION Golf managers would prefer turf that requires less herbicide and fungicide applications ( Lee 1996 ). Increasing seashore paspalum s resistance to pests like fall armyworm, would make seashore pasp alum a more popular turf. Due to the limited availability of insect resistance genes within the species, heterologous genes from non related sources are most promising for improvement of insect resistance in seashore paspalum For example it is very desi rable to introduce crystal protein encoding genes from Bacillus thuringiensis ( Bt C ry) as effective control against Lepidoptera However, a genetic transformation protocol was lacking for seashore paspalum Seashore paspalum is a vegetatively propagated turfgrass, assuring uniformity and avoiding variation caused by segregation of transgene loci. However before this turf can be bioengineered a tissue culture regime need ed to be established. Here a n efficient tissue culture protocol is presented, and for the first time genetic transformation in the species seashore paspalum is achieved The C ry1Fa endotoxin was detected in with a commercially available immuno chromathoraphic strip indicating t ransgenic plants of the commercially important SI1cultivar ex pressed high levels of the synthetic Bt c ry1F a gene This is expected to confer resistance to fall armyworm as it has already been demonstrated earlier for other transgenic grasses including bahiagrass (Luciani et al. 2007). A factor to take into considerat ion when developing a successful transformation protocol is the mutagenic effect that extended periods in tissue culture might have (Zaghmout and Torello 1992). Different auxin types might have different impact s on the frequency of undesirable somaclonal v ariation ( Deambrogio and Dale 1980 ; Murata 1989 ). A reduction in the tissue culture period has been reported as an effective means to reduce somaclonal variation in other

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59 grasses ( Altpeter et al. 1996 ; 2000). Seashore paspalum plants regenerated faster f rom dicamba containing media and grew more vigorously when transferred to soil, similar to wildtype. However dicamba is thought to lead to higher rates of somaclonal variation than 2,4 D ( Bregitzer et al. 1998 ) and some somaclonal variation was observed after extended periods in tissue culture This work has successfully shown that transformation of seashore paspalum can be achieved following callus induction on media containing dicamba or 2,4 D. Because of the presence of stable transformation events wi th SI1 plants induced to form callus with both 2,4 D and dicamba it is not clear as yet whether one of these growth regulators is superior to the other in terms of their use in a stable transformation system T h is is not surprising since significantly hi gher numbers of shoots regenerated per callus initiated with dicamba containing media but significantly higher level s of transient reporter gene expression events were observed with callus generated on 2,4 D media. These results confirm that transient ex pression and stable transformation results are not highly correlated due to f actors such as tissue damage from gold penetration which can affect plant regeneration response from callus and differences in tissue firmness. Additionally, t he finding that the medium with the far superior plant regeneration response did not promote higher transformation efficiencies than alternative media suggests that other factors such as genotype and selection might offer targets for further improvement of transformation effi ciency. Enhancement of commercially important seashore paspalum by genetic transformation is now achievable and is expected to contribute to genetic improvement of this environmentally friendly turf.

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60 APPENDIX LABORATORY PROTOCOLS USED FOR VECTOR CONS TRU CTION, TISSUE CULTUR E AND MOLECULAR ANALYS IS OF TRANSFORMED SE ASHORE PASPALUM PLAN TS Protocols For Molecular C loning Preparation o f E lectrocompetent E. coli Always sterilize LB broth, containers used to grow cultures, pip ette tips, and eppendorf tubes befo re use in an autoclave at 121C and 120kPa for 2 0 min Each centrifuge step was done with a Sorvall with GSA rotor unless otherwise noted. 1. Using a sterile glass tube at least 2 0 mL in volume, incubate a DH5 culture in 5 mL LB broth at 37 C and grow overnig ht with shaking at 22 0 2. Inoculate 2 25 0 mL LB broth in 1L sterile flasks with 2 mL culture. Incubate at 37C shaking at 22 0 0.8 (Approx 5 6 h). 3. Pellet cells for 1 5 min, 4000g at 4C (3x 25 0 mL bottles). 4. Pour off supernatant and resuspend in an equivalent volume of ice cold sterile water (3x 16 5 mL ). Handle cells gently at all times. 5. Centrifuge 1 5 min, 4000g, 4C. 6. Pour off supernatant and resuspend in 0.5 volume o f ice cold sterile water (3x 8 3 mL ). Divide cell culture between 2 bottles. 7. Centrifuge 1 5 min, 4000g, 4C. 8. Pour off supernatant and resuspend in 0.5 volume of ice cold sterile water (2x 12 5 mL ). 9. Centrifuge 1 5 min, 4000g, 4C. 10. Pour off supernatant and resus pend in 0.02 volume of ice cold sterile water (2x 5 mL ). Transfer cell culture to 3 0 mL sterile centrifuge tube. 11. Centrifuge 1 5 min, 4000g, 4C (Sorvall, SS 3 4 rotor). 12. Pour off supernatant and resuspend in 0.02 volume ice cold sterile 10% glycerol (1. 2 mL ). 13. Transfer 40 l aliquots to sterile, chilled 1. 5 mL eppendorf tubes with holes pierced in the lids using a sterile needle. Quick freeze in liquid nitrogen and store at 70C. Electroporation o f E. c oli 1. Chill 0. 1 cm gap cuvettes (BioRad; 1000 Alfred Nobel D rive, Hercules, CA 94547, US ) on ice. 2. Place DNA for transformation (usually 1 2 l 10 15 n g l igation) in 1. 5 mL sterile eppendorf tubes on ice. 3. Thaw 40 l aliquots of electrocompetent E. coli cells on ice. 4. Set BioRad MicroPulser Electroporator to Ec1 (1.8 kV, 5 m sec, 0. 1 cm gap). 5. Add DNA to chilled competent E. coli cells. 6. Immediately transfer competent E. coli cells with DNA to cuvette, being careful to evenly fill the gap of the cuvette with competent cells. 7. Place cuvette in slide and push slide into cham ber ensuring that cuvette is seated between contacts in the base of the chamber of the BioRad MicroPulser Electroporator. 8. Pulse once by pressing pulse button. Alarm signifies pulse is complete.

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61 9. Check and record pulse parameters (time constant should be clo se to 5 m sec, if not repeat procedure with a fresh batch). 10. Remove cuvette and immediately add 40 0 L SOC medium to the cuvette. Quickly transfer cells to a 2 mL eppendorf tube. Time is critical. 11. Incubate cells at 37C shaking at 22 0 rpm on an orbital 1 h before plating on appropriate antibiotic medium (50 l for circular plasmid, 100 200 l for ligation product). 12. To plate transformed E. coli use a blunt instrument that has been flame sterilized and cooled to spread bacteria even ly on a plate of freshly made LB agar with the appropriate antibiotic. (Depending on the antibiotic LB agar media can be stored at 4C between 1 w k and 1 m). Glycerol S tocks 1. Grow E. coli culture containing desired plasmid overnight at 37C in 2 mL sterile LB broth containing the appropriate antibiotic at 37C with constant shaking at 22 0 rpm on an orbital 2. Add 0.8 5 mL of the bacterial culture and 0.1 5 mL glycerol (previously autoclaved and cooled to room temperature) into a sterile epp endorf tube with a hole pierced in the lid. 3. Quickly mix by vortexing and immediately freeze in liquid nitrogen. Store 8 0 C Prepare several tubes for plasmid. Avoid successive freeze thaw cycles. Amplification and Purification o f P lasmid DNA U sing QIApre p Miniprep K it (Qiagen; 27220 Turnberry Lane, S uite 200, Valencia, CA 91355, US ) 1. Add the provided RNase A solution to Buffer P1, mix well by inverting and store at 4C. 2. Add 100% ethanol (volume provided on the bottle label) to the Buffer PE concentrate to prepare the working solution. 3. To dispose of any waste autoclave for 6 0 min (121C, 15psi). 4. To amplifying a large number of DNA plasmid one can grow plasmid in 10 to 4 0 mL sterile LB broth using 1 to 4 spin columns. Add a maximum of 1 0 mL of LB broth to e ach spin column. Procedure remains the same except for doubling the amount of P1, P2, and P3 buffers used (500, 500, and 75 0 L respectively mixed in a sterile 2 mL eppendorf tube. Also in the final step incubate the spin column after it has been washed at 65C in a heat block and add 5 0 L of heated sterile ddH 2 0 at 65C. Incubate an additional min at 65C before spinning into a sterile eppendorf tube. In the final step spin DNA plasmid from multiple columns into the same sterile eppendorf tube. 5. Precipi tation in Buffer P2 can occur in cold temperature, if precipitation occurs warm to room temperature before use. 6. Grow over night cultures each from a single colony of bacteria in 5 mL LB sterile broth containing the appropriate antibiotic at 37C with cons tant shaking at 22 0 rpm on an orbital 7. Centrifuge the cultures at 16,100g for 1 min to pellet the bacterial cells. Decant off the supernatant. 8. Resuspend the bacterial cells in 25 0 L Buffer P1 by vortexing and transfer the suspension t o a sterile 1. 5 mL eppendorf tube (Shake Buffer P1 well before use)

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62 9. Add 25 0 L Buffer P2 and mix well by inverting the tube gently 4 6 times, incubate for 4 min at room temperature (in some kits the mixture will now turn an even blue color) 10. Add 35 0 L Buff er P3 and mix immediately but gently by inverting the tube 4 6 times, a white cloudy precipitate should form (if previously mixture was blue the addition of Buffer P3 should make the mixture clear again, mix until there is no blue color left). 11. Centrifuge t he samples at 15,700g for 1 0 min. A compact white pellet will form. 12. Decant the supernatant onto QIAprep Spin Columns and centrifuge for 1 min at 15,700g using a table top centrifuge. Discard the flow through. 13. Pipette 75 0 L Buffer PB onto the columns and c entrifuge for 1 min at 15,700g to wash the columns (although this step is optional in the handbook we always do it) 14. Pipette 75 0 L Buffer PE onto the columns and centrifuge for 1 min at 15,700g to wash the columns 15. Discard the flow through and centrifuge fo r 1 min at 15,700g to remove all traces of the wash buffer (Buffer PE). 16. Place the QIAprep columns in clean 1. 5 mL eppendorf tubes. Add 5 0 L Buffer EB to the center of each QIAprep column to elute DNA. Let stand for 1 min and centrifuge at 15,700g for 1 mi n. 17. Estimate the DNA concentration using a spectrophotometer or by running on a 0.8% agarose gel. 18. Store the samples at 20C and avoid successive freeze thaw periods. Amplification a nd P urific ation o f P lasmid DNA Using t he QIAGEN Plasmid Midi Kit QIAGEN p lasmid purification protocol as provided by the manufacturer of the kit All buffers (P1, P2, P3, QBT, QC, QF, TE) are included in the QIAGEN kit 1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2 5 mL LB mediu m containing the appropriate selective antibiotic. Incubate for ~8h at 37C with vigorous shaking (~22 0 Use a tube or flask with a volume of at least 4 times the volume of the culture 2. Dilute the starter culture 1/50 0 to 1/1000 into selective LB medium. For high copy plasmids inoculate 2 5 mL medium. For low copy plasmids, inoculate 10 0 mL medium. Grow at 37C for 12 1 6 h with vigorous shaking (~ 22 0 ). Use a flask or vessel with a volume of at least 4 times the volume of the culture. 3. Harvest the bacteria cells by centrifugation at 600 0 g for 1 5 min at 4C. Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained. 4. Resuspend the bacteria l pellet in 4 mL or 1 0 mL Buffer P1. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. 5. Add 4 mL Buffer P2, mix gently but thoroughly by inverting 4 6 times and incubate at room temperature for 5 min. Do not vortex. 6. Add 4 mL of chilled buffer P3 (chill on ice), mix immediately but gently by inverting 4 6 times, and incubate on ice for 15 to 2 0 min. After the addition of buffer P3, a fluffy white material forms and the lysate becomes less viscous. If the material still appears viscous and brownish, more mixing is required to completely neutralize the solution. 7. Centrifuge at 20,000g for 3 0 min at 4C. Remove supernatant containing plasmid DNA promptly. Before loading the centrifuge, the sample shoul d be mixed again

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63 8. Centrifuge the supernatant again at 20,000g for 1 5 min at 4C. Remove supernatant containing plasmid DNA promptly. 9. Equilibrate a QIAGEN tip 100 by applying 4 mL Buffer QBT, and allow the column to empty by gravity flow. 10. Apply the supernata nt from step 8 to the QIAGEN tip and allow it to enter the resin by gravity flow. 11. Wash the QIAGEN tip with 2 1 0 mL Buffer QC. 12. Elute DNA with 5 mL buffer QF. Collect the eluate in a 1 0 mL tube. 13. Precipitate DNA by adding 3. 5 mL of room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at 150 0 g for 3 0 min at 4C. Carefully decant the super natant. All solutions should be at room temperature in order to minimize salt precipitation. 14. Wash DNA pellet with 2 mL of room temperature 70 % ethanol, and centrifuge at 150 0 g for 1 0 min. Carefully decant the supernatant without disturbing the pellet. 15. Air dry the pellet for 5 1 0 min and redissolve the DNA in a 20 0 L TE buffer (Allow the pellet to remain in contact with the TE buffer overnight at 4C before transferring to a sterile eppendorf tube) Gel E xtraction U sing QIAquick Gel Extraction K it Prepa re Buffer PE by adding 4 0 mL 100% ethanol to the provided concentrate. Always use protective wear to protect skin and eyes from harmful UV radiation. When running a gel ensure that all buffers were made fresh, when staining a gel make sure to use a fresh b atch of ethidium bromide so as to not contaminate your plasmid with foreign DNA or DNase. 1. Place stained gel onto a clear plastic sheet on top of an ultraviolet light source to accurately identify where plasmid DNA is located. 2. Excise the DNA fragment precis ely from the agarose gel using a clean sharp scalpel. Avoid excess agarose. 3. Weigh the gel slice in a clear sterile microcentrifuge tube. Restrict the volume of gel in each 2 mL eppendorf tube to 40 0 mg or less. 4. Add 3 volumes of Buffer QG to 1 volume of gel 5. Incubate at 50C in a heating block for 1 0 min or until the gel has completely dissolve. Vortex every 2 min during incubation to ensure complete dissolution of gel. 6. After incubation check that the color of the mixture remains similar to Buffer QG (yellow ), indicating that the pH has not changed. 7. Add 1 gel volume of isopropanol to the mixture and mix by inverting the tube several times. 8. Place a QIAquick spin column in a provided 2 mL collection tube and apply the sample to the column. Centrifuge at 16,100g for 1 min with a table top centrifuge. Repeat this step if the volume of the mixture is more than 80 0 L the maximum capacity of the QIAquick column. 9. Discard the flow through and place the column in the same collection tube. 10. Wash the QIAquick column by a dding 75 0 L Buffer PE to the column and centrifuging for 1 min at 15,700g. 11. Discard the flow through, place the column back in the same collection tube and spin for an additional 1 min at 15,700g to remove residual ethanol from Buffer PE. 12. Place the QIAquic k column in a clean, sterile 1. 5 mL microcentrifuge tube.

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64 13. Add 3 0 L Buffer EB (1 0 mM Tris Cl, pH 8.5) to the center of the QIAquick membrane to elute DNA, let stand for 1 min and then centrifuge for 1 min at 15,700g. 14. Store DNA at 20C. Vector C onstructio n Digestion of pUbiGUS Restriction Enzyme HindIII (Promega; 2800 Woods Hollow Road, Madison WI 53711, US ) pUbiGUS ( 5 g) 16.5 L Restriction Buffer 2 2 L HindIII 1.5 L Final volume 2 0 L Digest overnight at 37 C. L igation of backbone fragment from HindIII digested pUbiGUS Ligation kit (Promega) pUbiGUS 1 L Ligation Buffer 2X 5 L T4 ligase 1 L ddH 2 0 2.35 L Final volume 1 0 L Ligate overnight at room temp L inearization of HindIII digested pUbiGUS and self ligation prevention 1. Digest 5g pUbi GUS plasmid with Not1 overnight at 37C 2. Add 1l CIP directly to the Not1 digestion mixture then incubate for 2 h at 37C.

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65 3. Add 1.5l 0.5M EDTA (ph8.0) and incubate at 65C for 1hr to inactivate CIP. 4. Add 3 0 L phenol:chloroform:isoamyl alcohol 25:24:1. Mix b y tapping and inverting. 5. Centrifuge at 3,200g at 4C for 1 min. There will now be three layers. The top layer contains DNA, the middle layer contains precipitates, and the bottom layer contains phenol. Pipette out the bottom layer and discard into a waste bottle. 6. Spin at 3,200g at 4C for 5 min. Take the supernatant to a fresh 1. 5 mL eppendorf tube. 7. Add 1/10 volume (5 L ) 3M NaOAc, mix by tapping 8. Add 2 volume (10 0 L ) chilled absolute ethanol. Mix. DNA precipitation may become visible. 9. Wash the DNA pellet by adding 20 0 L chilled 75% ethanol and tap the tube to wash thoroughly 10. Spin at 3,200g at 4C for 5 min. disturb the DNA pellet. 11. Dry the DNA pellet by putting the tube on a clean bench for 1 0 min. 12. Ad d 2 0 L ddH 2 0 to dissolve the DNA pellet. Restriction D igestions The pHZ ubicry1Fa digestion Restriction Enzyme NotI (Promega) pHZ ubicry1Fa Vector ( 3 g) 12 L Restriction Buffer D 10X 3.5 L BSA 10X 3.5 L NotI 3.5 L DdH2O 12.5 L Final volume 35 L Digest overnight at 37 C Ligation of the ubiquitin promoter and c ry1Fa coding sequence from pHZ ubicry1Fa into the pUbiGUS linearized backbone Ligation kit (Promega) Ligation reaction pUbiGUS backbone 1 L c ry1Fa insert 0.5 L T4 ligase 1 L DdH2O 6.5 L Final volume 1 0 L Ligate overnight at room temp erature

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66 Protocols F or Seashore P aspalum C allus I nduction, T ransformation, A nd R egeneration Protocol for Se ashore P aspalum I mmature I nflorescence I n V itro C ultivat ion We maintained high quality donor plant tissue with 16hr photoperiods using 100 0 Watt sodium vapor lights. Greenhouse temperature was controlled with air conditioning to approximately 30C during day and 25C during night. An ebb and flow system for irr igation was used, irrigating 5 min every morning and fertilization was done every two wk using the recommended rate of Bloom Booster plant food from Miracle Gro (P.O. Box 606 Marysville, OH 43040). Plants were maintained in 6 inch pots with Fafard No2 soi l. 1. Remove immature inflorescences before or just upon the emergence of the fla g l eaf. Cut the stem of the grass two to three nodes down from the uppermost node 2. Maintain harvested tissue in a moist environment before sterilization, moisten a cloth or paper towel and place in a bag with harvested tissue 3. Before surface sterilization, remove excess leaf, root, or soil material from the harvested immature inflorescence still wrapped in the sheath 4. Place up to 30 stalks into a 5 0 mL tube and fill tube with 70% Eth anol 5. Shake for 4 min by hand 6. Remove excess Ethanol and add pool bleach with active ingredient 10% sodium hypochlorite 7. Add 0.01% tween and stir 2 0 min at a low stir setting on any shaker 8. After sterilization tissue should be rapidly rinsed with sterile disti lled water three time followed by two rinses with emersion periods in sterile distilled water for 1 0 min all in a clean bench. (When rinsing recap the 5 0 mL tube to ensure dilution of active ingredients on the lid itself) 9. The immature inflorescences can no w be excised from the surface sterilized sheath and inflorescences of up to 1 cm can be cut and placed onto callus induction media. Tissue Culture Conditions Tissue culture was initiated on either IF medium or Treatment Medium and transferred every 7 1 5 d to fresh medium. Tissue Culture was maintained with Low light intensity (3 0 Em 2 s 1 light) at 25C with a 1 6 h photoperiod in a Percival incubator. Shoot regeneration was done on Regeneration Medium at a high light intensity (15 0 Em 2 s 1 light) at 25C with a 1 6 h photoperiod. Tissue culture was maintained on fresh media biweekly to monthly depending on growth stage of newly formed shoots. Regenerated shoots were transferred to fresh medium less frequently than callus that had not yet formed shoots. Prot ocol for Particle B ombardment Gold s tock (6 0 mg mL 1 ) preparation 1. Weigh 3 0 mg 1. 0 g gold into a sterile 1. 5 mL eppendorf tube. 2. Add 1 mL of 70% ethanol. (Prepare 70% ethanol with autoclaved sterile ddH 2 0) 3. Vortex for 3 5 min. 4. Incubate at room temperature fo r 1 5 min.

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67 5. Pellet the microparticles using a table top centrifuge for 5 s 6. Discard supernatant then wash three times by adding 1 mL sterile ddH 2 0. 7. Vortex for 1 min and let particles settle an additional min. 8. Pellet the microparticles with a brief 5 s spin i n a table top centrifuge then remove supernatant. 9. After third wash add 50 0 L sterile 50% glycerol. Store at 20C. Sterilization of B iolistic G ene D elivery D evice (PDS 1000, BioRad ) C omponents 1. Autoclave macrocarrier holders, stopping screens, macrocarrie rs, and a device capable of securing macrocarriers into the macrocarrier holders. 2. Lay out in laminar flow hood to dry. 3. Sterilize 1100psi rupture discs by dipping in Absolute Ethanol and dry in flow hood on the rim of a sterile petri dish. 4. Clean biolistic g ene delivery device (PDS 1000, BioRad ) chamber, assembly and flow hood thoroughly with 70% ethanol and thoroughly dry before use. 5. For large scale bombardments allow time to resterilize the biolistic gene delivery device (PDS 1000, BioRad ) and its component s with 70% ethanol to reduce risk for contamination. Preparation of DNA Co ated M icroparticles 1. Mix 30 l 1 m gold stock and 30 l DNA by vortexing 1 min. 2. Add 20 l 0.1M spermidine and 50 l 2.5M CaCl 2 consecutively and immediately while continuing to vortex fo r an additional min. 3. Centrifuge briefly to settle gold. 4. Wash in 250 l Absolute Ethanol without disturbing the pellet. 5. Spin and remove supernatant. 6. Resuspend gold in 90 l Absolute Ethanol by sonication (Branson 2200, Branson Ultrasonics, Danbury, CT 06813 1 961, US ) for 2 s. 7. Ensure there are no large clumps of DNA by placing eppendorf tube against a light source and tapping the tube. 8. Use 5 l per shot enough for 12 shots (Upon placing DNA solution onto macrocarriers be sure that DNA and gold are in solution by rubbing briefly against an empty pipette rack) Biolistic B ombardment Use the same stopping screen for 15 20 shots. 1. Turn on PDS 1000/He Particle Delivery System and vacuum pump, than increase helium pressure to the tube attached to the biolistic gene deli very device (PDS 1000, BioRad ) to just slightly over 1100psi. 2. Place macrocarriers into holders securely using a sterile blunt object. 3. Spread 5 macrocarriers at a time as to min imize DNA degradation from exposure to the air). 4. Place rupture disc into holder and screw tightly into place.

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68 5. Place stopping screen into shelf assembly and put inverted macrocarrier assembly on top. 6. Place shelf containing the macrocarrier at the highest le vel. 7. Place tissue culture plate on shelf 2 levels below gold or 6 cm below the macrocarrier shelf. 8. Pull a vacuum to 27. 5 Hg then press and hold the fire button until the disc ruptures at 1100psi. 9. Vent vacuum and remove petri dish. 10. Dismantle assembly and se t up for next shot. The GUS Assay (Jefferson et al. 1987) Solution 1 1. Add 7 0 mg X gluc to 2 mL DMSO in a small beaker (cover beaker in foil to minimize exposure to light) Solution 2 2. Mix 15 0 mL of 100mM Na 3 Po 4 to 5 mL of 0.5M EDTA and add 20 0 l Trinton X 100. 3. Mix solution 1 and 2 and make final volume up to 20 0 mL with ddH 2 0 and aliquot in 1 5 mL tubes and store at 20C in the dark (wrap in foil). The GUS stock solutions 100mM Na 3 PO 4 (pH 7): Dissolve 7.60 2 g Na 3 PO 4 12H 2 0 and adjust pH to 7 with concentra ted HCl 0.5M EDTA (pH 8): Dissolve 46.52 5 g EDTA disodium salt in 20 0 mL ddH 2 0. Adjust pH to 8 with NaOH and make up to 25 0 mL Sterilize by autoclaving. The GUS Assay P rocedure 1. Bombard tissue using procedure described previously for biolistic transformat ion of callus. 2. Allow tissue to recover on fresh callus induction media after bombardment for 3 to 4 d 3. Place bombarded tissue into a 24 well microplate and place plate into an empty pipette box to prevent spilling of assay into biolistic gene delivery devi ce (PDS 1000, BioRad ) or incubator. 4. Add enough GUS assay mix to completely cover the callus material in the microplate and place into the biolistic gene delivery device (PDS 1000, BioRad ) to draw a vacuum for 1 0 min. 5. Afterwards incubate for 16hrs at 37C i n the dark. 6. Blue foci should be visible on callus and can be counted using a microscope.

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69 Buffers And M edium Bacterial G rowth The SOC medium SOB: 4 g tryptone, 1 g yeast extract and 0. 1 g NaCl. Dissolve in 18 0 mL ddH 2 O. Add 2 mL 250mM KCl. Adjust to pH7 wit h 5N NaOH. Make up to 20 0 mL Just before use, add 1 0 L 1M MgCl 2 and 2 0 L 1M glucose per 1 mL SOB. Antibiotics Ampicillin: Weigh 10 0 mg ampicillin. Dissolve in 2 mL of ddH 2 O. Filter sterilize into autoclaved eppendorf tubes. Freeze at 2 0C. Stock concen tration: 5 0 mg mL 1 Use 2l (10 0 mg mL 1 ) LB. Kanamycin: Weigh 10 0 mg kanamycin. Dissolve in 1 0 mL of ddH 2 O. Filter sterilize into autoclaved eppendorf tubes. Freeze at 20C. Stock concentra tion: 1 0 mg mL 1 Use 5l (5 0 mg mL 1 ) LB. Rifampicin: Weigh 10 0 mg rifampicin. Dissolve in 4 mL of DMSO. Filter sterilize into autoclaved eppendorf tubes. Wrap tubes in aluminum foil. Freeze at 2 0C. Stock concentration: 2 5 mg mL 1 Use 6l (15 0 mg mL 1 ) LB. Spectinomycin: Weigh 10 0 mg spectinomycin. Dissolve in 1 0 m L of ddH 2 O. Filter sterilize into autoclaved eppendorf tubes. Freeze at 20C. Stock concentra tion: 1 0 mg mL 1 Use 5l (5 0 mg mL 1 ) LB. Seashore Paspalum Tissue Culture M edium The IF medium 1. Add 4. 3 g L 1 MS salts, 12.4 5 mg L 1 CuSO4, 2 0 g L 1 sucrose. 2. Adj ust pH to 5.8. 3. Add 3 g L 1 phytagel. 4. Media was made with a final volume of 40 0 mL per bottle. 5. Sterilize at 121C and 120kPa in an autoclave for 2 0 min. 6. Before the media became solid add 3 mg L 1 Dicamba, 103. 2 mg L 1 MS 1000X vitamins, and 1. 1 mg L 1 BAP ( thoroughly mix all stock solutions before use) Treatment medium 6 treatments made just like IF media except for the auxin used and BAP level. Media was made with either 2,4 D or dicamba both at 3 mg L 1 and of the two auxin types levels of BAP were either 0, 0.1, or 1. 0 mg L 1 BAP.

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70 Sorbitol medium Medium was made with the same constituents of the medium callus was induced on with the inclusion of sorbitol at 72.8 5 gL 1 i.e. IF medium with 72.8 5 g L 1 sorbitol added before sterilization in autoclave. Selec tion medium Hygromycin at 4 0 mg L 1 was added to both IF medium and the six treatment medium and 6 g L 1 of the gelling agent agarose was used instead of phytagel when callus was under selection. All medium made with hygromycin must be stored in the dark. Regeneration medium Shoot regeneration medium was made following the protocol for IF or any of the six treatment medium with the exclusion of any auxin or cytokinin. Selection on regeneration medium was done at both 2 0 and 1 0 mg L 1 hygromycin again with t he substitution of 6 g L 1 agarose instead of phytagel. Stock Solutions f or Tissue C ulture The 2,4 dichlorophenoxyacetic acid (2,4 D) Weigh out 0. 1 g 2,4 D. Dissolve in very little 1N K0H and then add 5 0 mL ddH 2 0. Filter sterilize and store in 1 mL aliquot s at 20C. The 3,6 dichloro 2 methoxybenzoic acid (dicamba) Weigh 10 0 mg dicamba. Dissolve in 0. 5 mL of 100% ethanol with heat. Add 49. 5 mL ddH 2 O with heat. Filter sterilize the stock solution and aliquot into autoclaved eppendorf tubes and freeze at 20 C. Stock concentration: 2 mg mL 1 Use 60 0 L (120 0 mg)/40 0 mL IF. The 6 benzylaminopurine (BAP) Weigh 82 5 mg BAP. Dissolve in 0. 5 mL of NaOH (1N). Add 19. 5 mL ddH 2 O. Filter sterilize the stock solution and aliquot into autoclaved eppendorf tubes, then f reeze at 2 0 C Stock concentration: 1 mg mL 1 Use 44 0 L (120 0 mg)/40 0 mL IF. Hygromycin Dissolve hygromycin in ddH 2 0 making final volume 12 5 mg mL 1 Filter sterilize and store in aliquots at 20C in the dark. Paromomycin Weigh 0. 5 g paromomycin in 1 0 mL ddH 2 0. Filter sterilize and store in 1 mL aliquots in eppendorf tubes at 20C.

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71 Molecular Techniques used in the Confirmation of Stable Transformants Immunochromatographic Cry1F a QuickStix T est EnviroLogix (500 Riverside industrial parkway, Portland, Maine 04103 1486, US ) QuickStix Kit for Cry1F a protocol as provided by the manufacturer of the kit 1. Sandwich a section of leaf tissue between the cap and body of the disposable tissue extractor tube (We used 4 leaf segments all equal in length to a 1. 5 mL e ppendorf tube. Push the leaf punches down into the tapered bottom of the tube with the pestle. Sample identification should be marked on the tube with a waterproof marker. 2. Insert the pestle into the tube and grind the tissue by rotating the pestle against the sides of the tube with twisting motions. Continue this process for 20 to 3 0 s or until the leaf tissue is well ground. 3. Uncap the bottle of extraction buffer and invert it directly over the tissue extractor tube. Carefully squeeze 1 0 drops (we used 5 dr ops) into the tube containin g l eaf sample. 4. Repeat the grinding step to mix tissue with extraction buffer. Dispose of the pestle (do not re use pestles on more than one sample). 5. Allow refrigerated canisters to come to room temperature before opening. Remove the QuickStix Strips to be used. Avoid bending the strips. Reseal the canister immediately. 6. Place the strip into the extraction tube. The sample will travel up the strip. Use a rack to support multiple tubes if needed. 7. Allow the strip to develop for 1 0 mi n before making final assay interpretations. Positive sample results may become obvious much more quickly. Development of the control line within 1 0 min indicates that the strip has functioned properly. If the sample extract contained Cry1F a endotoxin, a s econd line (test line) will develop on the membrane strip between the control line and the protective tape, within 1 0 min of sample addition The ELISA f or Presence o f Cry1F a P rotein Cry1F a high sensitivity protocol as prov ided by the manufacturer of the kit Will detect 0.17% Herculex I corn in ground grain/seed, and requires 2. 5 h of total assay incubation time. Dilute the positive control ground corn extract 1:3 in negative control ground corn extract for this protocol. 1. Ad extract (S) to their respective wells. 2. Thoroughly mix the contents of the wells by moving the plate in a rapid circular motion on the benchtop for a full 20 3 0 s Be careful not to spill the contents! 3. Cover the wells with tape or parafilm to prevent evaporation and incubate at ambient temperature for 3 0 m in. If an orbital plate shaker i s available, shake plate at 200rpm. 4. Add Cry1F enzyme conjugate to each well. Thoroughly mix the contents of the wells, as in step 2. 5. Cover the wells with tape or parafilm to prevent evaporation and incubate at ambient temperature for 9 0 min. If an orbital plate shaker is available, shake pl ate at 20 0 rpm.

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72 6. After incubation, carefully remove the covering and vigorously shake the contents of the wells into a sink or other suitable container. Flood the wells completely with wash buffer, then shake to empty. Repeat this wash step three times. 7. Add 8. Thoroughly mix the contents of the wells, as in step 2. Cover the wells with new tape or parafilm and incubate for 3 0 min at ambient temperature. Use orbital shaker if available. Caution: stop solution is 1.0N hydrochloric acid. Handle carefully. 9. yellow NOTE: Read the plate within 3 0 min of the addition of stop solution. I nterpret ion of results (Spectrophotometric measurements and general test criteria) 1. Set the wavelength of the microtiter plate reader to 45 0 nm (If it has dual wavelength capability, use 600, 630 or 65 0 nm as the reference wavelength.) 2. Set the plate reader to blank on the extraction buffer blank wells (this should a utomatically subtract the mean optical density (OD) of the blank wells from each control and sample OD). If the reader cannot do this, it must be done manually. The mean OD of the blank wells should not exceed 0.2. The mean, blank subtracted OD of the posi tive control wells should be at least 0.15. The coefficient of variance (%CV) between the duplicate positive control wells should not exceed 15%: % 100 mean pos.ctl. OD Calculate the positive control ratio Divide the OD of each sample extract by the mean OD of the positive control wells. This Single corn leaf and seed samples: If the positive control ratio calculated for a sample is less than 0.5, the sample does not contain Cry1F a at the levels normally found in Herculex I corn. If the positive control ratio of a sample is greater than or equal to 0.5, the sample does contain Cry1Fa at the levels normally found in Herculex I corn Preparation of S olutions for ELISA Wash buffer Add the con tents of the packet of wash buffer salts (phosphate buffered saline, pH 7.4 tween 20) to 1 L of distilled or deionized water and stir to dissolve. Store refrigerated when not in use; warm to room temperature before assay. If more wash buffer is needed, o rder item # P 3563 from Sigma Chemical Co. (St. Louis, MO 63118), or prepare the equivalent. Extraction buffer Add 0. 5 mL tween 20 to 10 0 mL of prepared wash buffer, and stir to dissolve. Store refrigerated when not in use; warm to room temperature before assay. Sample preparation Positive and negati ve control ground corn extracts

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73 Extracts of these controls must be run in every assay. To extract, add 5 mL of extraction buffer to each tube containing 2 g of ground control corn. Cap and shake vigorously by ha nd or vortex for 20 3 0 s Let stand at room temperature for 1 h to extract. Mix again at the end of the hour, then clarify by settling 1 0 min or by centrifuging 5 min at 5000g. The high sensitivity protocol requires that the positive control ground corn ex tract be diluted 1:3 before use. Leaf testing 1. Take a single leaf punch of approximately 5 mm diameter (We used two leaf segments from the second and third uppe rmost leaves of length equivalent to that of a 1. 5 mL eppendorf tube), using a micro tube cap or a paper punch. Mash the leaf tissue with a pestle matched to the micro tube, or with a disposable pipette tip, or a Hypure cutter (HCT 200; PerkinElmer, 940 Wi nter Street, Waltham, Massachusetts 02451, US ) in a 96 well plate (Costar #3370; Cornin g l ife Sciences, Tower 2, 4 th floor, 900 Chelmsford street, Lowell, Massachusetts 01851, US ; or equivalent). 2. Add 0.2 5 mL of extraction buffer per leaf punch. Mix for at least 3 0 s then allow particles to settle. Take extreme care not to cross contaminate between leaf samples. Dispensing particles into the test plate can cause false positive results. The DNA Extraction Using the CTAB M ethod Procedure Sterilize mortar and pestle and eppendorf tubes in an autoclave for 2 0 min before use. 1. Collect 0. 1 g of youn g l eaf tissue 2. Freeze and grind the sample to a fine powder in liquid nitrogen (grind three times, grind when nitrogen has evaporated to prevent loss of sample) 3. Add fresh ly ground sample to 50 0 L CTAB buffer in a 1. 5 mL eppendorf tube. Mix by vortexing briefly. 4. Add 3l RNase 5. Incubate at 65C for 1 h mixing every 1 5 min. 6. Cool to room temperature then add an equal volume (50 0 L ) chloroform /isoamylalchohol 24:1. (Work in a fume hood) 7. Mix Thoroughly 8. Shake for 3 0 min gently 9. Spin at 3,200g at room temperature for 5 min 10. Transfer Supernatant to fresh tubes 11. Add equal volume (50 0 L ) chloroform /isoamylalchohol 24:1. (Work in a fume hood) 12. Mix thoroughly 13. Shake gently for 1 5 min 14. Spi n 3,200g at 4C for 5 min 15. Discard the bottom chloroform layer into the waste bottle in the fume hood 16. Spin 3,200g at 4C for 5 min 17. Carefully take the supernatant and transfer to fresh 1. 5 mL eppendorf tubes. 18. Add 2/3 volume isopropanol 19. Mix thoroughly, should be able to visually see DNA

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74 20. Wash 2X with 70% ethanol spin 3,200g at 4C for 5 min 21. Dry with a vacuum centrifuge for five min 22. Dissolve the DNA in 5 0 L sterile ddH 2 0 The CTAB b uffer (50 0 mL ) 5 0 mL 1M Tris HCl (100mM final) 2 0 mL 0.5M EDT A (disodium salt) ( 20mM final) 14 0 mL 5M NaCl (1.4M final) 1 0 g CTAB (2% w/v final) The PCR R eactions Basic PCR set u p u sing HotStarTaq DNA Po lymerase (Qiagen) 1. Determine the number of samples to be used for PCR analysis. Also include the wild type, negative control (react ion with all components but no template) and positive control (reaction containing the plasmid as template). 2. Prepare a master mix for all the samples together as follows: 10x Buffer 2.5 0 L 5x Q solution 5.0 0 L 50x dNTP mix 0.5 0 L 50x MgCl 2 0.5 0 L 1 0 M Forward primer 1.0 0 L 1 0 M Reverse primer 1.0 0 L HotStarTaq 0.1 5 L Sterile ddH 2 O 13.3 5 L Final volume 24.0 0 L 3. Label the tubes for PCR and dispense 2 4 L of the master mix into each tube. 4. Add 1 L DNA to the samples (5 0 ng), 1 L ste rile ddH 2 O to the negati ve control and 1 L plasmid (5 0 pg L 1 ) to the positive control. Mix well by pipetting, spin briefly and start the PCR program. Note: Remember to start the PCR program with 1 5 min at 95C to activate the HotStarTaq polymerase.

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75 A mp lif ication of hph in m odified phph c ry1Fa PCR kit PCR Core System II (Promega) Primer sequences Forward CCC GAT ATG AAA AAG CCT GA Reverse GAT GTT GGC GAC CTC GTA TT PCR program Isaac hph 5 C for initial denaturation, 30 cycles w 5 C for 8 C 2 C for extension, 2 C for final extension and hold at 4C PCR reaction DNA template 1. 0 L PCR Buffer 10 X 2. 0 L Q Buffer 4. 0 L DNTPs 0.4 l Forward primer 1. 0 L Reverse primer 1. 0 L Taq polymerase 0.1l DdH 2 O 10.5l Final volume 20. 0 L Amplification of c ry1F in modified phph c ry1Fa PCR kit PCR Core System II (Promega) Primer sequences Forward CCG GGA CCA TTG ACT CTC TA Reverse CAC TTC GTT GCC TGA ACT GA PCR program Isaac c ry 5 C for initial denaturation, 30 5 C for C for extension, 2 C for final extension and hold at 4 C PCR reaction DNA template 1. 0 L PCR Buffer 10 X 2. 0 L Q Buffer 4. 0 L DNTPs 0.4 l Forward primer 1. 0 L Reverse primer 1. 0 L Taq polymerase 0.1l DdH 2 O 10.5l Final volume 20. 0 L Agarose G el Electrophoresis Procedure 1. Prepare a 0.8% agarose gel by weighing 0. 4 g agarose. Transfer this to a 25 0 mL Erlenmeyer flask. Add 5 0 mL 0.5x TBE and boil in a microwave oven for 3 0 s followed by stirring and boiling another 3 0 s

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76 2. Allow to cool to 55 6 0 C. 3. Get a gel mold and seal both ends in the holder. Place in a level platform and attach a comb. 4. Pour agar ose into the gel mold and solidify. 5. Pour 0.5 TBE buffer into the electrode tank. Mount the gel mold on to the electrode tank with the comb oriented toward the cathode end. Remove bubbles with a pipette tip 6. Gently remove the comb. 7. Load 1 0 L 1 Kb ladder on the first well and load 1 2 L each of the 3different concentrations of lambda DNA (100, 50, 2 5 ng L 1 ) on the next 3 wells. 8. Apply sample in other wells with each well having a final volume if 12l 2 of which are a 6X loading dye. Load the mixtures in the succeeding wells. 9. Close the tank and attach the electrodes to the power supply. Run at 9 0 V for 4 5 min. 10. After the run, turn off the electric current and remove the gel mold from the tank. Transfer the gel in a staining tray with 10 0 L of EtBr solutio n in a 100 0 mL ddH 2 O. Stain for 15 2 0 min. 11. Photograph the gel under UV light and estimate each DNA sample by comparing with the lambda DNA made to concentrations of 25, 50, and 10 0 ng L 1 The TBE stock 5x stock TBE: 5 4 g Tris base 27. 5 g boric acid 2 0 mL 0.5M EDTA (pH8) Make up to 1L with ddH 2 O Dilute stock to 0.5x for use in gels and running buffer. Allow 50 0 mL for small gel tank Using t he Nanodrop ND 1000 Spectrophotometer (NanoDrop Technologies, 3411 Silverside Rd Bancroft Building, Wilmington, DE 1 9810) 1. Pipette 2l of water on the upper and lower pedestals and wipe off using a soft laboratory wipe to clean the pedestals. 2. Pipette 1 L water on the lower pedestal, close the sampling arm, initialize the instrument using the operating software and then set the blank. 3. Open the sampling arm and wipe the two pedestals using a laboratory wipe. 4. With the sampling arm open, pipette 1 L sample onto the lower pedestal (ensure complete covering of the lower pedestal) 5. Close the sampling arm and initiate the measur ement using the operating software. 6. On completion of the measurement, open the sampling arm and wipe the sample from both the upper and lower pedestals using a laboratory wipe. 7. Repeat from step 5 for subsequent samples. 8. Clean the pedestals as described in step 2 after completion of all measurements.

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77 LIST OF REFERENCES Able JA, Rathus C, Godwin ID (2001) The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum. In Vitro Cell Dev Biol 37:341 348 Adamczyk J J, Gore J (2004) Development of bollworms, Helicoverpa zea (Boddie), on two commercial Bollgard cultivars that differ in overall Cry1Ac level. J Insect Sci 4 ( 32 ):1 5 Ahn BJ, Huang FH, King JW (1985) Plant regeneration through somatic embryogenesis in comm on bermudagrass tissue culture. Crop Sci 25 : 1107 1109 Ahn BJ, Huang FH, King JW (1987) Regeneration of bermudagrass cultivars and evidence of somatic embryogenesis. Crop Sci 27:594 597 Akashi R, Adachi T (1992a). Somatic embryogenesis and plant regeneratio n from cultured immature inflorescences of apomictic dallisgrass ( Paspalum dilatatum Poir.). Plant Sci 82:213 218 Akashi R, Adachi T (1992b) Plant regeneration from suspension culture derived protoplasts of apomictic dallisgrass ( Paspalum dilatatum Poir.). Plant Sci 82:219 225 Alstad DN and Andow DA (1999) Implementing management of insect resistance to transgenic crops. http://www.agbiotechnet.com/review/misc/alstad.html Altpeter F (2006) Rye ( Secale cereale L.). Methods Mol Biol 343:223 231 Altpeter F, Va sil V, Srivastava V, Stoeger E, Vasil IK (1996) Accelerated production of transgenic wheat ( Triticum aestivum L.) plants. Plant Cell Rep 16:12 17 Altpeter F and Posselt UK (2000) Improved plant regeneration from cell suspensions of commercial cultivars bre eding and inbred lines of perennial ryegrass ( Lolium perenne ). J Plant Physiol 156:790 796 Altpeter F and Xu J (2000) Rapid production of transgenic turfgrass (Festuca rubra L.) plants. J Plant Physiol 157:441 448 Altpeter F, Xu J, and Ahmed S (2000) Gene ration of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage and turf type cultivars. Mol Breeding 6 : 519 528 Altpeter F, Positano M. (2005) Efficient plant regeneration from mature seed derived embry ogenic callus of turf type bahiagrass ( Paspalum notatum Flugge). Intern Turfgrass Soc Res J 10 : 479 484

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78 Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta S, Dix PJ, Fauquet C, Huang N, Kohli A, Mooibroek H, Nicholson L, Nguyen TT, Nugent G, Raemakers K, Romano A, Somers DA, Stoger E, Taylor N, Visser R (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15 : 305 327 Arizona Department of Water Resources (1995) Modification s to the second management plan:1999 2000. Phoenix, AZ, pp.74 Armstrong JS, Leser J, Kraemer G (2000) An inventory of the key predators of cotton pests on Bt and non Bt cotton in west Texas. In Proceedings of the Beltwide Cotton Conference Volume 2 (Dugger CP and Richter DA, eds) Memphis TN: National Cotton Council of America, pp 1030 1033 Artunduaga IR, Taliaferro CM, Johnson BL (1988) Effect of auxin concentration and growth of embryogenic callus from young inflorescence explants of old world bluestem (B othriochloa spp.) and Bermuda (Cynodon spp.) grasses. Plant Cell Tiss Org Cult 12 : 13 19 Artunduaga IR, Taliaferro CM, Johnson BL (1989) Induction and growth of callus from d 2,4 D concentration. In vitro Cell Devel Bio 25 : 753 756 Ashley YR, Wiseman BR, Davis FM, Andrews KL (1989) The fall armyworm: a bibliography. Fla Entomol 72:152 202 Bai Y and Qu R (2000) An evaluation of callus induction and plant regeneration in twenty five turf type tall fescue (Festuca arundinacea Schreb.) cultivars. Grass and Forage Science 55:326 330 Bai Y and Qu R (2001) Factors influencing tissue culture responses of mature seeds and immature embryos in turf type tall fescue (Festuca arundinacea Sc hreb.). Plant Breeding 120:239 242 Baja YLS, Sidhu BS, Dubey VK (1981) Regeneration of genetically diverse plants from tissue cultures of forage grass Panicum sps.. Euphytica 30:135 140 glucuronidase reporter gene expression analysis in turfgrass. Biochemical and Biophysical Research Communications 320:7 10 Bauer LS (1995) Resistance: a threat to the insecticidal crystal proteins of Bacillus thuringiensis Fla Entomol 78:414 443 Beard JB (1973) Turfgrass: Scien ce and culture. Prentice Hall, Englewood Cliffs, NJ Beard JB, Batten SM, Reed SR, Kim KS, Griggs SD (1982) A preliminary assessment of Adalayd Paspalum vaginatum for turfgrass characteristics and adaptation to Texas characteristics. P33 34. Texas Turfgrass research 1982. PR 4039

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79 Beard JB, Sifers SI, Hall MH (1991a) Cutting height and nitrogen fertility requirement of Adalayd seashore paspalum (Paspalum vaginatum) 1988 1989 p107 109. Texas Turfgrass Research 1991. PR 4291 Beard JB, Sifers SI, Menn GW (1991b) Cultural strategies for seashore paspalum. Grounds Maintenance 40(8):32,62 Becker D, Brettschneider R, Lorz H (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. The Plant J 5(2):299 307 Bernhard K, Jarrett P, Meadows M, Butt J, Ellis DJ, Roberts GM, Pauli S, Rodgers P, Burgess HD (1997) Natural isolates of Bacillus thuringiensis : worldwide distribution, characterization, and activity against insect pests J Inver. Pathol 70, 59 68 Bettany A, Dalton S, Timms E, Manderyck B, Dhanoa M, Morris P (2003) Agrobacterium tumefaciens mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.). Plant Cell Rep 21:437 444 Bevan M (1984 ) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 1 2:8711 8721 Bhaskaran S and Smith RH (1990) Regeneration in cereal tissue culture: a review. Crop Sci 30:1328 1336 Birch RG (1997) Plant transformation: Problems and strategies for practical application. Annu Rev Plant Physiol. Plant Mol Biol 48:297 326 Bo horova N, Frutos R, Royer M, Estaol P, Pacheco M, Rascon Q, Hoisington D (2001) Novel synthetic Bacillus thuringiensis cry1B gene and cry1B cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in tran sgenic tropical maize. Theor Appl Genet 103:817 826 Bovo OA and Mroginski LA (1985) Tissue culture in Paspalum (Gramineae). Plant regeneration from cultivated inflorescences. J Plant Physiol 124:481 492 Bradley DE, Bruneau AH, Qu R (2001) Effects of cultiv ar, explant treatment, and medium supplements on callus induction and plantlet regeneration in perennial ryegrass. Int Turfgrass Soc Res J 9:152 156 Braman SK, Pendley AF, Carrow RN, Engelke MC (1994) Potential resistance in zoysiagrass to tawny mole crick ets (Orthoptera: Gryllotalpidae) Fla Entomol 77:302 305 Braman SK, Duncan RR, Hanna WW, Engelke MC (2004) Turfgrass species and cultivar influences on survival and parasitism of fall armyworm. J Econ Entomol 97(6):1993 1998 Bregitzer P (1992) Plant regener ation and callus type in barley: effects of genotype and culture medium. Crop Sci 32:1108 1112

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80 Bregitzer P, Halbert SE, Lemaux PG, (1998) Somaclonal variation in the progeny of transgenic barley. Theor Appl Genet 96:421 425 Burson BL (1981) Genome relation ship among four diploid Paspalum species. Botanical Gazette (Chicago) 142:431 434 Cai T and Butler L (1990) Plant regeneration from embryogenic callus initiated from immature inflorescences of several high tannin sorghums. Plant Cell Tissue Organ Cult 20 : 1 01 110 California State Water Resources Control Board (1993). Porter Cologne Act provisions on reasonableness and reclamation promotion. California Water code, Section 13552 13557 Can E, Celiktas N, Hatipoglu R, Yilmaz S, Avci S (2004) Effects of genotype and concentrations of dicamba on callus induction and plant regeneration from young inflorescences of perennial ryegrass (Lolium perenne L.). Biotechnology and biotechnological equipment 18(2):52 57 Cannon RJC (2000) Bt transgenic crops: Risks and benefits Integrated Pest Management Reviews 5:151 173 Cardona CA and Duncan RR (1997) Callus induction and high efficiency plant regeneration via somatic embryogenesis in paspalum. Crop Sci 37 : 1297 1302 Carrow R (2005) Seashore paspalum ecotypes responses to drou ght and root limiting stresses. USGA Turfgrass and Environmental Research Online 4(13):1 9 Castle LA, Wu GS, McElroy M (2006). Agricultural input traits: past, present and future. Curr Opin Biotech 17:105 112 Castillo AM, Vasil V, Vasil IK (1994) Rapid pro duction of fertile transgenic plants of rye ( Secale cereale L.). Bio/Technology 12:1366 1371 Castillo AM, Egaa B, Sanz JM, Cistu L (1998) Somatic embryogenesis and plant regeneration from barely cultivars grown in Spain. Plant Cell Rep 17:902 906 Caswell Ki, Leung NL, Chibbar RN (2000) An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars. Plant Cell Tiss Org Cult 60:69 73 Chai B and Sticklen M (1998) Applications of Biotechnology in Turfgrass Genet ic Improvement. Crop Sci 38:1320 1338 Chaudhury A and Qu R (2000) Somatic embryogenesis and plant regeneration of turf type bermudagrass effect of 6 benzyladenine in callus induction medium. Plant Cell Tiss Org Cult 60:113 120 Chen CH, Senberg NE, Ross JG (1977) Clonal propagation of big bluestem by tissue culture. Crop Sci 17:847 850

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81 Chibbar RN, Kartha KK, Leung N, Qureshi J, Caswell K (1991) Transient expression of marker genes in immature zygotic embryos of spring wheat ( Triticum aestivum ) through microp article bombardment. Genome 34:453 460 Cho MJ, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138:229 244 Cho MJ, Ha CD, Lemaux PG (2000) Production of trans genic tall fescue and red fescue plants by particle bombardment of mature seed derived highly regenerative tissues. Plant Cell Rep 19:1084 1089 Choukr Allah et al) pp 3 13 (Marcel Dekke r, New York) Christensen AH, Sharrock RA, and Quail PH (1992) Maize polyubiquitin genes: Structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol 18:6 75 689 Colomba EL, Grunberg K, Griffa S, Ribotta A, Mroginski L, Biderbost E (2006) The effect of genotype and culture medium on somatic embryogenesis and plant regeneration from mature embryos of fourteen apomitic cultivars of buffel grass (Cenchrus cilia ris L.). Grass and Forage Science 61:2 8 Constable GA, Llewellyn DJ, Reid PE (1998) Biotechnology risks and benefits: the Ingrad cotton example. http://life.csu.edu.au/agronomy/papers/invite/const/agronsoc.html Crickmore N, Zeigler DR, Schnepf E, Van Rie J Lereclus D, Baum J, Bravo A, Dean DH (2000) Bacillus thuringiensis toxin nomenclature http://www.biols.susx.ac.uk/Home/NeilCrickmore/ Bt /index. html Croughan SS and Quisenberry SS (1989) Enhancement of fall armyworm (Lepidoptera: Noctuidae) resistance in bermudagrass through cell culture. J Econ Entomol 82:236 238 Cullings KW (1992) Design and testing of a plant specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1:233 240 Dale PJ (1980) Embryoids from cultured immature embryos o f Lolium multiflorum. Z Pflanzenphysiol 100:73 77 Dale PJ, Thomas E, Brettell RIS (1981) Embryogenesis from cultured immature inflorescences and nodes of Lolium multiflorum. Plant Cell Tiss Org Cult 1 : 47 55 Dale PJ and Dalton SJ (1983) Immature inflorescen ce culture in Lolium, Festuca, Phleum and Dactylis. Z Pflanzenphysiol 111 : 39 45 Dalton SJ, Bettany AJE, Timms E, Morris P (1995) The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arund inacea Schreb.) Plant Sci 108:63 70

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82 Dalton SJ, Bettany AJE, Bhat V, Gupta MG, Bailey K, Timms E, Morris P (2003) Genetic transformation of Dichanthium annulatum (Forssk) an apomitic tropical forage grass. Plant Cell Rep 21:974 980 Deambrogio E and Dale PJ (1980) Effect of 2,4 D on the frequency of regenerated plants in barley and on genetic variability between them. Cereal Res Commun 8:417 423 De Cleene M and Deley J (1976) The host range of crown gall. Bot Rev 42:389 466 Dornelles ALC, Carvalho FIF, Fed erizzi LC, Handel CL, Bered F, Sordi MEB, Schneider F (1997) Callus induction and plant regeneration by Brazilian triticale and wheat genotypes. Brazilian Journal of Genetics 20: (online): http://www.scielo.br/scielo.php Doyle JJ, Doyle JL (1987) A rapid D NA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19:11 15 Duble (1989) Seashore paspalum p86 89. In Southern turfgrasses: Their management and use. TexScape, College Station, TX Dudeck AE and Peacock CH (1985) Effec ts of salinity on seashore paspalum turfgrass. Agronomy Journal 77:47 50 Dulmage HT (1981) Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In Microbial Control of Pests and Plant Diseases 1970 1980 (Burgess HD, ed) New York, NY: Academic Press, pp 193 222 Duncan RR (1996) The environmentally sound turfgrass of the future. USGA Green Sect, Rec 34(1):9 11 Duncan RR and Carrow RN (2000a) Soon on golf courses: new seashore paspalums. Golf Course Manag 68(5):65 67 Duncan RR and Carrow RN (2000 b ) Seashore paspalum: a turfgrass for tomorrow. Diversity 16:45 46 Eapen S and Rao PS (1982) Callus induction and plant regeneration from immature embryos of rye and triticale. Plant Cell Tissue Organ Cult 1:221 227 Eapen S and Rao PS (1985) Plant regeneration from immature inflorescence callus culture of wheat, rye and triticale. Euphytica 34:153 159 Eizenga GC and Dahleen LS (1990) C allus production, regeneration and evaluation of plants from cultured inflorescence of tall fescue. Plant Cell, Tissue and Organ Culture 22:7 15 EPA (1998a) EPA Registration Eligibility Decision (RED) Bacillus thuringiensis EPA 738 R 98 004, March 1998. W ashington DC: Environmental Protection Agency

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83 EPA (1998b) (RED Facts) Bacillus thuringiensis EPA 738 F 98 001. Washington DC: Environmental Protection Agency Fennell S, Bohorova N, Ginkel MV, Grossa J, Hoisington D (1996) Plant regeneration from immature embryos of 48 elite CIMMYT bread wheat. Theor Appl Genet 92:163 169 Ferr J, Escriche B, Bel Y, Van Rie J (1995) Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins. FEMS Microbiol Lett 132:1 7 Ferr J and Van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis Annu Rev Entomol 47:501 533 Ferry N, Edwards MG, Gatehouse J, Capell T, Christou P, Gatehouse AMR (2006) Transgenic plants for insect pest control: a forward lookin g scientific perspective. Transgenic Res. 15:13 19 Filippov M, Miroshnichenko D, Vernikovskaya D, Dolgov S (2006) The effect of auxins, time exposure to auxin and genotype on somatic embryogenesis from mature embryos of wheat. Plant Cell Tissue and Organ C ulture 84:213 222 Finer JJ, Vain P, Jones MW, McMullen MD (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11 : 323 328 Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink C L, Fry JS, Galluppi GR, Goldberg SB, Hoffmann NL, and Woo SC (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci US 80:4803 4807 Frame BR, Shou HS, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Peg SEK, Li B, Nettleton DS, Pei D, Wang K (2 002) Agrobacterium tumefaciens mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13 22 Franklin CL, Trieu TN, Gonzales RA (1990) Plant regeneration through somatic embryogenesis in the forage grass Caucasian bluestem (Bothriochloa caucasica). Plant Cell Rep 9:443 446 Frutos R, Rang C, Royer M (1999) Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit Rev Biotechnol 19:227 276 Fu D, Tisserat Ned A, Xiao Y, Settleb D, Muthukrishnan c S, Liang GH (2005) Overexpression of rice TLPD34 enhances dollar spot resistance in transgenic bentgrass. Plant Sci 168:671 680 Ge Y, Wang ZY (2006) Bermudagrass ( Cynodon spp.). Methods Mol Biol 344:47 54 George L and Eapen S (1990) High frequency plant regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn). Euphytica 48:269 274

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84 Gill SS, Cowles EA, Pietrantonio PV (1992) The mode of action of Bacillus thuring iensis endotoxins. A Rev Entomol 37:615 636 Goldman JJ, Hanna WW, Fleming GH, Ozias Akins P, (2004) Ploidy variation among herbicide resistant bermudagrass plants of cultivar TifEagle transformed with the bar gene. Plant Cell Rep 22:553 560 Gondo T, Tsurut a S I, Akashi R, Kawamura O, Hoffma n F (2005) Green herbicide resistant plants by particle inflow gun mediated gene transfer to diploid bermudagrass (Paspalum notatum). J Plant Physiol 162:1367 1375 Gonzlez JM, Freiro E, Jouve N (2001) Influence of genoty pe and culture medium on callus formation and plant regeneration from immature embryos of Triticum turgidum Desf. Cultivars. Plant Breeding 120:513 517 Greenplate JT (1999) Quantification of Bacillus thuringiensis insect control protein Cry1Ac over time in Bollgard cotton fruit and terminals. J Econ Entomol 92, 1377 1383 Ha SB, Wu FS, Thorne TK (1992) Transgenic turf type tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts. Plant Cell Rep 11 : 601 604 Ha CD, Lemaux PG, Ch o M J (2001) Stable transformation of a recalcitrant Kentucky bluegrass (Poa pratensis L.) cultivar using mature seed derived highly regenerative tissues. In Vitro Cell Dev Biol Plant 37:6 11 Hagio T, Hirabayashi T, Machii H, et al. (1995) Production of fe rtile transgenic barley (Hordeum vulgare L.) plant using the hygromycin resistance marker. Plant Cell Rep 14:329 334 Allah et al ) pp147 180 (Marcel Dekker, New York) Hardwood WA, Ross SM Cilento P, Snape JW (2000) The effect of DNA/gold particle preparation technique, and particle bombardment device, on the transformation of barley ( Hodeum vulgare ). Euphytica 111:67 76 Hartman CL, Lee L, Day PR, Tumer NE (1994) Herbicide resistant turfgr ass (Agrostis palustris Huds.) by biolistic transformation. Bio/Tech 12:919 923 Hauptmann RM, Vasil V, Ozias Akins P, Tabaeizadeh Z, Rogers SG, Fraley RT, Horsch RB, Vasil IK (1988) Evaluation of selectable markers for obtaining stable transformants in the Gramineae. Plant Physiol 86:602 606 Haydu JJ and Hodges AW (2002) Economic impacts of the Florida golf course industry. Institute of Food and Agricultural Sciences, Gainesville, Florida: University of Florida Hiei Y, Ohta S, Komari T, Kumashiro T (1994) E fficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T DNA. Plant J 6:271 282

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85 Hoffman GJ and Rawlins SL (1971) Growth and water potential of root crops as influenced by salinity and rela tive humidity. Agronomy Journal 63:877 880 Hoffman C, Luthy P, Hutter R, Pliska V (198 8a) Binding of the delta endotoxin from Bacillus thuringiensis to brush border membrane vesicles of the cabbage butterfly (Pieris brassicae). Eur J Biochem 173:85 91 Hoff man C, Vanderbruggern H, Hofte H, Van Rie J, Jansen S, Van Mellaert H (1988b) Specificity of Bacillus thuringiensis delta endotoxins is correlated with the presence of high affinity binding sites in the brush border membranes of target insect midguts. Proc Natl Acad Sci US 85:7844 7848 Horn ME, Shil lito RD, Conger V, Harms CT (198 8) Transgenic plants of orchardgrass (Dactylis glomerata L.) from protoplasts. Plant Cell Rep 7:469 472 Ibitayo OO and Butler JD (1981) Cold hardiness of bermudagrass and Paspalum vaginatum Sw.. HortScience 16:683 684 Inokuma C, Sugiura K, Imaizumi N, Cho C (1998) Transgenic Japanese lawngrass ( Zoysia japonica Steud.) plants regenerated from protoplasts. Plant Cell Rep 17:334 338 Ishida y, Saito H, Ohta S, Hiei Y, Komari T, Kumashir o T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens Nature Biotechnol 14:745 750 Jain M, Chengalrayan K, Gallo M, Mislevy P (2005) Embryogenic callus induction and regeneration in a pentaploid hybrid be rmudagrass CV. Tifton 85. Crop Sci 45:1069 1072 Jain M, Chengalrayan K, Abouzid A, Gallo M (2007) Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants. Plant Cell Rep 26:571 590 J ames C (2005) Global status of commercialized Biotech/GM crops. ISAAA Briefs No. 34. ISAAA: Ithaca, New York James C (2006 ) Global status of commercialized Biote ch/GM crops. ISAAA Briefs No. 35 ISAAA: Ithaca, New York James VA, Altpeter F, Positano MV (20 04) Stress inducible over expression of transcription factor DREB1A in bahiagrass ( Paspalum notatum Flugge). Poster Abstract 149. Plant Biology Conf. Lake Buena Vista, FL. 24 28 July 2004. Am. Soc. of Plant Biologists, Rockville, MD James VA, Neibaur I, Al tpeter F (2007) Stress inducible expression of the DREB1A transcription factor from xeric, Hordeum spontaneum L. in turf and forage grass (Paspalum notatum Flugge) enhances abiotic stress tolerance. Transgenic Res (in press)

PAGE 86

86 Jefferson RA (1987) Assaying c himeric genes in plants: The GUS gene fusion system. Plant Mol Biol Re 5 : glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901 3907 Jogeswar G, Ran dadheer D, Anjaiah V, Kavi Kishor PB (2007) High Frequency somatic embryogenesis and regeneration in different genotypes of Sorghum bicolor (L.) Moench from immature inflorescence explants. In Vitro Cell Dev Biol Plant doi: 10.1007/s11627 007 9033 x Johnso n BB and Worthington M (1987) Establishment of suspension cultures from seeds of Plains bluestem [Bothriochloa ischaemum (L.) Keng] and regeneration of plants via somatic embryogenesis. In vitro Cell Dev Biol 23:783 787 Kaur S (2006) Molecular approaches f or identification and construction of novel insecticidal genes for crop protection. World J of Micro & Biotech 22:233 253 Kavi Kishor PB, Rao AM, Dhar AC, Naidu KR (1992) Plant regeneration in tissue cultures of some millets. Ind J Exp Biol 30 : 729 733 Kjel gren R, Rupp L, Kilgren D (2000) Water conservation in urban landscapes. HortScience 35(6):1037 1040 Krishnaraj S and Vasil IK (1995) Somatic embryogenesis in herbaceous monocots. In: TA Thorpe (ed), In vitro Embryogenesis in Plants pp417 470. Kluwer, Dord recht Krumbiegel Schroeren G, Finger J, Schroeren V, Binding H (1984) Embryoid formation and plant regeneration from callus of Secale cereal. Z Pflanzenzucht 92:89 94 Kumar, P.A., Sharma, R.P. and Malik, V.S. (1996) The insecticidal crystals proteins of Ba cillus thuringiensis Adv Appl Microbiol 42, 1 43 Lazer MD, Collins GB, Vian WE (1983) Genetic and environmental effects on the growth and differentiation of wheat somatic cell cultures. J Heredity 74:353 357 Lee L (1996) Turfgrass biotechnology. Plant Sci 115:1 8 Lee L, Hartman C, Laramore C, Tumer N, Day P (1995) Herbicide resistant creeping bentgrass. USGA Green Selection Record 33:16 18 Lee G, Duncan RR, Carrow RN (2002). Initial selection of salt tolerant seashore paspalum ecotypes. Turfgrass and Envir onmental Research Online, 1(11), 1 7 http://usgatero.msu.edu/currentpastissues.htm Lee G, Duncan RR, Carrow RN (2004a) Salinity tolerance of seashore paspalum ecotypes: Shoot growth responses and criteria. HortScience 39:1138 1142

PAGE 87

87 Lee G, Duncan RR, Carrow RN (2004b) Salinity tolerance of selected seashore paspalums and bermudagrasses: Root and verdue response criteria. HortScience 39:1143 1147 Lee S H, Lee D G, Woo H S, Lee K W, Kim D H, Kwak S S, Kim J S, Kim H, Ahsan N, Myung SC, Yang J K, Lee B H (2006) Production of transgenic orchardgrass via Agrobacterium mediated transformation of seed derived callus tissues. Plant Sci 141(3):408 414 Li L, and Qu R (2002) In vitro somatic embryogenesis in turf type bermudagrass: roles of abscisic acid and gibberellic acid, and occurrence of secondary somatic embryogenesis. Plant Breeding 121:151 158 Li L and Qu R (2004) Development of highly regenerable callus lines and biolistic transformation of turf type common bermudagrass [Cynodon dactylon (L.) Pers.]. Plant Cell Rep 22:403 407 Li R, Bruneau AH, Qu R (2006) Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]. Plant Breeding 125:52 56 Liu CA, Zhang H, Vargas J, Penner D, Sticklen M (1998) Preve ntion of fungal diseases in transgenic, bialaphos and glufosinate resistant creeping bentgrass (Agrostis palustris L) Weed Sci 46:139 146 Lo PF, Chen CH, Ross JG (1980) Vegetative propagation of temperate forage grasses through callus culture. Crop Sci 20: 363 367 Lu C and Vasil IK (1982) Somatic embryogenesis and plant regeneration in tissue cultures of Panicum maximum Jacq. Am J Botany 69:77 81 Lu C Y, Chandler SF, Vasil IK (1984) S omatic embryogenesis and plant regeneration from cultured embryos of rye (Secale cereale L.) J Plant Physiol 115:237 244 Lu S, Wang Z, Peng X, Guo Z, Zhang G, Han L (2006) An efficient callus suspension culture system for triploid bermudagrass (Cynodon tra nsvaalensis X C. dactylon) and somaclonal variations. Plant Cell Tiss Organ Cult 87:77 84 Luciani G, Altpeter F, Yactayo Chang J, Zhang H, Gallo M, Meagher RL, Wofford D (2007) Expression of a minimal cry1Fa expression cassette in bahiagrass ( Paspalum nota tum var. Fluggge) confers resistance to fall armyworm [ Spodoptera frugiperda (J E Smith)]. Crop Science (in press) Maddock SE, Lancaster VA, Risiott R, Franklin J (1983) Plant regeneration from cultured immature embryos and inflorescences of 25 cultivars o f wheat (Triticum aestivum). J Exp Bot 144:915 926

PAGE 88

88 Malcom CV (1969) Saltland pastures. J Agric West Austr 10:119 122 Malcom CV and Laing IAF (1969) Paspalum vaginatum for salty seepages and lawns. Journal of Agriculture, Western Australia 11:474 475 Maximo va SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacterium mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T DNA transfer are rate limitin g. Plant Mol Bio 37:549 559 McCarty LB and Dudeck AE (1993) Salinity effects on bentgrass germination. HortScience 28:15 17 McClintock JT, Schaffer CR, Sjoblad RD (1995) A comparative review of the mammalian toxicity of Bacillus thuringiensis based pestici des. Pestic Sci 45:95 105 McGaughey WH (1985) Insect resistance to the biological insecticide Bacillus thuringiensis Science 229:193 195 Meagher RL, Gallo Meagher M (2003) Identifying host strains of fall armyworm (Lepidoptera : Noctuidae) in Florida using mitochondrial markers. Florida Entomal 86:450 455 Meagher RL, Nagoshi RN (2004) Population dynamics and occurrence of Spodoptera frugiperda host strains in southern Florida. Ecological Entomology29(5):614 620 Metzinger BD, Taliaferro CM, Johnson BB (1987) In vitro regeneration of apomitic bluestem grasses. Plant cell Tiss Organ Cult 10:31 38 Montaigne F (2002) Water Pressure. National Geographic 2002(3):2 33 Morton JF (1973) Salt tolerant silt grass (Paspalum vaginatum Sw.). 86th Proc Florida State Herr S ection 6 8:482 490 Mller Cohn J, Chaufaux J, Buisson C, Gilos N, Sanchis V, Lereclus D (1996) Spodoptera littoralis (Lepidoptera: Noctuidae) resistance to CryIC and cross resistance to other Bacillus thuringiensis crystal toxins. J Econ Entomol 89:791 797 Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473 497 Murata M (1989) Effect of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Trit icum aestivum L.) Theor Appl Genet 78:521 524 Murdock CL (1987) Water: the limiting factor for golf course development in Hawaii. USGA Green Section Record 25:11 13

PAGE 89

89 Nagoshi RN, Meagher RL (2004a) Seasonal distribution of fall armyworm (Lepidoptera: Noctuid ae) host strains in agricultural and turf grass habitats. Environ Entomol 33(3):881 889 Nagoshi RN, Meagher RL (2004b) Behavior and distribution of the two fall armyworm host strains in Florida. Florida Entomologist 87(4):440 449 Nagoshi RN, Meagher RL, Ad amczyk JJ, Braman SK, Brandenburg RL, Nuessly G (2006) New restriction fragment length polymorphism in the cytochrome oxidase I gene facilities h o st strain identification of fall armyworm (Lepidoptera: Noctuidae) populations in the southeastern United Stat es. J of Economic Entomology 99(3):671 677 methyl D glucuronidase ( uidA ). Mol Gen Genet 120:319 335 Palm CJ, Seidler RJ, Donegan KK, Harris D (1993) Transgenic plant pesticides: fate and persistence in soil. Plant Physiol S102:166 Palm CJ, Donegan KK, Harris D, Seidler RJ (1994) Quantization in soil of Bacillus thuringiensis var. kurstaki delta endotoxin from transgenic plants. Mol Ecol 3:145 151 Palm CJ, Schaller DL, Donegan KK, Seidler RJ (1996) Persistence in soil of transgenic plant produced Bacillus thuringiensis var. kurstaki delta endotoxin. Can J Microbiol 42:1258 1262 Procee dings Florida Turfgrass Management Conf 23:13 36 Peacock CH and Dudeck AE (1985) Physiological and growth responses of seashore paspalum to salinity. Hort Sci 20;111 112 Poeaim A, Matsuda Y, Murata T (2005) Plant regeneration from immature inflorescences o f zoysiagrass (Zoysia spp.). Plant Bio 22(3):245 248 Popelka JC, Altpeter F (2001) Interactions between genotypes and culture media components for improved in vitro response of rye (Secale cereale L.) inbred lines. Plant Cell Rep 20:575 582 Popelka JC, Xu J, Altpeter F (2003) Generation of rye ( Secale cereale L.) plants with low transgene copy number after biolistic gene transfer and production of instantly marker free transgenic rye. Trans Res 12:587 596 Purnhauser L (1991) Stimulation of shoot and root re generation in wheat Triticum aestivum callus cultures by copper. Cereal Res Commun 19:419 423 Qi C H, Han L B, Liang X H, Zeng H M, Liu J (2006) Transgenic Zoysia japonica plants obtained by biolistic bombardment transformation. J Beijing Forestry Universi ty 28:71 75

PAGE 90

90 Qu R and Chaudhury A (2001) Improved young inflorescence culture and regeneration of `Tifway' bermudagrass (Cynodon transvaalensis C. dactylon). Int. Turfgrass Soc. Res. J 9,198 201 Quisenberry SS (1990) Plant resistance to insects and mites in forages and turf grasses. Fla Entomol 73:411 421 Ramos Leal R, Maribona RH, Ruiz A et al. (1996) Somaclonal variations a source of resistance to eyespot disease of sugarcane. Plant Breed 115:37 42 Rao AM, Padma Sree K, Kavi Kishor PB (1995) Enhanced pla nt regeneration in grain and sweet sorghum by asparagines, proline and cefotaxime. Plant Cell Rep 15 : 72 75 Renner, R. (1999) Will Bt based pest resistance management plans work? Environ. Sci. Technol. News (1 October 1999), 410 415 Ritala A, Aikasalo R, As pegren K, Salmenkallio Martila M, Akermen S, Mannonen L, Kurtn U, Puupponen Pimiand R, Teeri TH, Kauppinen V (1995) Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristic of the transgenic barley plants. Euphyti ca 85:81 88 Ross AH, Manners JM, Birch RG (1995) Embryogenic callus production, plant regeneration, and transient gene expression following particle bombardment, in the pasture grass Cenchrus ciliaris (Gramineae). Aus J Bot 43:193 199 Roush RT (1996) Can w e slow adaptation by pests to insect transgenic crops? In G.J. Persley (ed) Biotechnology and Integrated Pest Management, pp. 242 263. Wallingford, Oxon, UK: CAB International Roush RT (1997) Bt transgenic crops: just another pretty insecticide or a chance for a new start in resistance management? Pestic Sci 51:328 334 Sachs ES, Benedict JH, Stelly DM, Taylor J, Altman DW, Berberich SA, Davis SK (1998) Expression and segregation of genes encoding CryIA insecticidal proteins in cotton. Crop Sci 38:1 11 Sacchi VF, Parenti P, Hanozet GM, Giordana B, Luthy P, Wolfersberg MG (1986) Bacillus thuringiensis toxin inhibits K gradient dependent amino acid transport across the brush border membranes of Pie res brassicae midgut cells. FEBS 204:213 218 Sanford JC, De Vit MJ, Russel JA, Smith FD, Harpending PR, Roy MK, Johnson SA (1991) An improved, helium driven biolistic device. Technique 3:3 16 Sanford JC, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzymol 217:483 509 SAS Institute Inc. (2005) Version 9.1, SAS Institute Inc., Cary, NC, US

PAGE 91

91 Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, et al. (1998) Bacillus thuringiensis and its pesticid al crystal proteins. Microbiol. Mol Biol Rev 62:775 806 Shortman SL, Braman SK, Duncan RR, Hanna WW, Engelke MC (2002) Evaluation of turfgrass species and cultivars for potential resistance to two lined spittlebug, Prosapia bicincta (Say) (Homoptera: Cerco pidae) J Econ Entomol 95:478 486 Sims SR and Martin JW (1997) Effect of Bacillus thuringiensis insecticidal proteins Cry1A (b), Cry1A (c), CryIIA CryIIIA on Folsomia candida and Xenylla grisea (Insecta: Collembola). Pedobiologia 41:412 416 Smith RL, Grand o MF, Li YY, Seib JC, Shatters RG (2002) Transformation of bahiagrass (Paspalum notatum Flugge). Plant Cell Rep 20:1017 1021 Somers DA, Rines HW, Kaeppler HF, Bushnell WR (1992) Fertile, transgenic oat plants. Bio/Technology 10:1589 1594 Somleva MN, Tomasz ewski Z, Conger BV (2002) Agrobacterium mediated genetic transformation of switchgrass. Plant Crop Sci 42:2080 2087 Songstad DD (1983) Tissue culture of the forage grass little bluestem [Schizachyrium scoparium (Michx) Nash.] MSc thesis, South Dakota State University Songstad DD, Chen CH, Boe AA (1986) Plant regeneration in callus cultures derived from young inflorescences of little bluestem. Crop Sci 26:827 829 Spangenberg G, Wang ZY, Nagel J, Potrykus I (1994) Protoplast culture and generation of transgen ic plants in red fescue ( Festuca rubra L.). Plant Sci 97:83 94 Spangenberg G, Wang Z Y, Wu X, Nagel J, Potrykus I (1995a) Transgenic perennial ryegrass (Lolium perenne) plants from microparticle bombardment of embryogenic suspension cells. Plant Science 10 8:209 217 Spangenberg G, Wang ZY, Wu XL, Nagel J, Iglesias VA, Potrykus I (1995b) Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells. J Plant Physiol 145:693 701 Sp angenberg G and Wang Z Y (1998) Biolistic transformation of embryogenic cell suspensions. In Celis JE ed. Cell biology: a laboratory handbook, vol 4, 2nd edn. New York, NY: Academic Press:162 168 Spangenberg G, Wang Z Y, Potrykus I (1998) Biotechnology in forage and turf grass improvement. Berlin: Springer Sparks AN (1979) A review of the fall armyworm. Florida Ent 62:82 87 Straub PF, Decker DM, Gallagher JL (1989) Tissue culture and regeneration of Distchlis spicata (Gramineae). Amer J Bot 76:1448 1451

PAGE 92

92 Str aub PF, Decker DM, Gallagher JL (1992) Characterization of tissue culture initiation and plant regeneration in Sporobolus virginicus (Gramineae). Am J Bot 79:1119 1125 Street HE (1979) Embryogenesis and chemically induced organogenesis. In: Plant Cell and Tissue Culture. Principles and Applications. W. R. Sharp, P. O. Larsen, E. F. Padock, and V. Raghavan (Eds.). Ohio State University Press, Columbus, OH Tabashnik BE (1994) Evolution of resistance to Bacillus thuringiensis Anuu Rev Entomol 39:47 79 Tabashn ik BE, Groeters FR, Finson N, Johnson MW (1994) Instability of resistance to Bacillus thuringiensis Biocontrol Sci Technol 4:419 426 Tabashnik BE, Liu Y B, Finson N, Masson L, Heckel DG (1997 a ) One gene in diamondback moth confers resistance to four Bacil lus thuringiensis toxins. Proc Natl Acad Sci US 94:1640 1644 Tabashnik BE, Liu F B, Malvar T, Heckel DG, Masson L, et al. (1997 b ) Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis Proc Natl Acad Sci US 94:12780 12785 Tabashnik BE, Liu Y B, Malvar T, Heckler DG, Masson L, Ferr J (1998) Insect resistance to Bacillus thuringiensis : uniform or diverse? Phil Trans R Soc Lond B 353:1751 1756 Tiidema A and Truve E (2004) Efficient regeneration of ferti le barley plants from callus cultures of several Nordic cultivars. Herediats 140:171 176 Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thomton S, Brettel R (1997) Agrobacterium tumefaciens mediated barley transformation. Plant J 11:1369 1376 Toyama K, Bae CH, Kang JG, Lim YP, Adachi T, Riu KZ, Song PS, Lee HY (2003) Production of herbicide tolerant zoysiagrass by Agrobacterium mediated transformation. Mol Cells 16:19 27 Trenholm LE, Duncan RR, Carrow RN (1999) Wear tolerance, shoot performance, and spectral reflectance of seashore paspalum and bermudagrass. Crop Sci 39:1147 1152 Trenholm LE, Carrow RN, Duncan RR (2000) Mechanisms of wear tolerance in seashore paspalum and bermudagrass. Crop Sci 40:1350 1357 Trenholm LE, Unruh JB (2002) Seashore paspalum for Florida lawns. University of Florida, Florida Cooperative Extension Service CIR 1244 Trifonova A, Madsen S, Olesen A (2001) Agrobacterium mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:8 71 880

PAGE 93

93 Van der Valk PM, Zaal MACM, Creemers Molenaar (1989) Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass) Plant Cell Rep 7:644 647 Van Rie J, Jansens S, Hofte H, Degh eele D, Van Mellaert H (1990a) Specificity of Bacillus thuringiensis delta endotoxins: importance of specific receptors on the brush border membranes of the midgut of target insect. Eur J Biochem 186:239 247 Van Rie J, Jansens S, Hofte H, Degheele D, Van M ellaert H (1990b) Receptors on the brush border membranes of the insect midgut as determinants of the specificity of the Bacillus thuringiensis delta endotoxins. Appl Environ Microbiol 56:1378 1385 Van Rie J, Ferr J (2000) Insect resistance to Bacillus th uringiensis insecticidal crystal proteins. In Entomopathogenic Bacteria: From Laboratory to Field Application, ed. JF Charles, A Delcluse, C Nielsen LeRoux, 4:219 236. Dordrecht/Boston/London: Kluwer. 524pp Vasil IK and Vasil V (1994) In vitro culture of cereals and grasses. P293 312. In IK Vasil and TA Thorpe (ed) Plant cell and tissue culture. Kluwer Academic Publ, Hingham, MA Vasil IK (1995) Cellular and molecular genetic improvement of cereals. p5 8. In M Terzi et al. (ed) Current Issues in plant molec ular and cellular biology. Kluwer Academic Publ, Hingham MA Wang ZY, Takamiza T, Iglesias VA, Osusky M, Nagel J, Potrykus I, Spangenberg G (1992) Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts Bio/Technology 10:691 696 Wang ZY, Hopkins A, Mian R (2001) Forage and turfgrass biotechnology. Crit Rev Plant Sci 20:573 619 Wang ZY and Ge Y (2006) Recent advances in genetic transformation of forage and turf grasses. In Vitro Cell. Dev. Biol Plant 42: 1 18 Warkentin D, Chai B, Hajela RK, Zhong H, Sticklen MB (1997) Development of transgenic creeping bentgrass (Agrostis palustris Huds.) for fungal disease resistance p153 161. In MB Sticklen and MP Benna (ed) Turfgrass biotechnology Cell and molecular g enetic approaches to turfgrass improvement. Ann Arbor Press, MI Whalon ME and Norris DL (1996). Resistance management for transgenic Bacillus thuringiensis plants. Biotechnol Dev Monitor 29:8 12 Xiao L and Ha SB (1997) Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment. Plant Cell Rep 16:874 878 Xu ZH, Wang DY, Yan g L J, Wei ZM (1984) Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica. Plant Cell Rep 3:149 1 50

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94 Yu TT, Skinner DZ, Liang GH, Trick HN, Huang B, Muthukrishnan S (2000) Agrobacterium mediated transformation of creeping bentgrass using GFP as a reporter gene. Hereditas 133 : 229 233 Zaghmout OMF, Torello WA (1992) Restoration of regeneration potential of long term cultures of red fescue (Festuca rubra L.) by elevated sucrose levels. Plant Cell Rep 11:142 145 Zhang G, Lu S, Chen TA, Funk CR, Meyer WA (2003) Transformation of triploid bermudagrass (Cynodon dactylon C. transvaalensis cv TifEagle) by means of biolistic bombardment. Plant Cell Rep 21:860 864 Zhong H, Bolyard MG, Srinivasan C, Sticklen M (1993) Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic ca llus. Plant Cell Rep 13:1 6 Zimmy J and Lrz H (1989) High frequency of somatic embryogenesis and plant regeneration of rye (Secale cereal L.). Plant Breeding 102:89 100

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95 BIOGRAPHICAL SKETCH Isaac Neibaur was born in Reno, Nevada in 1980. He grew up in J acksonville F L where he graduated from Episcopal High School in 1998. H e obtained his bachelors degree s at the University of Florida where he dual majored in East Asian languages and literature and b otany. While completing his undergraduate degree he visited China on three occasions. He graduated from the University of Florida in December of 2003. He began his MS studies at the University of Florida in 2005.