Generation, Characterization and Risk Assessment of Transgenic Apomictic Bahiagrass

Permanent Link: http://ufdc.ufl.edu/UFE0022475/00001

Material Information

Title: Generation, Characterization and Risk Assessment of Transgenic Apomictic Bahiagrass
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Sandhu, Sukhpreet
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008


Subjects / Keywords: apomictic, assessment, bahiagrass, biolistic, flow, gene, herbicide, resistant, risk, transgenic
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Bahiagrass (Paspalum notatum Flugge) is a warm season perennial species distributed in tropical and subtropical regions. It supports the cattle beef production system in Florida, where diploid sexual and tetraploid apomictic cultivars are grown widely as pasture and utility turf. Apomictic cultivars like 'Argentine' represent promising targets for genetic transformation due to their asexual seed production. The major objective of this research was to study pollen-mediated gene flow from transgenic to non-transgenic bahiagrass using herbicide resistance as a marker. Experiment involving the generation and molecular characterization of herbicide resistant transgenic bahiagrass, pollen-mediated gene transfer, breeding behavior of hybrids, and the fate of transgene(s) are described. Unlinked, minimal transgene expression cassettes (MCs) were introduced into Argentine bahiagrass by biolistic transformation. Co-expression of unlinked nptII and bar genes occurred in 19 of 20 co-transformed lines (95% co-expression frequency). Transgenic lines were morphologically identical to wild-type bahiagrass, produced viable pollen, and were resistant to 0.6% glufosinate ammonium in the greenhouse and field. Uniform seed progeny with stable integration and expression of nptII and bar was confirmed by Southern blot hybridization, ELISA and herbicide application, respectively. Open-pollination of transgenic and non-transgenic bahiagrass was conducted in the field and greenhouse at a close physical distance (0.5 to 2.5m), to ensure a detectable level of gene transfer. Gene transfer from transgenic apomictic tetraploid bahiagrass to sexual diploid bahiagrass or to apomictic tetraploid bahiagrass was 0.03% and 0.17%, respectively, under field conditions, and 0.16% for both cases under greenhouse conditions. A total of 56 hybrids were identified from 44,205 germinated seedlings derived from non-transgenic pollen receptors. Twelve of 13 hybrids examined by flow cytometer and karyotypic analysis were triploids. An apospory-linked RFLP marker was detected in 15 of 17 triploid hybrids and all 12 tetraploid hybrids analyzed. The majority of hybrids showed reduced vigor. During the first year, five of 10 hybrids flowered with viable pollen while two of the non-flowering hybrids did not persist under greenhouse conditions. Gene transfer between transgenic and non-transgenic bahiagrass can occur under field conditions; however, hybridization frequency is low compared to other transgenic cross-pollinating grasses, and the resulting hybrids display reduced fitness.
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 Sukhpreet Sandhu.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Altpeter, Fredy.
Local: Co-adviser: Blount, Ann R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022475:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022475/00001

Material Information

Title: Generation, Characterization and Risk Assessment of Transgenic Apomictic Bahiagrass
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Sandhu, Sukhpreet
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008


Subjects / Keywords: apomictic, assessment, bahiagrass, biolistic, flow, gene, herbicide, resistant, risk, transgenic
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Bahiagrass (Paspalum notatum Flugge) is a warm season perennial species distributed in tropical and subtropical regions. It supports the cattle beef production system in Florida, where diploid sexual and tetraploid apomictic cultivars are grown widely as pasture and utility turf. Apomictic cultivars like 'Argentine' represent promising targets for genetic transformation due to their asexual seed production. The major objective of this research was to study pollen-mediated gene flow from transgenic to non-transgenic bahiagrass using herbicide resistance as a marker. Experiment involving the generation and molecular characterization of herbicide resistant transgenic bahiagrass, pollen-mediated gene transfer, breeding behavior of hybrids, and the fate of transgene(s) are described. Unlinked, minimal transgene expression cassettes (MCs) were introduced into Argentine bahiagrass by biolistic transformation. Co-expression of unlinked nptII and bar genes occurred in 19 of 20 co-transformed lines (95% co-expression frequency). Transgenic lines were morphologically identical to wild-type bahiagrass, produced viable pollen, and were resistant to 0.6% glufosinate ammonium in the greenhouse and field. Uniform seed progeny with stable integration and expression of nptII and bar was confirmed by Southern blot hybridization, ELISA and herbicide application, respectively. Open-pollination of transgenic and non-transgenic bahiagrass was conducted in the field and greenhouse at a close physical distance (0.5 to 2.5m), to ensure a detectable level of gene transfer. Gene transfer from transgenic apomictic tetraploid bahiagrass to sexual diploid bahiagrass or to apomictic tetraploid bahiagrass was 0.03% and 0.17%, respectively, under field conditions, and 0.16% for both cases under greenhouse conditions. A total of 56 hybrids were identified from 44,205 germinated seedlings derived from non-transgenic pollen receptors. Twelve of 13 hybrids examined by flow cytometer and karyotypic analysis were triploids. An apospory-linked RFLP marker was detected in 15 of 17 triploid hybrids and all 12 tetraploid hybrids analyzed. The majority of hybrids showed reduced vigor. During the first year, five of 10 hybrids flowered with viable pollen while two of the non-flowering hybrids did not persist under greenhouse conditions. Gene transfer between transgenic and non-transgenic bahiagrass can occur under field conditions; however, hybridization frequency is low compared to other transgenic cross-pollinating grasses, and the resulting hybrids display reduced fitness.
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 Sukhpreet Sandhu.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Altpeter, Fredy.
Local: Co-adviser: Blount, Ann R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022475:00001

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2 2008 Sukhpreet Sandhu


3 To my husband, parents, sist er and brother-in-law for their love and unfaltering faith in me.


4 ACKNOWLEDGMENTS I first thank God Al mighty for His inspiration, support and bliss. I am especially grateful to my major advisor, Dr. Fredy Altpeter, for his continual guidance, patience, encouragement and financial support. His knowledge an d enthusiasm for work has been a great source of inspiration for me. I thank my committee members for their time and effort. Sincere thanks go to Dr. Ann Blount whose positive attitude and appreciation helped me to accomplish my goals. I thank Dr. Kenneth Quesenberry and Dr. Maria Gallo fo r their keen observations and meticulous suggestions. I especially thank Dr. Megh Singh for providing financial support, and for his commitment and belief in my capabilities. I thank all lab members for their help and cam araderie that made it an enjoyable working environment. I thank Dr. Victoria James Hurr a nd Dr. Walid Fouad for their generous assistance in research. I especially apprec iate the friendship of Isaac Neibaur, Loan Ngo, Paula Lomba and John Leeds. I thank Dr. Hangning Zhang, Jeff Seib, Eric Ostmark, Richard Fetheire, Melissa Thorpe and Jeff Jones for their assistance with labor atory and field work. I thank Dr. Kenneth Quesenberry, Dr. Lynn Sollenberger, Dr. Ernest He ibert, Dr. Victoria James Hurr and Carlos Acuna for providing necessary labor atory equipment and materials. I am grateful to Dr. Greg MacDonald for his unconditional support throughout my studies at UF. I am thankful to Bob and Harrioet Soffes for providing accommodation during my field study in Marianna, FL. I appreciate my family for their affection and encouragement to pur sue my ambitions in life.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........8LIST OF FIGURES.........................................................................................................................9ABSTRACT...................................................................................................................................10CHAPTER 1 INTRODUCTION..................................................................................................................122 APOMICTIC BAHIAGRASS EXPRESSING THE BAR GE NE IS HIGHLY RESISTANT TO GLUFOSINA TE UNDER FIELD CONDITIONS................................... 16Introduction................................................................................................................... ..........16Materials and Methods...........................................................................................................17Callus Induction...............................................................................................................17Gene Expression Cassettes..............................................................................................17Biolistic Transformation, Plan t Regeneration and Propagation...................................... 18Glufosinate Application................................................................................................... 19Greenhouse application............................................................................................ 19Field application....................................................................................................... 20Immuno-detection of PAT using a Lateral Flow Membrane Strip..................................20Southern Hybridization...................................................................................................21Results.....................................................................................................................................21Regeneration of Transgenic Plants..................................................................................21Evaluation of Herbicide Resistance................................................................................. 21Molecular Analysis of the Herbicide Resistant Lines..................................................... 23Discussion...............................................................................................................................243 CO-INTEGRATION, CO-EXPRESSION AND INHERITANCE OF UNLINKE D MINIMAL TRANSGENE EXPRESSION CASSETTES IN AN APOMICTIC TURF AND FORAGE GRASS ( Paspalum notatum Flugge)...........................................................31Introduction................................................................................................................... ..........31Materials and Methods...........................................................................................................33Gene Constructs...............................................................................................................33Plant Material, Transformation and Regeneration..........................................................33Screening of Putative Transgenic Lines for Transgene Expression................................ 34Characterization of Transgene Integrati on, Genomic DNA Isolation, Southern Blot Analysis, Polymerase Chain Reaction (PCR) and Sequence Analysis of PCR Products........................................................................................................................35


6 Quantification of Transgene Expression.........................................................................36T1 Progeny Characterization: Transgene Integration, Expression and Herbicide Resistance....................................................................................................................36Results.....................................................................................................................................37Transformation Efficiency and Transgene Integration.................................................... 37Transgene Expression...................................................................................................... 39Correlation of Transgene Expression and Transgene Integration................................... 40T1 Progeny Analysis........................................................................................................ 40Discussion...............................................................................................................................414 POLLEN-MEDIATED GENE FLOW FROM TRANSGENIC APOMICTIC TETRAPLOID BAHIAGRASS AND BREEDI NG BEHAVIOR OF INTRA-SPECIFIC HYBRIDS ...............................................................................................................................56Introduction................................................................................................................... ..........56Materials and Methods...........................................................................................................59Pollen Donor....................................................................................................................59Greenhouse Experiment Layout...................................................................................... 60Field Experiment Layout.................................................................................................61Open-pollination and Seedhead Collection..................................................................... 62Screening of Seed Progeny..............................................................................................62Hybrid Confirmation....................................................................................................... 63Transgene expression............................................................................................... 63Transgene integration............................................................................................... 63Hybrid Characterization: Phenotype...............................................................................64Hybrid Characterization: Cytogenetic.............................................................................64Flow cytometric determination of ploidy................................................................. 64Karyotypic analysis using root-tip preparations.......................................................64Hybrid Characterization: Reproductive...........................................................................65Embryo sac analysis................................................................................................. 65Pollen viability......................................................................................................... 65F2 seed production and viability............................................................................... 65RFLP marker analysis for apomixis......................................................................... 66Results.....................................................................................................................................66Transgenic Pollen Source................................................................................................ 66Anthesis and Synchrony of Flowering............................................................................67Pollen-mediated Gene Transfer from Transg enic Tetraploid to Diploid Bahiagrass...... 67Pollen-mediated Gene Transfer from Transgenic Tetraploid to Tetraploid Bahiagrass.................................................................................................................... 67Hybrids: Transgene Characterization..............................................................................68Hybrids: Cytogeneti c Characterization........................................................................... 68Hybrids: Growth and Repr oductive Characterization..................................................... 69Discussion...............................................................................................................................705 CONCLUSIONS.................................................................................................................... 84


7 APPENDIX LABORATORY PROTOCOLS USED FO R BI OLISTIC TRANSFORMATION, TISSUE CULTURE AND CHARACTERIZATION OF TRANSGENIC PARENT AND HYBRID BAHIAGRASS PLANTS............................................................................. 87Protocols for Molecular Cloning............................................................................................ 87Protocol for Particle Bombardment using PDS-1000/He....................................................89Tissue Culture Induction, Maintenance, Selection and Plant Regeneration........................... 90Molecular Techniques used in the Confir mation of Putative Transgenic Plants.................... 92Cytological Techniques used in the Character ization of Herbicide Resistant Hybrids.......... 98Buffers and Solutions.......................................................................................................... ...99LIST OF REFERENCES.............................................................................................................104BIOGRAPHICAL SKETCH.......................................................................................................117


8 LIST OF TABLES Table page 3-1 Southern blot expected band size from potential MC/concatemers................................... 47 3-2 Expression analysis of unlinked transgenes....................................................................... 47 3-3 Transgene integration and expression level based on Southern blot and E LISA.............. 48 4-1 Pollen-mediated gene transfer freque ncy from herbicide resistant apomictic tetraploid bahiagrass to diploid bahiag rass following open pollination at 0.5 to 2.5m distance between the pollen donor and recipient............................................................... 77 4-2 Pollen-mediated gene transfer freque ncy from herbicide resistant apomictic tetraploid bahiagrass to tetraploid bahi agrass following open pollination at 0.5 to 2.5m distance between the po llen donor and recipient...................................................... 77 4-3 DNA ploidy determination through flow cy tom eter and chromosome counting of F1 hybrids obtained from sexual diploid pollen r eceptors O.P. with transgenic apomictic tetraploid bahiagrass as pollen donor.................................................................................78 4-4 Embryo sac types in the F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass........................................................ 78 4-5 Growth, habit and fertility of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass........................................79


9 LIST OF FIGURES Figure page 2-1 Diagrammatic representation of the npt II and bar gene expression cass ette used for genetic transformation of bahiagrass.................................................................................282-2 Generation and evaluation of herbicide resistant bahiagrass............................................. 292-3 Southern blot analysis of transgenic bahiagrass................................................................303-1 Expression cassettes and Southern blot analysis of 21 transgenic lines............................ 493-2 PCR analysis of putative bar npt II 3-3 concatemers......................................................513-3 Sequence analysis of transgenic bahiagrass lines.............................................................. 523-4 The npt II and bar transgene expression for T0 transgenic lines........................................ 533-5 Southern blot analysis of T1 progeny plants...................................................................... 543-6 Transgene expression analysis in the T1 progeny..............................................................554-1 Pollination experiments under greenhouse a nd field conditions and screening of O.P. seed....................................................................................................................................804-2 Transgene expression and integration in F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass............................. 814-3 Karyotypic analysis of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetrap loid bahiagrass as pollen donor......................................824-4 Reproductive characterization of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass as pollen donor............... 824-5 Molecular marker analysis of F1 hybrids using RFLP marker linked to apospory in tetraploid Paspalum notatum .............................................................................................83


10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GENERATION, CHARACTERIZATION AND RISK ASSESSMENT OF TRANSGENIC APOMICTIC BAHIAGRASS By Sukhpreet Sandhu August 2008 Chair: Fredy Altpeter Co-chair: Ann R. Blount Major: Agronomy Bahiagrass ( Paspalum notatum Flugge) is a warm season pere nnial species distributed in tropical and subtropical re gions. It supports the ca ttle beef production system in Florida, where diploid sexual and tetraploid apomictic cultivar s are grown widely as pasture and utility turf. Apomictic cultivars like Argentine represent pr omising targets for genetic transformation due to their asexual seed production. The major obj ective of this research was to study pollenmediated gene flow from transgenic to non-transg enic bahiagrass using herb icide resistance as a marker. Experiment involving the generation and molecular characte rization of herbicide resistant transgenic bahiagrass, pollen-mediated gene transfer, br eeding behavior of hybrids, and the fate of transgene(s) are described. Unlinked, minimal transgene expression cassett es (MCs) were introduced into Argentine bahiagrass by biolistic transfor mation. Co-expression of unlinked npt II and bar genes occurred in 19 of 20 co-transformed lines (95% co-exp ression frequency). Tr ansgenic lines were morphologically identical to wild-t ype bahiagrass, produced viable pollen, and were resistant to 0.6% glufosinate ammonium in the greenhouse a nd field. Uniform seed progeny with stable


11 integration and expression of npt II and bar was confirmed by Southern blot hybridization, ELISA and herbicide app lication, respectively. Open-pollination of transgenic and non-tran sgenic bahiagrass was conducted in the field and greenhouse at a close physical distance (0.5 to 2.5m), to ensure a detectable level of gene transfer. Gene transfer from transgenic apom ictic tetraploid bahiagrass to sexual diploid bahiagrass or to apomictic tetraploid bahiag rass was 0.03% and 0.17%, re spectively, under field conditions, and 0.16% for both cases under green house conditions. A total of 56 hybrids were identified from 44,205 germinated seedlings deri ved from non-transgen ic pollen receptors. Twelve of 13 hybrids examined by flow cytometer and karyotypic analysis were triploids. An apospory-linked RFLP marker was detected in 15 of 17 triploid hybrids and all 12 tetraploid hybrids analyzed. The majority of hybrids showed reduced vigor. During the first year, five of 10 hybrids flowered with viable pollen while two of the non-flowering hybrids did not persist under greenhouse conditions. Gene transf er between transgenic and non-transgenic bahiagrass can occur under field conditions; how ever, hybridization frequency is low compared to other transgenic cross-pollinating grasses, and th e resulting hybrids display reduced fitness.


12 CHAPTER 1 INTRODUCTION Bahiagrass ( Paspalum notatum Flugge) is a perennial, warm season grass native to South America. It is a primary component of native grasslands in southern Brazil, Paraguay, Uruguay and northeastern Argentina (Gat es et al. 2004). In Florida, it is a popular forage grass characterized by drought tolerance, excellent persistence under cont inuous grazing, and few disease and pest problems (Gates et al. 2004) Pseudogamous apomictic tetraploid (2n=4x=40) races are the most common form of this specie s found near its center of genetic diversity. The sexual self-incompatible diploid (2n=2x=20) cy totypes, commonly called Pensacola inhabit only limited areas in its native ha bitat (Daurelio et al. 2004). Pens acola bahiagrass has become one of the major forage grasses in the southeas tern United States since its introduction in 1945 (Burton 1967; Gates et al. 2004). Bahiagrass spreads by short rhizomes. Internod es are short and leaves are crowded at the base with overlapping keeled sh eaths. The characteristic V-shaped inflorescences are the two racemes which can be ascending, recurved-divergent in some races. Solitary spikelets are borne in two rows on the rachis (Gates et al. 2004). Flowering starts from the middle and advances towards the terminal portion of th e raceme. Anthesis is completed in four to five days (Burton 1942). Anthesis in most florets occurs in the morning between 6:00-8:00AM. Weather conditions including temperature and humi dity influence the timing and rate of anthesis. Warm temperatures hasten anthesis, whereas humid conditions delay dehiscence of exerted anthers (Burton 1942). Herbage accumulation in bahiagrass shows a seasonal pattern determined by temperature, rainfall and daylength (Gates et al. 2001; Mislevy et al. 2001; Sinclair et al. 2001). Strong support from the beef industry sector has prom pted several breeding programs to target


13 bahiagrass improvement and variety development. Traditional breeding fo r bahiagrass cultivar development is based on recurrent selection for cold tolerance, earlyand late-season forage growth, and ample stolon development within th e original Pensacola germplasm. Recurrent Restricted Phenotypic Selecti on (RRPS) for yield has resu lted in altered morphology with upright growth habit and reduced rhizome developm ent in diploid cytotypes (Blount et al. 2001). Although traditional breeding procedures have been successful in obtaining higher yields and other desirable traits in the diploid sexual fo rm (Pensacola) of bahiag rass, breeding tetraploid apomictic cultivars has been difficult. Forage impr ovement of apomictic cultivars is limited due to the lack of genetic recombin ation during asexual seed produc tion (Blount et al. 2001). Sexual tetraploid bahiagrass have been artificially generated by colchine treatment of diploid seeds or calli. This novel germplasm represents valuable parent material for genetic improvement of tetraploid bahiagrass (Forbes and Burton 1961; Qu arin et al. 2001; Quesenberry and Smith 2003; Acuna et al. 2007). Genetic transf ormation offers the opportunity to utilize heterologous genes for bahiagrass improvement (Altpeter et al. 2008). Transgenic plants of the commercially important apomictic cultivar Argentine were generated to improve turf quality, drought and cold tolerance and herbicide resistance (Agharkar et al. 2007; Sandhu et al. 2007; Zhan g et al. 2007; James et al. 2008). A viable seed industry in the southeastern United States is supporte d by locally grown bahiag rass; however pasture establishment is often slow due to seed dormancy and reduced seedling vigor. These characteristics, together with the open growth habit of bahi agrass encourage weed growth. Therefore, chemical weed control or mechanical mowing is often necessary to achieve uniform weed-free pastures. However, bahiagrass is often damaged by commercially available post-


14 emergence herbicides. Hence, genetic transformati on to produce herbicide tolerant bahiagrass is a desirable approach to enhance bahi agrass utility as forage and turf. Biolistic transformation using minimal linear expression cassettes (MCs) reduces transgene loci complexity and copy number whil e supporting stable expr ession (Fu et al. 2000; Altpeter et al. 2005). However, a detailed analysis of transgen e integration, expression and inheritance in an apomictic progeny is lacking. Su ch information is also important for the risk assessment of transgenic plants, since introduc ed trait(s) can have a large effect on the introgression of transgen e(s) in nature (Felber et al. 2007). By employing vector-free constructs, the clean DNA technology enhances the bio-safety of genetically modified plants (GMPs) (Fu et al. 2000; Agrawal et al. 2005). Apomictic seed production maintains the maternal genotype in the progeny without transgene segreg ation. This is a desirable feat ure for seed production in both hybrid varieties (Hanna 1995; Sa vidan 2000) and GMPs. The obligat e or facultative nature of apomixis in P. notatum has been determined by embryo sac observations (Quarin and Hanna 1980; Quarin et al. 1982). However this met hod provides limited information on the expression of apomixis in the seed proge ny of facultative apomicts. Prog eny analysis using molecular markers overcomes this limitation (Ortiz et al. 1997; Martinez et al. 2003), although this approach, like the embryo sac analysis can be re stricted by sample size. Progeny analysis with a phenotypic marker (such as herbic ide resistance) is a better a pproach to determine percent apomictic seed production. The use of a herbicide resistance marker allows rapid screening of large populations. This feature has been exploited in pollen-mediated gene flow studies of other transgenic crops such as rice ( Oryza sativa, Messeguer et al. 2001;Chen et al. 2004), tall fescue ( Festuca arundinaceae ; Wang et al. 2004) and creeping bentgrass ( Agrostis stolonifera ; Watrud et al. 2004;).


15 A major environmental concern relating to transgenic plants is their gene flow to wild relatives. For an assessment of eco logical risks associated with tr ansgenic plants, it is important to understand the mechanism and consequences of transgene flow (Chapman and Burke 2006). Gene flow and introgression of transgenes de pend on the hybridization rate, the fitness of hybrids, and the behavior of transgene(s). Pollen -mediated gene flow studies provide important data on hybridization frequency, the potential of introgression, and further direct transgene containment strategies. Objectives The goal of this research was to evaluate the potential of gene flow in transgenic bahiagrass. The specific objectives were: 1. Generation of transgenic herbicide resistan t bahiagrass using biolistic transformation. 2. Molecular analysis of the herbicide resistant bar gene and the selectable marker antibiotic resistance gene npt II in transgenic bahiagrass and in seed progeny. 3. Determination of gene transfer rate and fate of hybrids from herbicide resistant transgenic bahiagrass to non-transgenic bahiagrass under field and greenhouse conditions.


16 CHAPTER 2 APOMICTIC BAHIAGRASS EXPRESSING THE BAR GE NE IS HIGHLY RESISTANT TO GLUFOSINATE UNDER FIELD CONDITIONS (Re-printed from Sandhu et al. 2007 with permission from Crop Science1) Introduction Bahiagrass is an im portant turf and forage gra ss in the southeastern United States and in subtropical regions around the world. Commercially important cytotypes include sexually reproducing, cross pollinating diploids (2n=2x=20 ) like Pensacola or Tifton 9 and apomictic, tetraploid (2n=4x=40) genotypes like Argentine (Gates et al. 2004). Genetic improvement of tetraploid bahiagrass cultivars has been compro mised by the absence of ge netic recombination in apomictic cytotypes. Genetic manipulation through cellular and molecular techniques will overcome this hurdle. The obligate apomict Ar gentine (Burton 1948) re presents a preferred target for genetic transformation to incorporat e herbicide resistance. Its apomictic mode of reproduction will allow generation of uniform tran sgenic seed progeny with a reduced risk of unintended gene dispersal by polle n. Bahiagrass is drought and heat tolerant and resists most insects and diseases. However, its open growth habit supports weed encroachment and its very low tolerance to commercially available herbicides make weed management a difficult task. Hence, development of herbicide resistant Argentine bahiagrass will reduce weed management problems for this low input grass. Glufosinate ammonium is a post-emergence he rbicide widely used for broad-spectrum weed control. The short half life (seven days) of glufosinat e ammonium limits soil residual activity and makes it environmentally benign. Co mmercially available glufosinate ammonium resistant crops include cotton, co rn and canola (Castle et al. 2006). Transgenic glufosinate 1 Sandhu S, Altpeter F, Blount AR (2007) Apomictic bahiagrass expressing the bar gene is highly resistant to glufosinate under field conditions. Crop Sci. 47:1691-1697


17 ammonium herbicide resistant turf and forage grasses have been reported from several species including creeping bentgrass ( Agrostis palustris Huds., Hartman et al 1994; Luo et al. 2004), bermudagrass (Cynodon dactylon Goldman et al. 2004; Li et al. 2005) and Kentucky bluegrass ( Poa pratensis L.; Gao et al. 2006). Transg enic plants expressing the bar gene in the experimental apomictic genotype Tifton 7 and the diploid bahiagrass cultivar Pensacola have been recently reported (Smith et al. 2002; Gondo et al. 2005). However, herbicide resistance was not investigated in these transgenic bahiagrass lines, other than regeneration of plants from bialaphos-containing culture medi um (Gondo et al. 2005) or leaf pa inting of primary transgenic plants with 0.4% glufosinate am monium (Smith et al. 2002). This is the first report of bar transgene expression in apomictic progeny of the commercially important bahiagrass cultivar Argentin e and identification of transgenic lines with high level herbicide resistance under bot h greenhouse and field conditions. Materials and Methods Callus Induction Em bryogenic callus was induced from germinati ng seedlings as described by Altpeter and James (2005). The callus induction medium (CIM) consisted of MS salts (Murashige and Skoog 1962), 30 g l-1 sucrose, 1.1 mg l-1 6Benzylaminopurine (BAP), 3 mg l-1 3,6-Dichloro-2methoxy benzoic acid (dicamba) and 6 g l-1 agarose (Sigma, St. Louis, Mo.), supplemented with filter sterilized MS v itamins (Murashige and Skoog 1962), which were added after the medium was autoclaved for 20 min. Calli were kept in darkness at 28C and subcultured to fresh CIM biweekly. Gene Expression Cassettes Plasm id p35S-int-nptII contains the selectable marker gene npt II gene (Bevan 1984) under the transcriptional control of the constitutive CaMV 35S promoter (Odell et al. 1985) with the


18 HSP70 intron (Rochester et al. 1986) and CaMV 35S 3UTR (Dixon et al. 1986). The pJFbar plasmid contains the bar gene (encoding phosphinothricin acetyltransferase) (Thompson et al. 1987), under transcriptional contro l of the constitutive maize ubi quitin promoter, first intron (Christensen and Quail 1996) and CaMV 35S 3UTR (Dixon et al. 1986) (Fig. 2-1). The npt II gene expression cassette was exci sed from plasmid p35S-int-nptII using Not I and Alwn I to generate a 2.5 kb fragment. The bar gene expression cassette was excised from pJFbar plasmid with IsceI flanking the expression cassettes, to produce a fragment of 2.7 kb. Following restriction digests, transgene expression cassettes were isolat ed by gel electrophoresis and the corresponding bands were excised and purified by the Wizard SV Gel and PCR cleanup system (Promega U.S., Madison, WI) to remove vector backbone sequences. Biolistic Transformation, Plant Regeneration and Propagation The npt II an d bar gene expression cassettes were us ed in a 1:2 molar ratio and coprecipitated on 1.0 m diameter gold particle s as described (Altpet er and James 2005). Embryogenic calli were placed on CIM medium s upplemented with 0.4M sorbitol, for 4-6 hours prior to gene transfer. The Bi oRad PDS-1000 / He device (Sanford et al. 1991) was used for biolistic gene transfer at 1100 psi and 28 mm Hg The bombarded calli were transferred to fresh CIM medium following the gene transfer and kept in the dark for 10 days. Then they were transferred to low li ght conditions (30Em-2s-1), with 16/8h (light/dark) photoperiod, at 28C, on selection CIM medium containing 50 mg l-1 of paromomycin. After 4 weeks, the calli were subcultured on shoot regeneration medium, si milar to CIM except for containing 0.1 mg l-1 BAP, and no dicamba. These cultures were transferred to an illuminated (150Em-2s-1) incubator with a 16/8h (light/dark) photoperiod at 28C. To indu ce root formation, calli were transferred to hormone-free CIM medium. The regenerated plantle ts were transplanted into Fafard 2 potting mix (Fafard, Inc. USA, Apopka, FL) and acclimatiz ed in growth chambers set at the conditions


19 described above, and later moved to an air-c onditioned greenhouse at 30/20C (day/night) and natural photoperiod. Plants were fertilized bi-w eekly with Miracle-Gro Lawn Food (Scotts Miracle-Gro Products Inc., Marysville, OH) at the recommended rate. Visual observations of plant morphology were recorded. Seed viability was determined by germination. Lemma and palea were removed with a scalpel to break se ed dormancy and seedlings were evaluated for transgene expression. Glufosinate Application Greenhouse application Ignite (18.1% glufosinate amm onium, activ e ingredient, AgrEvo Inc., USA, Wilmington, DE) was used for herbicide applications. Thirty-t wo putative transgenic lines were sprayed with 0.2% glufosinate ammonium using a hand sprayer. Wild-type (WT) bahiagrass (non-transgenic cultivar Argentine) was used as negative contro l and a known herbicide re sistant bahiagrass plant was used as a positive control. Visual observations of plant in jury or other symptoms were recorded 14 days after he rbicide application. Eighteen of the a bove lines were further tested at three different concentrations of glufosinate a mmonium. A completely randomized experimental design (CRD) with two replications per line and 3 treatment concentrat ions, 0.2%, 0.5% or 1.0% glufosinate ammonium was use d. One liter of the herbicide mixture containing 8 drops of Tween (Fisher Biotech, NJ, USA), was used to treat 38 individual plants. Using a hand sprayer, each plant was dosed until the herbicide ran off. Visual observations for plant injury were scored 14 days after herbic ide application. The degree of pl ant injury was scored on a 1-5 scale as follows: 1: Plant with no injury; 2: 2 to 3 leaves per plant display necrosis at the leaf tip (less than 10% of the leaf area); 3: Most leaves display between 10 to 40% leaf necrosis; 4: Most leaves display between 40 to 80% leaf necrosis; 5: All leaves display 80 to 100% leaf necrosis.


20 Field application USDA-APHIS (USDAAPHIS permit 05-365-01r) allowed evaluation and seed production of transgenic herbicide resistant lines under fiel d conditions in a designa ted plot in Marianna, Florida. Highly resistant transgenic lines 2, 3 and 33, along with the wild-type were vegetatively propagated in the greenhouse and transplanted into the field by the end of May 2006. Transgenic lines were transplanted in the center of 1m diamet er circular plots with wild-type plants located to the outside. There were 4 plants of each transgenic line per plot and each circular plot set up was replicated four times. Viable seeds were pr oduced on wild-type and transgenic bahiagrass plants during July and August following a sing le nitrogen applica tion of 30 kg hectare-1. Herbicide resistance of the above lines was tested by applying 0.3% and 0.6% glufosinate ammonium application in two pl ots per treatment on September 1st 2006. Applying 0.3% glufosinate ammonium is the recommended field ra te for control of broadl eaf and grassy weeds. The herbicide was applied till run off using a hand sprayer. Visual observations were recorded 14 days after herbicide application. Immuno-detection of PAT using a Lateral Flo w Membrane Strip An immuno-chromatographic detection kit th at uses lateral flow membrane strips (Envirologix, Quick Stix for LibertyL ink/bar) for the analysis of the bar gene product, phosphinothricin acetyltransferase (PAT) was employed in this study. Total protein was extracted with the buffer solution provided in the kit, and the prot ein concentration was estimated using the Bradford assay (Bradford 1976). Protein extracts (50l) at a concentration of 1 g l-1 were used for the assay. Protein extracts from non-transformed wild-type bahiagrass were used as a negative control. The lateral fl ow membrane assay was performed on all lines that showed no injury after application of 0.2% glufosinate a mmonium and on T1 progeny from selected lines.


21 Southern Hybridization Fresh leaf tissue (3 g) w as used to extract genomic DNA by th e CTAB method (Murray and Thompson 1980). Genomic DNA (20 g) digested with Bgl II was electrophoresed overnight at 20V on a 1.0% agarose gel in 1x TAE buffer (Sambrook and Russell 2001) and then transferred to Hybond N membrane (Amersham Ph armacia Biotech, Piscataway, N. J.) with 0.4N NaOH (Sambrook and Russell 2001). The full-length coding region of the bar gene, (0.56 kb) used as a probe was obtained by re striction digestion of pJFbar with Mlu I and XhoI followed by gel extraction (QIAquick gel ex traction kit, Qiagen Inc., Va lencia, CA). Labeling of the probe with [32P] dCTP was done using the Prime-a-gene labeling system (Promega, U.S., Madison, WI). The membrane was pre-hybridized for 4 hours at 65C and then hybridized overnight in the hybridization buffer cont aining denatured salm on sperm DNA (1 g l-1) and the labeled probe (Sambrook and Russell 2001). The membrane was washed with 0.1X SSC and 0.1% SDS at 65C prior to expos ure to Kodak Biomax MS autoradiography film at -80C for 30hrs. Results Regeneration of Transgenic Plants Growth, regeneration of plants (Fig. 2-2A) and root form a tion on paromomycin containing culture medium allowed for the selection of 32 in dependent putative transgenic plants (Fig. 22B) from a total of 300 bombarded callus pieces and a total of 10 bombardments. Evaluation of Herbicide Resistance Screening for expression of the co-transfor med bar gene initially was done by applying a foliar application of a low concentration (0.2 %) of glufosinate amm onium to the putative transformants. Preliminary experiments had indi cated that 0.2% glufosin ate ammonium was the lowest concentration that resulte d in 90-100% leaf necrosis in wild-type Argentine bahiagrass.


22 Therefore, this herbicide resistance screening was expected to contain lines with a low level of bar gene expression. Thirty lines (93.3% of the putative transgen ic plants) survived the 0.2% glufosinate ammonium application and displayed no necrosis or onl y minor leaf tip necrosis. In contrast, two lines (lines 16 and 40) displayed extreme injury symptoms similar to those shown by wild-type plants. Further testing of the level of herbicide tolerance was performed by applying 0.2%, 0.5% and 1.0% glufosinate ammonium to 18 randomly se lected herbicide tolera nt lines, as well as wild-type and one susceptible lin e (16). These lines showed differences in the response to applications of higher concentr ations of glufosinate ammonium (0.5% and 1.0%) (Fig. 2-2C and D). Highly resistant lines incl uding 2, 3, 4, 11, and 33 did not display any injury or stress symptoms even at the highest application rate of at 1.0% glufosinate ammonium (injury score 1, Fig. 2-2C). Lines 7, 10, 19, 28, and 52 showed no injury following an application of 0.2% glufosinate ammonium, and only mild or no inju ry at 0.5% glufosinate ammonium as well as minor injury symptoms at 1.0% glufosinate ammonium (scored 1.5-2). Lines 6, 21, 22, 24, 32, 36, 38 and 39 displayed moderate leaf injury (score s between 2 and 3) at the lowest glufosinate ammonium concentration, but sti ll survived applications of 0.5% or 1.0% glufosinate ammonium (Fig. 2-2D). Herbicide leaf injury in line 24 was moderate. Howeve r, this line displayed a growth inhibition following application of 0.5 or 1.0% of glufosinate ammonium. Wild-type bahiagrass showed 100% leaf necrosis at all gluf osinate ammonium application levels. Under greenhouse and field conditio ns, transgenic bahiagrass plants did not differ from wild-type plants in appearance and produced seeds which germinated into seedlings (Fig. 2-2E). The field application of 0.3% (t he recommended application rate that is equivalent to 32 fl.oz./acre applied with 15 gal water) and 0.6% glufosinate ammonium (two times the


23 recommended application rate) did not produce any in jury symptoms in any of the replications of the transgenic lines. In contrast, all the su rrounding wild-type bahiagrass plants showed 100% leaf necrosis (Fig. 2-2F and G). Also, it wa s observed, that applica tion of 0.3% and 0.6% glufosinate ammonium fully suppressed comm on weeds which co-established with the transplanted bahiagrass, including common bermudagrass ( Cynodon dactylon ), goosegrass ( Eleusine indica ), crabgrass ( Digitaria sanguinalis ), crowfootgrass ( Dactyloctenium aegyptium ), pigweed ( Amaranthus spp.), teaweed ( Sida spinosa) and wild radish ( Raphanus raphanistrum ). Molecular Analysis of the Herbicide Resistant Lines Immuno-detection of the bar gene product phosphinothricin acetyltransferase (PAT) was accom plished by a lateral flow membrane assay (Quick Stix for Liberty Link, Envirologix). This is a qualitative assay with very high detection limit. The PAT detection band is often faint in most samples, but provides a quick and easy method of protein expression in transgenic lines. All bahiagrass lines that were resistant to 0.2% glufosinate ammonium application showed the PAT-sp ecific band in the immuno-chromat ographic assay (indicated with an arrow in Fig. 2-2H ), in cont rast to the wild-type and glufos inate ammonium susceptible lines 16 and 40 which did not display this band (data not shown). Stable bar expression in apomictic progeny plants was indicated by the presence of the PAT-specific band in the immunochromatographic assay (Fig. 2-2I). Southern bl ot analysis of the genomic DNA of herbicide resistant bahiagrass lines confirmed the stable integration of the bar gene (Fig. 2-3). All lines showed a different integration pattern indicating that they were obtained from independent transformation events. Most tran sgenic lines showed complex bar gene integration patterns. An exception from this trend was line 7 w ith approximately two copies of the bar gene.


24 Discussion The genera tion of fertile, bar transgenic bahiagrass lines fr om cultivar Argentine that display high levels of resistance to glufosin ate ammonium under controlled environment and field conditions is described here. Following biol istic gene transfer, a total of 30 independent transgenic plants expressing the bar gene, as detected by herb icide tolerance and immunochromatographic assays, were regenerated from 300 pieces of embryogenic callus. This transformation efficiency (10%) compares favorably to an earlier report with 2.2% transformation efficiency in bahiagrass (Gondo et al. 2005). Smith et al. (2002) reported that more than 90% of the putative transgenic li nes displaying tolerance to 0.4% glufosinate ammonium were non-transgenic escapes. Inte restingly, application of 0.2% glufosinate ammonium was an efficient screening tool in th e experiments presented here. Expression of the bar gene was confirmed with immuno-chromatography in all plants which displayed tolerance to 0.2% glufosinate ammonium. Genotypic differenc es and alterations in tissue culture and selection protocols may have contributed to th ese contrasting findings. With prolonged culture, bahiagrass callus tends to unde rgo somaclonal variation whic h leads to a reduction in regeneration potential and nega tively affects transformation efficiency (data not shown). The importance of a short tissue culture peri od has been emphasized in earlier monocot transformation reports (e.g. Altpeter et al. 1996; Zhang et al. 2003; Gao et al. 2006). Gondo et al. (2005) have reporte d the production of a herbicide resistant diploid bahiagrass cytotype ( Paspalum notatum cv. Pensacola). However, regeneration of plants on bialaphoscontaining culture medium was the only presented evidence of herbicide resistance. In contrast, the experiments described here, focused on th e production of transgenic bahiagrass with resistance to high concentra tions of glufosinate ammonium Application of glufosinate ammonium under greenhouse cond itions at different concentr ations (0.2%, 0.5% or 1.0%)


25 identified different levels of herbicide resist ance in independent transgenic bahiagrass lines ranging from no injury to moderate injury at the highest (1.0%) glufosinate ammonium application level. Wild-type bahi agrass showed 100% leaf necrosis at all glufosinate ammonium application levels. Glufosinate ammonium is an inhibitor of glutamine synthase (GS), a key enzyme in the nitrogen assimilation pathway. GS inhibition leads to ammonia accumulation, resulting in phytotoxicity. The bar gene encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate ammoni um by acetylating its active amino group. Low level herbicide resistance in some of the lines is probably a re sult of inadequate PAT levels, thus resulting in partial leaf necrosis. Glufosinat e ammonium has also been shown to have an indirect effect on plant photosynthesis, which may be responsible for the growth inhibition observed in line 24 (Wild and Wendler 1993). Representative lines from each resistance level were selected for Southern blot analysis. Multiple transgene copies have been associated with reduced expression levels, due to transgene silencing at the transcriptional or post-transcriptional level (Wang and Waterhouse 2000). The complex transgene integrati on pattern of transgenic line 52 resulted in a similar, moderate herbicide resistance level as in line 7 that contained approximately two copies of the bar gene. Further, line 52 stably expressed the transgene in progeny seedlings as detected by immuno-chromatography despite its complex tr ansgene integration pattern. On the other hand, highly resistant lines 4, and 33, had less copies of the bar gene compared to the moderate resistant lines 28 and 52. The obser ved line to line vari ation could be due to the position of transgene insertion and may be influenced by the transcriptiona l activity of neighboring regions or by flanking endogenous regulatory seque nces (Meyer 2000). A common band of approximately 3 kb was observed in all transgenic lines, which could be due to the formation of concatemers. This rather complex transgene inte gration pattern was observed despite the use of


26 minimal cassettes without vector backbone. In c ontrast Fu et al. (2000) observed a simple integration pattern and lower copy numbers with minimal cassettes than plasmids. However, consistent with our findings, Breitler et al. (2002) also describe d highly complex transgenic loci following the use of minimal cassett es. This result suggests that th e complexity of a transgenic locus depends on factors intrinsic to the plant and not to the transgen e as stated earlier by Agrawal et al. (2005). All naturally occurring tetraploid bahiagrass genotypes are considered apomictic (Burton 1948). The commercially important apomictic cultiv ar Argentine used in this study represents the preferred target for genetic transformation to incorporate herbicide resistance. Its apomictic mode of reproduction supports the generation of uniform transgenic seed progeny with a reduced likelihood of unintended gene dispersal by pollen. Transgenic lines 2, 3 and 33 were establishe d in the field under USDA-APHIS permit no. 05-365-01r and herbicide resist ance was evaluated by applic ation of 0.3% and 0.6% of glufosinate ammonium. The app lication of 0.3% glufosinate a mmonium is the recommended application rate, and it resulted in 100% leaf necrosis of non-tran sgenic bahiagrass and all the annual and perennial weeds that co-established during the field trail. Fig. 2-2G shows the difference between the glufosinate ammonium resistant transgenic lines 2, 3 and 33 in the center and the wild-type bahiagrass plants surrounding them that display 100% necrosis. All these, transgenic bahiagrass lines had a normal phenot ype and produced viable seeds under greenhouse and field conditions. Bahiagrass seedlings easily succumb to weed pres sure until they are 5-6 tall, and are very sensitive to phenoxy herbicides (Chambliss and Ad jei 2006). Bahiagrass pasture establishment is variable due to poor seedling vigor, slow germ ination and sensitivity to soil moisture. Poor


27 stands of bahiagrass encourage weed infestation and re-seeding a poorly esta blished pasture of bahiagrass without weed control is not an effective way to in crease economic returns (Gates 2000). Most problematic is the lack of a sel ective herbicide to cont rol grassy weeds in bahiagrass. The following weeds co-established during our field trial: common bermudagrass ( Cynodon dactylon ), goosegrass ( Eleusine indica ), crabgrass ( Digitaria sanguinalis ), crowfootgrass (Dactyloctenium aegyptium ), pigweed ( Amaranthus spp.), teaweed ( Sida spinosa), and wild radish ( Raphanus raphanistrum ). All these problematic weeds displayed 100% leaf necrosis following application of 0.3% gluf osinate ammonium. This demonstrates that glufosinate ammonium resistant ba hiagrass will work effectively in weed control programs and increase the value of this low input turf and forage grass.


28 Figure 2-1. Diagrammatic representation of the npt II and bar gene expression cassettes used for genetic transformation of bahiagrass. CaMV 35 S promoterNot I BamHI Not I nptII gene expression cassette (2554 bp) npt II CaMV 35S-polyA HSP-intron probeAubi-1 promoter bar gene expression cassette (2771 bp) ubi-1 intron CaMV 35S-polyA barIsce I Isce I EcoR I probeB Bgl II


29 To T1 Line 52 2h 2i FG BE 0.2% glufosinateC 0.5% glufosinate 1.0% glufosinateD A WT 4 WT 24 WT 4 11 19 38 22 28 33 2 3 52 24 1 2 3 4 5 6 7 8 910 HI To T1 Line 52 2h 2i FG BE 0.2% glufosinateC 0.5% glufosinate 1.0% glufosinateD A WT 4 WT 24 WT 4 11 19 38 22 28 33 2 3 52 24 1 2 3 4 5 6 7 8 910 HI Figure 2-2. Generation and evalua tion of herbicide resistant ba hiagrass. A) Regeneration of plants from embryogenic bahiagrass callus following gene transfer. B) Transgenic bahiagrass plants established in soil in the greenhouse. C and D) Response of nontransgenic wild-type bahiagrass (WT) and transgenic bahiagrass line 4 or line 24 expressing the bar gene, 14 days after application of 0.2%, 0.5% or 1.0% glufosinate ammonium under greenhouse conditions. E) Seed-derived progeny plants of transgenic bahiagrass. F and G) Tran sgenic bahiagrass lines expressing the bar gene (center) surrounded by non-transgenic bahiagra ss plants before and 14 days after application of 0.6% glufosinate ammoni um under field conditions. H and I) Immunodetection of bar encoded phosphinothricin acetyltransferase (PAT) in T0 transgenic lines and seed-derived (T1) progeny of transgenic plants by lateral flow membrane assay (Libertylink Stix Test). Protein extr acts with detectable le vels of PAT display a PAT-specific band indicated by the arrow, whereas extracts from non-transgenic, wild-type (WT) bahiagrass shows a separate band, common to all lines which indicates the functionality of th e lateral flow membrane strip.


30 Figure 2-3. Southern blot analysis of transg enic bahiagrass. Genomi c DNA (20 g) from nontransgenic wild-type (WT) and transgenic bahiagrass lines (4-52) was digested with Bgl II which cuts once in the expression cassette of the bar gene (Fig. 2-1). The full length coding region of the bar gene was used as a probe and labeled with 32 PCTP. Linearized pJFbar plasmid (20 pg) was used as a positive control (PC). Lambda HindIII was used as DNA marker. 9.4 6.5 4.3 2.0kb 2.3 23.1 WT PC4 7 11 19 22 24 2833 52


31 CHAPTER 3 CO-INTEGRATION, CO-EXPRESSION AND INHERITANCE OF UNLINKE D MINIMAL TRANSGENE EXPRESSION CASSETTES IN AN APOMICTIC TURF AND FORAGE GRASS ( Paspalum notatum FLUGGE) Introduction Biolistic gene transfer is one of the most versatile plant transform ation methods (reviewed by Altpeter et al. 2005). It is the preferred met hod for simultaneous transfer of multiple genes in applications like gene stacking or pathway e ngineering because it eliminates the need for multiple Agrobacterium strains, or sequential crossing (C hen et al. 1998a; Maqbool et al. 2001; Datta et al. 2003). In contrast to Agrobacterium -mediated gene transfer, biolistic gene transfer of MCs avoids the integration of vector backbone sequences into the recipient genome (Fu et al. 2000). Vector sequences are of bacterial origin and trigger gene silencing mechanisms because of their unusual sequence composition and the inability to bind eukaryotic nuclear proteins. They often acquire dense methylation that can spread into neighboring transgenes (Jakowitsch et al. 1999). Further, vector backbone sequences may bear recombination hotspots like AT-rich sequences or origins of replication which can stimulate transgene rearrangement by plasmidplasmid illegitimate recombination (Muller et al. 1999; Kohli et al. 1999a). Persistence of prokaryotic selectable marker e xpression cassettes from vector b ackbone sequences in transgenic crops is considered problematic by most biosaf ety regulatory authoriti es. Transgene expression following biolistic transfer of minimal, linear transgene expression cons tructs lacking vector backbone sequences (MCs), has been reported in ri ce (Fu et al. 2000; Breitle r et al. 2002; Loc et al. 2002; Agrawal et al. 2005; Zhao et al. 2007), wheat (Yao et al. 2006), soybean (Gao et al. 2007); potato (Romano et al. 2003), grapevine (Vidal et al. 2006) a nd bean (Vianna et al. 2004). Plants transformed with MCs displayed lower transgene copy numbers and higher expression stability than plants transformed with whole plasmids (Fu et al. 2000; Vidal et al. 2006).


32 However, several reports describe complex tr ansgene integration patterns following biolistic transfer of MCs (Breitler et al. 2002; Romano et al. 2003; Sandhu et al. 2007; Zhao et al. 2007). Co-expression frequencies of unlinked MCs were higher than previously reported for whole plasmid transformants (Fu et al. 2000; Agrawal et al. 2005). Two MCs were co-introduced into the apomictic bahiagrass cultivar Argentine. Bahiagrass is a primary forage grass in the southeastern Unit ed States and also a popu lar low-input turfgrass. Its apomictic cytotypes may provide several benefits compared to sexua lly reproducing grasses. These benefits include uniform seed progeny an d a drastically reduced gene flow by pollen. A bahiagrass tissue culture and transformation protoc ol developed in our la boratory (Altpeter and James 2005) has been successfully used to improve turf quality (Agharkar et al. 2007; Zhang et al. 2007) and to introduce other im portant traits includi ng abiotic stress resistance (James et al. 2008), insect (Luciani et al. 2007) and herbic ide resistance (Sandhu et al. 2007). Previously, genetic transformation was reported in apomictic bahiagrass genotype Tifton 7 (Smith et al. 2002) and sexual diploid cultivar Pensacola (G ondo et al. 2005). However, a detailed study of the behavior of MCs in an apomictic species is lacking. Apomictic species produce seeds by bypassing the meiotic reduction phase and hence no genetic recombination results in uniform seed progeny. This feature is desirable for tran sgenic crops as it avoids transgene segregation. However, mitotic recombination or somatic rear rangements which are often heritable can occur in plant cells (Das et al. 1990; Tovar and Lichtenstein 1992; Wang et al. 1999). Therefore it is important to investigate transgene transmission and expression in an apomictic seed progeny. Here the results from co-integration and quant itative co-expression analyses of two unlinked MCs are reported in a population of independent primary transfor mants and their apomictic seed progeny.


33 Materials and Methods Gene Constructs Minim al linear transgene expression cassettes were used for transformation with the selectable marker genes, npt II and bar The expression cassette containing the npt II gene under the transcriptional regulation of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter (Odell et al 1985) with HSP70 intron (Rochester et al. 1986) and CaMV 35S 3UTR (Dixon et al. 1986) was excised from plasmid pHZnptII by restriction digest with Not I (Fig. 3-1A). Plasmid pJFbar carrying the bar gene under the control of th e constitutive maize ubiquitin promoter and first intron (Christensen and Quail 1996) and CaMV 35S 3UTR (Dixon et al. 1986) yielded the minimal bar gene cassettes after re striction digest with IsceI (Fig. 3-1C). The digested MCs were subject to gel electrophoresis, excised and purified with the QIAquick gel purification kit (Qiagen Inc., Valencia, CA) acco rding to the manufacturers instruction. Plant Material, Transformation and Regeneration Em bryogenic callus of cultivar Argentine was in itiated from mature seeds as described by Altpeter and James (2005). Callus induc tion medium (CIM) contained 4.3 g l-1 MS salts (Murashige and Skoog 1962), 30 g l-1 sucrose, 1.1 mg l-1 6Benzylaminopurine (BAP), 3 mg l-1 3,6-Dichloro-2-methoxy benzoic acid (dicamba), MS vitamins and 3 g l-1 phytagel (Sigma, St. Louis, Mo.) and the pH was adjusted to 5.8. Call us was maintained in darkness at a temperature of 28C and subcultured to fresh CIM medium bi weekly. Six wks after cu lture initia tion, calli were transferred to CIM supplemented with 0.4 M sorbitol, for four to six hrs before transformation. Linear minimal npt II and bar gene expression constructs were co-precipitated on 1.0m gold particles in a 1:2 molar ra tio. Gold particles coated with npt II MC and bar MC were delivered to the target tissue, using a DuPont PDS-1000/He devi ce (Sanford et al. 1991) at 1100 psi and 28mmHg. The preparation of MCs, DNA coating of gold particles, and particle


34 bombardment were performed as described in Altpeter and Sandhu (2008). Briefly, DNA was precipitated onto 1.8 mg gol d particles (60 mg ml-1 stock) using 0.1 M spermidine and 2.5 M calcium chloride. The amount of DNA delivered to the target tissue on a per shot basis was 55.5 ng npt II MC and 120.4ng bar MC. Calli were transferred to CIM medium after microprojectile bombardment, and cultured for 10 days in darkness at 28C, followed by two biweekly subcultures on selective CIMmedium containing 50 mg l-1 paramomycin and 6 g l-1 agarose at 30mol-2s-1 light intensity with 16/8-h (light/dark) photoperiod, at 28C. Following four wks of selection, actively growing calli were transferred to shoot rege neration medium containing MS salts and vitamins, 30 g l-1 sucrose, 0.1 mg l-1 BAP, and no dicamba and cultured at 150mol-2s-1 light intensity with a 16/8-h (light/dark) photope riod at 28C. Two wks, later regenerating calli were placed on hormone-free shoot regeneration medium to enha nce root formation and shoot elongation. Regenerated plantlets with roots were transplant ed to pots filled with Fafard 2 potting mix (Fafard, Inc., Apopka, FL) and cultiv ated in an air-conditioned greenhouse at 28C/20C day/night temperatur e with a natural photoperiod. Screening of Putative Transgenic Lines for Transgene Expression Putativ e transgenic lines were screened fo r the expression of the selectable marker npt II gene using a commercially available enzymelinked immunoadsorbent assay (ELISA) assay (NPTII ELISA, Agdia Inc. Elkhart, IN). The as say was performed according to manufacturers instructions, using 20 g of total protein pe r well. Transgene expre ssion was determined by visual observation. Expression of the bar gene was determined by foliar application of 0.2% glufosinate ammonium as desc ribed in Sandhu et al. (2007).


35 Characterization of Transgene Integratio n, Genomic DNA Isolation, Southern Blot Analysis, Polymerase Chain Reaction (PCR) and Sequen ce Analysis of PCR Products Total genomic DNA was extracted from leaf ti ssue using the CTAB pr otocol (Murray and Thompson 1980). DNA (20g) from transgenic line s and nontransformed wild-type bahiagrass was digested with restriction enzymes having on e recognition site in the expression cassettes ( BamHI for npt II and EcoR I and BglII for bar ). The digested genomic DNA was electrophoresed overnight, and blotted onto Hybond N membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The complete coding sequences for npt II and bar were isolated from their respective plasmids by restriction digestion and gel purification, and used as probes for Southern blot analysis. The npt II probe was isolated from pHZnptII us ing restriction enzymes flanking the npt II CDS ( BamHI and XhoI). The bar probe was isolated from pJFb ar using restriction enzymes flanking the bar CDS ( Mlu I and XhoI). For probe labeling, the Pr ime-a-gene labeling system (Promega U.S., Madison, WI) was used with 25ng of probe DNA and [32P] dCTP. The hybridization procedure followed that de scribed in Sambrook and Russell (2001). For PCR analysis of potential concatemer s, primers designed to amplify 3-3 bar npt II integration from genomic DNA were: Forward primer 5CTCTACACCCACCTGCTGAAG3; Reverse primer 5CGTTGGCTACCCGTGATATT3. The PCR reaction was carried out with 200 ng genomic DNA using HotStarTaq DNA polymerase (Qiagen Inc., Valencia, CA) in a buffer supplied by the manufacturer. Optimized reaction conditions were as follows: denaturation at 95C for 15 min, followed by 35 cycles of 94C fo r 30 sec, 59.3C for 1 min, 72C for 30 sec and final extension at 72C for 10 min. PCR products were analyzed by electrophoresis on 1.2% agarose gels with an expected product size of 759 bp. Amplified PCR products were cloned into pDRIVE cloning vector (Qiagen Inc., Valencia, CA) using the Qiagen PCR cloning kit. Qiagen EZ competent cells were transfor med with the ligated pDRIVE ve ctor by electroporation using a


36 Biorad micropulser (Bio-Rad Laboratories In c., Hercules CA). Transformed cells were immediately placed in SOC medium and incubated at 37C for one hr following which the cells were plated on LB agar plate with the antibiotic kanamycin (50 mg l-1). Plasmids from individual colonies were purified using the QIA Mini prep kit (Qiagen Inc., Valencia CA) and further tested by PCR to confirm cloning. Samples were sequenced by MWG (MWG-Biotech Inc., High Point NC) using T7 and SP6 primer pairs and sequences were compared to the concatemeric 3-3 bar npt II template. Quantification of Protein Expression Quantita tive NPTII and PAT protein expression were analyzed with commercially available ELISA assay kits (Agdia nptII ELISA, Agdia Inc. Elkhart, IN; Envirologix LibertyLink pat/bar ELISA, Envirologix Inc ., Portland, Maine). Total protein was extracted from the second fully expanded leaf using a pr otein extraction buffer and quantified using the Bradford assay (Bradford 1976). Four independent protein extracts from two clonal plants were used for quantification and 20 g of the total protein per extract was loaded per well. The assay was performed using wild-type bahiagrass as a negative control and a known sample expressing the transgene as a positive control. Reaction ki netics were recorded at 655nm using an ELISA microplate reader (Model 680; Bi o-Rad Laboratories, Inc., Herc ules, CA). O.D values were compared within the linear rang e of the reaction kinetics at 5 00sec after additi on of the ELISA substrate. The O.D values indicative of relative expression of NPTII and PAT were plotted for 12 transgenic lines. T1 Progeny Characterization: Transgene Integration, Expression and Herbicide Resistance Ten T1 progeny plants derived from line 52 a nd 18 progeny plants derived from line 24 were used for Southern blot analysis. The pr ocedure for genomic DNA isolation, blotting and hybridization was the same as described earlier for T0 plants. Quantitative protein expression was


37 analyzed for 10 T1 progeny plants from line 52 using the T0 parent as a positive control and wildtype as a negative control. NPTII and PAT prot ein ELISAs were performed using commercially available kits (Agdia nptII ELISA, Agdia Inc. Elkhart, IN; Envirologix LibertyLink pat/bar ELISA, Envirologix Inc., Portland, ME) following the manufacturers instructions. The O.D. values read at 500 sec were plotted and statistically anal yzed using Tukeys Test ( =0.05). Herbicide resistance of T1 progeny was tested by applicati on of Ignite (18.1% glufosinate ammonium, active ingredient, AgrEvo Inc ., USA, Wilmington, DE) using line 52 T0, line 24 T0 and line 4 T0 as positive controls and wild-type as nega tive control. Ten progeny plants from line 52 and 18 progeny plants from line 24 were sprayed with 5.0% glufosinate (commercial application rate) and two wks following applicati on visual observations were made for percent necrosis compared to the T0 parent. Results Transformation Efficiency and Transgene Integration A total of 21 transgen ic plants expressing npt II were regenerated following paromomycin selection. Co-transformation of both npt II and bar was detected in 20 of the 21 transgenic lines, resulting in a 95% co-t ransformation frequency. Transgene integration patterns of all regenerated lines (lines 2, 3, 4, 6, 7, 10, 11, 16, 18, 19, 21, 22, 24, 28, 32, 33, 36, 38, 39, 40, and 52) were anal yzed. Southern blot analyses were performed following restriction digestion with e ndonucleases that cut at a single site in the transgene expression casse ttes. Unique hybridizati on patterns for individu al transgenic lines confirmed the independent nature of the transgenic lines (Fig. 3-1). Transgene integration for npt II and bar showed a weak positive linear correlation (R2=0.59). Four transgenic lines displayed a single npt II integration event, 15 transg enic lines had two to four npt II integration events and; 2 transgenic lines had more than four npt II integration events (lines 21 and 37; Fig.


38 3-1B). Fifteen transgenic lines had more than four bar integration events, five transgenic lines displayed 2-4 bar integration events and none of the bar lines had a single integration event (Fig. 3-1D and E). Line 16, not expressing the bar gene, displayed two integration events, while a non-expressing line (line 40) did not show co-integration of the bar gene, resulting in a cointegration frequency of bar and npt II of 95%. In contrast to the relatively simple integration patterns obtained for the npt II gene, Southern blots for the bar gene showed higher integration events and complex integration patter ns. Following restriction digest with EcoR I and hybridization with the bar probe, several lines showed a band of approximately 2.7 kb which suggests transgene concatemers. Restriction digestion with EcoR I was not able to differentiate between 3-3 or 3-5 homomultimerization due to similar expected hybridization band sizes of 2.72 kb or 2.77 kb for these two events. Table 3-1 summarizes the expected band size in Southern blot using different restriction enzy mes and transgene combinations. An additional Southern blot analysis using Bgl II (Fig. 3-1E) allowed differentiation between 3-3 and 3-5 homomultimerization. Bgl II like EcoR I, cleaves the entire bar expression cassette once (Fig. 31A; 3-1C), but at a differe nt position (1810 bp in the bar MC). Therefore, 3-3 orientation of bar concatemers results in a 3.6 kb band, whereas a 3-5 concatemer re sults in a 2.7 kb band, which was not detected. Transgenic lines 3, 4, 6, 7, 10, 11, 19, 21, 22, 24, 28, 33, and 52 (lines 2, 32, 36 and 39 were not included in the Southern blot) showed a 3.6 kb hybridization band, which suggests that they contain 3-3 bar gene homomultimers. PCR amplification of potential 3-3 bar or npt II homoconcatemers was not successful desp ite using various primer combinations. However, primers for heteromultimeric 3-3 bar-npt II concatemers supported generation of PCR amplification products of the expected product size in nine transgenic lines (3, 4, 18, 19, 21, 22 24, 32, and 39; Fig. 3-2). Cloning and sequence analysis of these amplification products


39 confirmed bar-npt II 3-3 concatemers in three lines (4, 18, 21). PCR products from the remaining five lines were not cloned. Sequence analysis of the PCR amplified transgene loci detected a 363 bp truncated sequence in line 18 (position 369-730) resulting in the bar 3 end missing 175 bp and the entire CaMV 3UTR. Sim ilarly a 125 bp deletion was detected in the CaMV 3UTR of line 4 (position 611-734; Fig. 3-3). In line 21 the 3 end of the bar gene was truncated by 76 bp and the entire CaMV 3 UTR was replaced by 264 bp from the ubiquitin promoter and first intron region of vector pJFbar (Fig. 3-3). Protein Expression ELISA confirm ed NPTII expression of in 21 independent transgenic lines. Co-expression of PAT in the 20 transgenic lines containing both transgenes was further studied by herbicide treatment using 0.2% glufosinat e ammonium. Nineteen of thes e 20 lines displayed herbicide resistance indicating a 95% co-expression frequency of bar and npt II. These transgenic lines had normal phenotypes, growth habits, and seed production. Twelve independent transgenic lines were randomly selected for quantitative ELISA analysis of NPTII and PAT expression. The relative protein expression wa s analyzed by plotting the O.D values over time. O.D values at the linea r phase of the curve ( 500 sec) are shown in Figure 3-4. Relative co-expression of NPTII a nd PAT was evaluated by subtracting the O.D value of the wild-type, used as negative control. The relative protein e xpression as shown in Figure 3-4, allowed clustering of the transgenic plants into th ree categories: High expressing lines (O.D of 0.6 and above); medium expre ssing lines (O.D between 0.2 and 0.6); low expressing lines (O.D less than 0.2). High NPTII expressors include lines 4, 33, 16, 19 and 24; medium expressors include lines, 11 and 33; and low expressors include lines, 52, 7, 28, 18 and 22. High PAT expressors include lines 4, 33, 11, 19, 52 and 28; medium express ors include lines 38, 7, 18 and 22; and low


40 expressors include line 24 (line 16, di d not show PAT expression). Protein expression of unlinked nptII and bar did show any correlation (R2=0.13; Table 3-3). Correlation of Transgene Integration and Protein Expression A clear correlation betw een integration ev ents and expression was not found for both npt II (R2=0.01) and bar (R2=0.1). Low or medium npt II integration events produced high protein expression in transgenic lines 19, 24 a nd 4, 33 respectively (Table 3-3). High bar integration events in transgenic lines 11, 19, 28 and 52 prod uced high PAT expression, while lines 4 and 33 with medium integration events, also displayed high protein expression (Table 3-3). Transgenic lines 11, 18, 22 and 28 with comparatively strong PAT and weak NPTII expression typically had five or more bar integration events and in all cases a lower number of npt II integration events. However, in line 24 with a single npt II integration event a nd more than five bar integration events, a high NPTII but low PAT expression was detected. Low NPTII was produced in both low (e.g. line 7) and medium npt II integration lines (e.g. line 52). In general, high protein expression was supported by both high and low transgene integration events (Table 3-3). Transgenic line 4 was the highest npt II and bar expressor despite the presence of at least one heteromultimeric 3-3 bar npt II concatemer (Fig. 3-3). NPT II expression in line 18 was 25 times lower than in line 19 with similar integration events. T1 Progeny Analysis Analysis of 10 seed-derived progeny plants from line 52 and 18 seed-derived progeny plants from line 24 showed identical bar and npt II hybridization patterns as found in their respective primary transformant (Fig. 3-5). Npt II expression level in 10 seed-derived progeny plants from line 52 showed no significant difference ( P <0.05) relative to their T0 parent (Fig. 36A). PAT expression was significantly higher fo r one of the progeny plants, but the remaining nine progeny plants showed no significant difference in expression ( P <0.05) relative to their T0


41 parent (Fig. 3-6A). In contrast to the wild-type, all 10 proge ny from line 52 survived 0.5% glufosinate without any visible injury symptoms. The progeny from line 24 showed leaf tip necrosis (Fig. 3-6B) which was comparable to the T0 parent (not shown) a nd consistent with the low PAT expression (Fig. 3-4). Discussion Behavior of two unlinked, linear m inimal gene constructs without vector backbone (MCs) is described for the apomic tic turf and forage grass Paspalum notatum Co-integration and coexpression frequenc y of the unlinked bar transgene in npt II transformants was 95%, as confirmed by Southern blot anal ysis and ELISA and herbicide re sistance screeni ng, respectively. This co-expression frequency is in the same range as reported earlier for co-integrated MCs in rice (88%; Fu et al. 2000). The co-expression fr equency of MCs in bahiagrass is higher compared to most earlier reports using unlinke d plasmids in other monocot species including 65% in Kentucky bluegrass (Gao et al. 2006), 50-79% in creeping bentgrass (Wang et al. 2003), 40% in wheat (Altpeter et al. 1996 ); 37-50% in perennial ryegrass a nd Italian ryegra ss (Dalton et al. 1999) and 18% in maize (Gordon-Kamm et al. 1990). Chen et al. (1998a) identified a clear co rrelation between molar ratios of the cotransformed, unlinked plasmids, with an incr ease from 37% to 85% co-expression when the plasmid amount of the target gene was increased fr om 1:1 to 1:12 with respect to the selectable marker gene. However, such high ratios of target gene to selectable marker gene could result in reduced transformation efficiency and higher complexity of transg enic target loci. The present study confirmed that even a 1:2 molar ratio of sel ected to non-selected MCs resulted in 95% cointegration (20 out of 21 lines showed co-integration of bar and npt II genes) and co-expression (19 out of 20 lines with co-integrated bar and npt II genes co-expressed both transgenes). Cotransformation with a 1:3 mola r ratio of two unlinked plasmids resulted in 87% co-


42 transformation and 59% co-expressi on frequency in transgenic ri ce (Vain et al. 2002). Complex transgenic loci containing multiple intact and/or truncated copies were observed in transgenic rice following biolistic transfer of plasmids (Vain et al. 2002). Such complex loci and higher copy number have been associated with low tr ansgene expression due to silencing or other transcriptional limitations (Matzke et al. 1994, Vain et al. 1999; Allen et al. 2000; Vain et al. 2002). Biolistic transfer of MCs has resulted in simp ler transgene integrati on patterns with higher expression stability than using whole plasmids (F u et al. 2000; Vidal et al. 2006). Southern blot analysis of bahiagrass revealed a more complex tr ansgene integration pattern for the non-selected bar than for the selected npt II MC. The high co-expression frequency of both transgenes (95%) suggests that there is an absence of a selectiv e advantage for low complexity. Interestingly, the lower quantity of npt II compared to bar MCs used during transformation seems to be correlated to the copy number as proposed earlier by Vain et al. (2002) for co-tra nsformation of unlinked plasmids. Transgene copy number could be posit ively or negatively related to its expression (Hobbs et al. 1990). Kohli et al. (1999b) also reported that in creased copy number does not always result in reduced expres sion levels or silencing. Integr ation of quantitative ELISA and Southern blot data showed that high expression of MCs in bahi agrass was independent of copy number. Most lines with high bar copy number also exhibite d high PAT expression (eg. 11, 19, 28 and 52); the only exception was low PAT expression in line 24 high bar copies. These results suggest a low gene silencing frequency in the high copy number transgenic bahiagrass lines. Functional analysis also showed th at the transgenic lines with complex integration were resistant to the commercial application rate of Ignite herbicide under greenhouse and field conditions (Sandhu et al. 2007).


43 Southern blot data suggested the presence of bar homoconcatemers in several transgenic bahiagrass lines. In addition, PCR analysis indica ted that nine out of 20 transgenic lines had heteromultimeric bar npt II concatemers. Sequence analysis of transgenic loci confirmed deletions and insertions in the 3 UTR region of the MCs consistent with the size of the PCR amplification products. The absence of geno mic DNA in these concatemers suggests that recombination events took place prior to integr ation and may be favored by the presence of potential recombination hot s pots in the CaMV 3UTR. The CaMV 3UTR was used in both, bar and npt II gene constructs which could have caused the frequent formation of heteromultimeric bar npt II concatemers. The presence of the CaMV 35S promoter resulting in concatemers and recombination hotspots in this sequence was also proposed (Kohli et al. 1999a). Elimination of the vector backbone may reduce gene clusteri ng and other transgene rearrangements by limiting the substrate availability for homologous recombination prior to transgene integration as proposed by Agrawal et al. 2005. However, the pr esence of recombinoge nic sequences within the MCs and larger quantities of MC molecules during transformation may enhance the complexity of transgenic loci. In agreement with the present fi ndings, several earlier reports also described complex transgene integration patterns following biolistic transfer of MCs (Breitler et al. 2002, Romano et al. 2003, Zhao et al. 2007). Tr ansgene loci with 3-3 concatemers may support the formation of double stranded RNA by read through transcription and RNAi induced gene silencing (reviewed in Iyer et al. 2000). In terestingly, 3-3 concatemers were confirmed in both line 18 and line 4. Despite the presence of 3-3 npt IIbar heteroconcatemers, both transgenes were highly expressed in line 4. In contrast, line 18 showed a low bar expression. Other possible sources of variati on in transgene expression include position effects, and effects caused by somaclonal variation and regulatory se quences (Butaye et al. 2005). Despite the


44 complex integration of MCs a high co-expressi on frequency was observed in bahiagrass and these earlier studies. This suggest s that most of the MCs may inse rt at independent loci, while plasmid sequences on the other hand tend to b ecome physically and gene tically linked (Perucho et al. 1980; Lyznik et al. 1989). Co-transformed, unlinked plasmids typically co -segregate as a singl e genetic locus (Hadi et al. 1996; Chen et al. 1998b). In contrast, the vast majority of co-integrated, unlinked MCs segregated as independent genetic loci (Romano et al. 2003; Agrawal et al. 2005). Cosegregation of multiple transgene expression casse ttes as a single genetic locus is critically important for gene stacking and pathway engineeri ng. Therefore, the report ed segregation of cointegrated unlinked MCs as independent genetic loci presents a severe limitation of the MC technology in sexually reproducing sp ecies. In contrast, transformation of apomictic species like bahiagrass with MCs allows gene stacking and overcomes this limitation. However, segregation of single transgenes from a co-transformed, unlinke d selectable marker gene may be desirable to simplify the regulatory process and improve consumer acceptance (Rosellini and Veronesi 2007). This approach has been used in generatin g marker free transgen ic plants (reviewed by Ebunima et al. 2001 and Darbani et al. 2007). Hen ce, concerns about selectable marker genes need to be addressed differently in apomictic species. Alternatives include marker removal by site-specific recombination (H are and Chua 2002), genetic progr amming (Verweire et al. 2007), marker free regeneration of transgenic plants (Popelka et al. 2003) and use of alternative selection markers (Joersbo et al. 1998; Erikson et al. 2004). Gene dosage is a critical factor determini ng transgene expression and stability in the progeny. Additive effects on transgene expression were reported in homozygous transgenic rice lines; the homozygous state may be associated with lower transgene expression levels, gene


45 silencing or counter selecti on of homozygous plants across generations (James et al. 2002). Hence, T0 parents may provide little information about future transgene behavior and stability. Transgene instability caused either by inher itance or expression limits the utility of early generation testing (Breg itzer and Tonks 2003). Obligate apomictic target species like the ba hiagrass cultivar Argentine, produce uniform seed progeny without segregation of genetic lo ci due to the bypassing of meiosis and hence maintain a hemizygous state of the transgene in the progeny. This system will increase transgene stability in the progeny and will be useful fo r multigene stacking. Gene tic transformation of other apomictic grass species has be en reported in Kentucky bluegrass ( Poa pratensis L.; Ha et al. 2001; Gao et al. 2006), Dichanthium annulatum (Dalton et al. 2003; Kumar et al. 2005), buffel grass ( Cenchrus ciliaris L.; Batra and Kumar 2003) and blue grama grass ( Bouteloua gracilis ; Aguado-Santacruz et al. 20 02). However, transgene transmission in the apomictic progeny has not been analyzed in most cases a nd so far only introduction of full plasmids was described in these apomicts. Dalt on et al. (2003) reported 68-100% hph transgene expression and identical hybridization patterns in seed derived progeny from thr ee transgenic lines. Identical uid A gene integration pattern and no transgene expression in the progeny were correlated to apomictic inheritance of transgen es, but lack of expression due to transgene silencing following biolistic transfer of plasmids. Gene integration patterns identical to the parent and 100% transgene transmission in seed derived progeny fr om bahiagrass showed apomictic inheritance of bar and npt II genes. Uniform expression levels of co-transformed MCs in apomictic seed progeny and high co-expression frequency (95%) obt ained here will likely facilitate multitransgene stacking in bahiagrass.


46 Agrobacteriummediated gene transfer has been reported to result in partial vector backbone integration with frequencies between 31 and 70% (Zhang et al. 2008; Afolabi et al. 2004; Shou et al. 2004; Lange et al. 2006). Transgene integration through either Agrobacteriummediated or biolistic transfor mation has been proposed to occur through a similar mechanism involving illegitimate recombination (Zhang et al. 2008; Kohli et al. 1999b). Complex transgene loci commonly associated with biolistic transformation have al so been reported in Agrobacterium -mediated transformation (Kononov et al 1997; Zhang et al. 2008). Clean DNA technology, employing the use of MCs for biolistic transformation is not only important to increase transgene expression stab ility but may also facilitate regulatory approval for commercial release. Our results demonstrate that biolistic co -transformation of MCs supports efficient transformation of the commercially important, apomictic turf and forage grass bahiagrass. High level of transgene co-expression, uniform transg ene transmission and expression in apomictic seed progeny plants that excluded vector backbo ne sequences from the bahiagrass genome were observed.


47 Table 3-1. Southern blot expected band size from MC/concatemers Orientation MC/concatemer Restriction enzyme Probe Expected size of band (kb) bar MC EcoRI bar CDS 1.36 bar MC Bgl II bar CDS 0.96 npt II MC BamHI Npt II CDS 1.47 3-3 bar-bar EcoR I bar CDS 2.72 5-5 bar-bar EcoR I bar CDS ND 3-5 bar-bar EcoR I bar CDS 2.77 3-3 bar-bar Bgl II bar CDS 3.62 5-5 bar-bar Bgl II bar CDS ND 3-5 bar-bar Bgl II bar CDS 2.77 3-3 npt IInpt II BamHI npt II CDS 2.15 5-5 npt IInpt II BamHI npt II CDS ND 3-5 npt IInpt II BamHI npt II CDS 2.56 3-5 bar npt II EcoR I bar CDS 3.92 3-5 npt IIbar BamHI npt II CDS 3.85 3-3 bar npt II EcoR I bar CDS 3.92 5-3 npt IIbar BamHI npt II CDS 3.85 NDnon-detectable in Southern blot. 5-5 concatemers cannot be detected using a restriction enzyme which cuts upstream of the probe. Table 3-2. Expression analysis of unlinked transgenes Line % transgene expression npt II bar 4 100 100 33 83.15 65.31 24 72.75 7.96 19 70.22 52.24 38 50.84 38.16 11 29.21 65.31 52 10.39 54.08 7 7.36 24.08 28 3.85 68.98 18 3.51 19.39 22 3.48 41.02 Line 16 was excluded, sin ce it did not express the npt II gene. Percent transgene expression of all transgenic lines was calculated from the O.D values and using the highest expressing line as 100%.


48 Table 3-3. Transgene integration and expressi on level based on Southern blot and ELISA Integration Low (1-2 hybridization signals) Medium (3-5 hybridization signals) High (> 5 hybridization signals) Expression npt II bar npt II bar npt II bar Low (O.D<0.2) 7, 16, 18 28 24 Medium (O.D 0.2-0.6) 22, 38 7, 38 11 18, 22 High (O.D>0.6) 19, 24 16 4, 33 4, 33 11,19, 28, 52


49 Figure 3-1. Expression cassettes and Southern blot analysis of 21 transgenic lines. A) npt II gene xpression cassette (2.5 kb) used for biolisti c transformation. Also shown is full-length npt II gene used as the probe. B) Southern blot for the npt II transgene. Genomic DNA from transgenic lines and wild-type were digested with Bam HI, which was unique in the npt II MCs. Full-length coding sequence of the npt II gene was used as probe. PC refers to the positive control which was lin earized plasmid pHZnptII (5.1 kb); NC, the negative control was untransfor med wild-type bahiagrass. C) bar gene expression cassette (2.7 kb) used for biolistic transf ormation. Full-length coding sequence of the bar gene was used as probe. D) Southern blot for the bar gene with Eco RI. Genomic DNA was digested which has a single site in the bar gene expression cassette. Linearized pJFbar plasmid (9.8 kb) was used as positive control and wild-type bahiagrass genomic DNA was the negative control. E) Southern blot for the bar gene prepared using restriction enzyme BglII, which has a unique site in the bar MC. The restricted genomic DNA was electrophores ed, blotted and hybridized with the bar gene CDS following standard procedures. Lamda HindIII DNA marker was used to assess band size .CaMV 35 S promoterNot I BamHI Not I npt II gene expression cassette (2554 bp) npt II CaMV 35S-polyA HSP-intron probeAPC 4 7 11 192224 2833 523 6 101618213239 38 40 362NC kb PC 4 7 1119 22242833523 6 10 16182132393840362 NC 2.3 9.4 6.5 4.3 23.1 B23.1 kb 9.4 6.5 4.3 2.3D Dubi-1 promoter bar gene expression cassette (2771 bp) ubi-1 intron CaMV 35S-polyA barIsce I Isce I EcoR I probeC Bgl II


50 Figure 3-1. contd. 346 7 10 111618 19 21 22 24 2833 38 4052PC NC kb 2.3 9.4 6.5 4.3 23.1 E


51 Figure 3-2. PCR analysis of putative bar npt II 3-3 concatemers. Prim er pairs were designed to amplify 3-3 bar npt II concatemers from the genomic DNA of T0 transgenic lines. Forward primer 5CTCTACACCCACCT GCTGAAG3; Reverse primer 5CGTTGGCTACCCGTGATATT3. The e xpected product size was 759bp. L1kb ladder;WT-wild-type and; NC-negative control L 3 4 6 7 10 11 16 181921 2224 L28323339 38 252 WTNC L 3 4 6 7 10 11 16 181921 2224 L28323339 38 252 WTNC


52 Figure 3-3. Sequence analysis of transgenic bahiag rass lines.The primers were designed for a 33 barnpt II gene concatemer with forward primer in the bar gene and reverse primer in the npt II gene. The PCR product sequence was aligned with the concatemer sequence (as described in the diagram) using BLAST and mismatched sequence stretches were separately a ligned to configure rearrange ments in the vector DNA. probe 1 310 468 731 925 1068 1751 Ubi-1 promoter ubi-1 intron bar CaMV 35S poly A EcoRI 3.9kb npt II HSPintron CaMV 35S promoter probe 1 310 468 731 925 1068 1751 Ubi-1 promoter ubi-1 intron bar CaMV 35S poly A EcoRI 3.9kb npt II HSPintron CaMV 35S promoter line 21310ctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccga gcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactg468 468[gggagatctggttgtgtgtgtgtgcgctccgaacaacacgaggttggggaaagagggtgtggagggggtgtctat ttattacggcgggcgaggaagggaaagcgaaggagcggtgggaaaggaatcccccgtagctgccggtgccgtgaga ggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgccacgcaatttctggatgccgacagcggagca agtccaacggtggagcggaactctccgccccaattc] 730 731tagggataacagggtaatgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaa atacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcac acatattatggagccagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcg ataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacg1068line 18310 ctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtc369 369{ atcgggctgcccaacgacccgagcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccg gcttcaagcacgggaactggcatgacgtgggtttctggcagctggacttcagcctgccggtaccgccccgtccggtcctgcccgtca ccgagatttgactcgagtttctccataataatgtgtgagtagttcccagataagggaattagggttcctatagggtttcgctcatgtgttga gcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaaat ccagatcccc } 730 731ctagggataacagggtaatgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttaca aatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactc acacattattatggagccagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcgg cgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacg1068line 4 310tctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccgag cgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgt gggtttctggcagctggacttcagcctgccggtaccgccccgtccggtcctgcccgtcaccgagatttgactcgagtttctccataataa tgtgtgagtagttcccagataagggaattagggttcctataggg610 611{ gtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaa ccaaaatccagtactaaaatccagatccccctag } 734 735gataacagggtaatgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatac aaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacat tattatggagcagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgatacc gtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacg1068[ ] parentheses indicate inserted base pairs where bold base pairs indicate an insertion matching a 264bp sequence from the ubiquitinpromoter and first intron region of vector pJFBarin replacement of the region 468 to 731 shown in the graph above. { } parentheses indicate deleted base pairs where underlined basepairsare the missing base pairs


53 Figure 3-4. The npt II and bar transgene expression levels for T0 transgenic lines. NPTII and PAT ELISAs were performed as per manufacturers instructions using wild-type nontransformed bahiagrass as the negative c ontrol and known transgene expressing plant as positive control. The O.D values (655nm) obtained after 500 seconds of addition of ELISA substrate are plotted for relative expression of npt II and bar transgenes. 0 0.3 0.6 0.9 1.2 1.54163324193811527281822 nptII bar Independent Transgenic LinesTransgene Expression O.D. (655nm) at 500 sec.


54 Figure 3-5. Southern blot analysis of T1 progeny plants. Southern blot analysis for characterization of T1 progeny plants was as described for T0 plants in Fig. 3-1. A) npt II transgene integration pattern of T0 and 10 T1 progeny plants obtained from line 52). B) bar transgene integration pattern of T0 and 10 T1 progeny plants obtained from line 52. C) npt II transgene integration pattern of T0 and 18 T1 progeny plants obtained from line 24. D) bar transgene integration pattern of T0 and 18 T1 progeny plants obtained from line 24 Lambda HindIII was used as DNA marker. 23.1 6.5 4.3 9.4 2.3 WT T01 2 3 4 56 78 9 10PC A kb 23.1 6.5 4.3 9.4 2.3 WT T01 2 3 4 56 78 9 10PC A 23.1 6.5 4.3 9.4 2.3 WT T01 2 3 4 56 78 9 10PC A kbPCT01 2 3 4 56 78 910WT 4.3 9.4 2.3 6.5 B kb PCT01 2 3 4 56 78 910WT 4.3 9.4 2.3 6.5 B kb23.1 6.5 9.4 PC T01 2 3 4 56 78 91011 12 13 14 15 16 17 18 WT C 4.3 2.3 kb 23.1 6.5 9.4 PC T01 2 3 4 56 78 91011 12 13 14 15 16 17 18 WT C 4.3 2.3 kbkb 9.4 PC T01 2 3 4 56 78 91011 12 13 14 15 16 17 18 WT D 23.1 6.5 4.3 2.3 kb 9.4 PC T01 2 3 4 56 78 91011 12 13 14 15 16 17 18 WT D 23.1 6.5 4.3 2.3


55 Figure 3-6. Transgene expression analysis in T1 progeny. A) Npt II and bar transgene expression for T1 progeny plants from line 52. NPTII a nd PAT ELISA was performed following manufacturers instructions using wild-type non-tr ansformed bahiagrass plant as negative control and known transgene expressing plant as positive control. Tukeys test at =0.05 showed no significant difference between the T0 parent and T1 progeny except when indicated by asterisk (*). B) Herbicide resistance of T1 progeny plants. Progeny plants from line 52 and line 24 in comparison to wild-type bahiagrass 2 weeks after treating with 0.5% glufosinate. 0 0.1 0.2 0.3 0.4 T0T1T2T3T4T5T6T7T8T9T10 nptII bar ALine 52 T1 progeny=0.05O.D. (655nm) at 500 sec.Transgene Expression in T1 progeny WT 52 T124 T1B 0 0.1 0.2 0.3 0.4 T0T1T2T3T4T5T6T7T8T9T10 nptII bar ALine 52 T1 progeny=0.05O.D. (655nm) at 500 sec.Transgene Expression in T1 progeny 0 0.1 0.2 0.3 0.4 T0T1T2T3T4T5T6T7T8T9T10 nptII bar ALine 52 T1 progeny=0.05O.D. (655nm) at 500 sec.Transgene Expression in T1 progeny WT 52 T124 T1B


56 CHAPTER 4 POLLEN-MEDIATED GENE FLOW FROM TRANSGENI C APOMICTIC TETRAPLOID BAHIAGRASS AND BREEDING BEHAVIOR OF INTRA-SPECIFIC HYBRIDS Introduction Bahiagrass ( Paspalum notatum ) is the single most importa nt seed propagated pasture species in Florida (Chambliss a nd Sollenberger 1991). The most dominant cytotypes in its native South American habitat are te traploid (2n=4x=40) which produce seed asexually through apomixis (Pozzobon and Walls 1997). Naturally occu rring sexual tetr aploid plants have not been found in P. notatum (Espinoza et al. 2006). A diploid cy totype known as Pensacola bahiagrass was introduced in Florida in 1945 and is sexual a nd cross pollinating due to self-incompatibility (Quarin et al 2001). This diploid race of bahiagrass represents th e dominant cytotype naturalized in the southeastern US, but naturally occurs onl y in a small area in the Santa Fe province in Argentina (Burton 1967; Blount et al. 2002). They are allogamous self-incompatible and wind pollinated (Burton 1955). Apomixis in Paspalum notatum is characterized by apospor y, where unreduced embryo sacs develop from nucellar somatic cells. Sexual or apomictic plants can be differentiated on the basis of structure, number of embryos sacs pe r ovule and orientation of the egg cell in the embryo sac. A sexual plant has a characteristic si ngle embryo sac, with the egg cell towards the micropylar end and antipodals towards the chala zal end. Apomictic plants show multiple embryo sacs with an unreduced egg cell a nd central nuclei, but lack the an tipodal cells (Ortiz et al. 1997; Quarin et al. 2001). There are two hypotheses to explain the co mplete absence or lack of expression of apomixis in diploid cytotypes. The first hypothesis proposed by Nogler (1984) is based on the assumption that monoploid (n=x) gametes cannot transfer the apomictic trait. Apomixis is thus a monogenic dominant trait th at can only be transmitted in the heterozygous state through diploid or pol yploid gametes. Apospory in P. notatum and P. simplex is inherited as


57 a single dominant gene with tetrasomic inheritanc e and incomplete penetrance (Martinez et al. 2001). A pleiotropic lethal effect is proposed to prevent transmission of apospory through monoploid gametes (Martinez et al. 2001; Caceres et al. 2001). The second hypothesis proposed by Mogie (1988) assumes a dosage requirement for the expression of apomixis. The absence of apomixis in diploids is hence an expression effect rather than non-transmission of apomictic genes (Quarin et al. 2001). Triploid hybrids obtained from sexual 2x and apomictic 4x crosses are ideal plant material to study inheritance of apomixis in P. notatum and also to evaluate the lack of apomixis at the diploid level (Martinez et al. 2007). Molecular markers such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) completely linked to apospory have been described (Ortiz et al. 1997; St ein et al. 2004; Martinez et al. 2003). RFLP rice C1069 locus is a codominant marker mapping to the telomeric lo ng arm of chromosome 12 in rice. It is considered to be completely linked to apospory in P. simplex and P. notatum (Pupilli et al. 2001; Martinez et al. 2003). The apomictic bahiagrass cultivar Arge ntine is a preferred target for genetic transformation since its apomictic mode of reproduction should allow the production of uniform seed progeny and potentially reduce the risk of unintended gene dispersal by pollen. It is also a commercially important cultivar and genetic im provement through traditional breeding has not been possible due to the lack of female meio tic recombination during seed production. Genetic transformation of bahiagrass has been reported fro m several laboratories and improvement of the species following introduction of transgenes wa s documented. Smith et al. (2002) first reported the recovery of transgenic bahiagrass plants after bio listic introduction of bar gene into the apomictic bahiagrass accession Tifton 7. Altpeter and James (2005) described a highly efficient


58 biolistic transformation protocol for the commerci ally important, apomictic cultivar Argentine. This protocol has now been successfully used to introduce a number of important traits to Argentine including herbicide resist ance (Sandhu et al. 2007); abiotic stress tolerance (James et al. 2008) and improved turf quality (Agharkar et al. 2007; Zhang et al. 2007). Transgenic, herbicide resistant bahiagrass pl ants of the sexual diploid cul tivar Pensacola (Gondo et al. 2005) and insect resistant plants from diploid cultivar Tifton 9 have also been reported (Luciani et al. 2007). Pollen-mediated gene transfer is an e nvironmental concern regarding genetically modified (GM) plants. Transgenic crops can be broadly categorized into high, medium and low risk crops based on the reproductive biology, mo de of pollination, locat ion of the crop and production area. Crop species which are genetically distinct and in trogression is limited to the geographic center of diversity are considered very low risk crops. Wind cross-pollinated species that have a prevalence of relatives in the same genus are considered high risk crops (Stewart et al. 2003). Tetraploid to diploid gene flow may occur through the formation of triploids. Naturally occurring triploids have been found near the center of diversity for Paspalum notatum (Quarin et al. 1989; Tischler and Burson 1995) Because of the ploidy barrier very low gene transfer frequencies are expected from tetraploid to diploid bahiagrass. Detailed reproductive and cytogenetic characterization of hybr id plants will add valuable in formation for risk assessment of transgenic bahiagrass. Using herbicide resistance as a screenable ma rker allows efficient identification of rare pollen-mediated gene transfer events and can also help to monitor the mo de of reproduction in apomictic tetraploids. Embryo sac analysis using an ovule clearing technique enables the


59 differentiation of apomictic or meiotic embryo sacs (Young et al. 1979; Quarin et al. 1982; Acuna et al. 2007). However, whether meiotic em bryo sacs present in apomictic plants form viable seeds is unclear. Seed pr ogeny analysis is hence important to determine the frequency of functional apomixis or amphimixis (Ozias -Akins 2006). Any sexual reproduction in a heterozygous apomictic plant will produce off-type individuals. A quantitative assessment of the degree of sexuality versus apomixis can be estimated by the use of a dominant phenotypic marker. Flow cytometry is a widely used tech nique for rapid detecti on of DNA ploidy (Valkonen 1994; Taliaferro et al. 1997; Dolezel et al. 1997; Roux 2003; Dolezel et al. 2007). It has been applied for large-scale comparative analyses of genome size, repr oductive biology, taxonomic identification and delineation (reviewed by Kron et al. 2007 and Ochatt 2008). The flow cytometeric seed screen (FCSS; Matzk et al. 2000) is an alternative method to establish apomixis or amphimixis mode of reproduction. The objectives of this work were to determin e the frequency of gene flow from transgenic apomictic tetraploid bahiagrass to sexual diploi d or apomictic tetraploid bahiagrass cultivars under field and greenhouse conditions, and charac terize intra-specific hybrids for transgene integration, ploidy, fertility and mode of reproduction. Materials and Methods Pollen Donor The pollen donor for this gene flow study wa s a transgenic herbicide resistant bahiagrass line (B9). This line was generate d by Dr. Victoria James Hurr (P ost-doctoral Fellow, Department of Agronomy, University of Florida, Gainesvi lle) in our laboratory by the transformation of apomictic tetraploid bahiagrass cultivar Argent ine with pJFbar. Plasmid pJFbar contains the bar gene (Thompson et al. 1987) under the control of the constitutive maize ubiquitin promoter and first intron (Christensen and Qu ail 1996) and CaMV 35S 3UTR (D ixon et al. 1986). Transgenic


60 calli and regenerating shoots were selected fo r herbicide resistance by inclusion of 3mg l-1 bialaphos (Phytotechnologies In c., Shawanee Mission, KS). Furthe r characterization of line B9 was carried out as part of this st udy. Southern blot analysis of the bar gene was carried out by Bgl II digested genomic DNA from line B 9. Blotting and hybridization with the bar probe was carried out according to sta ndard procedures (Sambrook and Russell 2001). Viable pollen is required for apomictic seed production in Argen tine. Formation of viable seed by complete exclusion of pollen (selfing) from other plants is indicative of viable pollen production by the mother plant. The emerging inflorescences were bagged in a glassine po llination bag to exclude foreign pollen. The bags were shaken every morning (between 6:30 to 7:30 AM) to ensure pollination. The viability of self ed seeds was checked by germination. In order to confirm the apomictic seed production of line B9, a set of 30 seed-derived progeny plants were sprayed with 0.3% glufosinate i.e the recommended a pplication rate of the herbicide. Greenhouse Experiment Layout Non-transgenic bahiagrass plants were placed at a distance of 0.5 to 2.5m from transgenic bahiagrass (B9). Open-pollination under air-conditioned greenhouse conditions allowed transgene containment. The greenhouse environment also facilitated the synchronization of flowering of transgenic pollen donor plants and non-transgenic pollen receptor plants. A flex duct system ensured continuous and even airflo w supporting pollen dispersal. An ebb and flow irrigation system increased the humidity and su pported the production of high quality seed. The plants were maintained at 28C day and 23 C night temperature and natural photoperiod. Equal numbers of B9 pollen donor plants (10) were used in all experiments to generate similar pollen sources. The greenhouse experiment s to assess the hybrid ization potential of transgenic apomictic bahiagrass with non-transgenic bahiagrass were carried out in five sets in different greenhouses:


61 1) pollen-mediated gene transfer from transg enic apomictic tetraploid to a population of 30 different non-transgenic sexual di ploid bahiagrass genotypes; 2) pollen-mediated gene transfer from transgenic apomictic tetraploid to 30 clonal plants of a single diploi d genotype i.e. clone1; 3) pollen-mediated gene transfer from transgenic apomictic tetraploid to 30 clonal plants of a second single diploid genotype i.e clone21; 4) pollen-mediated gene transfer from transgenic apomictic tetraploid to three nontransgenic highly apomictic tetraploids i.e. cv. Argentine, cv. Paraguay22 and the experimental hybrid Tifton 7; and 5) pollen-mediated gene transfer from transgenic apomictic tetraploid to a non-transgenic facultative apomictic tetraploid accession with a known high degree of sexuality(determine d by embryo sac analysis). This accession was provided by Carlos Acuna (Graduate student, Department of Agr onomy, University of Florida, Gainesville, FL) for this study and is referred to as facultative tetr aploid for simplicity. Field Experiment Layout The field study was conducted for tw o consecuti ve years (2005 and 2006) at North Florida Research and Education Center (NFREC), Mari anna, FL. Transgenic apomictic pollen donor plants (24 plants plot-1) were planted in a circle of 0.5m radius. An adjace nt concentric zone of 1m was left without plants, followed by a concen tric 0.5m strip of non-transgenic bahiagrass. Two plots represented 2 replicati ons of each of the two experime nts. The first experiment was similar to greenhouse set 1 with 64 non-transgenic diploid cultivar Pensacola plants as pollen receptors planted in the outer 0.5m strip. The se cond experiment was similar to greenhouse set 4 with 64 non-transgenic highly apomictic tetrap loid bahiagrass genotypes planted as pollen receptors in the 0.5m strip. The tetraploid pollen recipients including Argentine, Paraguay 22 and 1 Clonal plants for the second and third experiment set were generated by clonal propagation of a single seedling of cultivar Pensacola in liquid culture media containing 4.3 g l -1 MS salts with vitamins (Murashige and Skoog 1962), 30 g l-1 sucrose and 0.3 mg l-1 6Benzylaminopurine (BAP).


62 Tifton 7 were selected based on their abundance and/or availability in Florid a. Total diameter of each plot was 4m. All plants were clonally propa gated in greenhouses and transplanted to the field in May, 2005. Transgenicand non-transgenic plants were ca 0.5m apart at full maturity, and maximum distance between the pollen recep tor and pollen donor was 2.5m. The field study was carried out in compliance with USDA-AP HIS guidelines (Notification no. 05-076-11n and Permit No.05-364-01r). In order to prevent seed dispersal by birds or animals, cage-like structure were built on a wooden scaffold with mosquito ne t. The four transgenic plots were separated by a distance of 30 to 88m. An isolation zone of 100 m surrounding the transgenic plots was mowed weekly. Open-pollination and Seedhead Collection Transgenic pollen donor and non-transgenic poll en receptor plants were allowed to open pollinate (O.P.) in the greenhouse and field. At an thesis, the inflorescences were marked with a colored tape to monitor synchronous flowering of transgenic pollen donors and non-transgenic pollen receptors. O.P. seed heads were bagged in glassine pollination bags 10 days after pollination to prevent seed loss (Fig. 4-1A-B). Mature seeds were harvested 30 days after pollination, air dried, and stored in double containe rs at room temperature. Seed head collection continued until the end of flowering. Screening of Seed Progeny The seeds obtained from non-transgenic plants were germ inated following acid treatment to break seed dormancy. Seedlings were grow n in germination flats containing Fafard 2 potting mix (Fafard Inc., Apopka FL) and were ma intained in the greenhouse at 28/23C day / night temperature and natural phot operiod. The herbicide concentr ation was optimized for this study to identify even those tr ansgenic hybrids expressing the transgene at a low level. A concentration of 0.14% glufosin ate ammonium (Ignite, ArgEvo Inc., Wilmington, DE) was


63 applied to seedlings until runoff at six weeks afte r germination (Fig. 4-1C-D). This is the lowest concentration of glufosinate ammonium that st ill produces 80 to 100% necrosis in non-transgenic bahiagrass. Herbicide resistant seedlings (F1 hybrids) were scored for necrosis three weeks after glufosinate application. The herbic ide resistant putative hybrid s eedlings were transplanted to pots for further characterization. Hybrid Confirmation Transgene expression Protein extracts of the herbicid e resistant putative hybrid plan ts were analyzed with the immuno-chrom atographic lateral membrane Quick Stix (LibertyLink/ bar Envirologix, Portland, ME) assay for the determination of bar gene product phosphinothricin acetyl transferase (PAT). Total protein was extracted from 300mg leaf tissue using 150 l extraction buffer (supplied with the kit). Protein extracts from non-transformed wild-type bahiagrass were used as a negative control (NC) and known transgen ic plant(s) expressing PAT (B9) was used as positive control. Transgene integration Southern blot analysis wa s carried out to evalu ate bar gene integration into the genome of the putative hybrid plants. Genomic DNA was extracted fr om fresh leaf tissu e according to the CTAB method (Murray and Thompson 1980). DNA wa s subjected to electrophoresis following digestion with Bgl II which has a unique site in the pl asmid pJFbar. Blotting and hybridization procedures were carried out following a sta ndardized protocol (Sambrook and Russell 2001). The full-length coding sequence of the bar gene was radio-labeled with [32P] dCTP using the Prime-a-gene labeling system (Promega, U. S., Madison, WI) and used as a probe.


64 Hybrid Characterization: Phenotype Herbicide resistant hybrid (F1) plants verified by Southern bl ot analysis were maintained under greenhouse conditions at 28C day, 23 C night and natural photoperiod. Visual observations for phenotypic character istics such as leaf width and growth habit (prostrate or upright) were recorded from two vege tative clones per hybrid plant. Hybrid Characterization: Cytogenetic Flow cytometric determination of ploidy F1 hybrid plants obtained from the seed progeny of O.P. sexual diploids were characterized for DNA ploidy using a Partec Ploidy Analyzer P.A (Partec North Am erica Inc., Mt. Laurel, NJ). Shoot apical meristems (ca. 5mm section) were macerated in 500l Partec CyStain UV solution A (supplied with the kit) using a clean razor blade. The material was incubated for 3 min at room temperature followed by filtrati on through 1 micron nylon mesh. Th e filtrate was stained with 1500l Partec CyStain UV solution B (supplied with the kit) for 3 min before reading. Mean peak values were recorded when cell count were ca. 10,000. The analyzer was arbitrarily calibrated using a known tetraploid bahiagrass (Argentine), and a known diploid bahiagrass cultivar (Pensacola). The peak values indicate the average of four inde pendent sample extracts. Karyotypic analysis using root-tip preparations Cleaned healthy roots (ca. 5cm ) were pretreated with 8-hydroxyquinoline followed by overnight treatment with fixative (95% ethanol: acet ic acid, 3:1 v/v). Fixe d material was stored in 70% ethanol. For root-tip preparations, ca. a 2mm section of the root-tip (excluding the root cap) was excised and chopped in 45% acetic acid followed by addition of aceto-orcein stain. A cover slip was carefully placed, a nd gently tapped to spread the cells. The material was allowed to stain for 30 min before observing under a compound microscope. Chromosomes were counted at 500x or 1000x magnification.


65 Hybrid Characterization: Reproductive Embryo sac analysis Inflorescences were co llected at anthes is and fixed in FAA (70% ethanol: 37% formaldehyde: glacial acetic acid, 18 :1:1) for 24 hrs. Pistils were di ssected from the spikelets and prepared following the ovule clear ing technique of Young et al (1979). Ovules were observed under a differential interference contrast (DIC) microscope (Nikon Melville, NY). A minimum of 20 ovules were observed for each plant analyz ed. Meiotic embryo sacs were characterized by a single embryo sac containing the egg cell and the central nuclei at the micropylar end and mass of anitpodals at the chalazal end. Apomictic embryo sacs were differentiated from sexual embryo sacs by the presence of multiple embryo sacs and also by the position of the egg cell. Pollen viability A clean glas s slide containing a drop of aceto-orcein stain was dusted with pollen from a dehiscing inflorescence. A cover slip was gently placed and observations on pollen viability were recorded using a microscope fitted with line tran sects. Viable pollen appeared dark red, while non viable pollen appeared white or faint pink. F2 seed production and viability O.P. seeds from F1 hybrid plants were collected from greenhouse grown plants. Seed heads were thrashed and fully developed seeds were manually separated from the empty seeds (i.e. seeds that are devoid of embryo, endosperm and nu cellus). Germination was facilitated by partial removal of the lemma and palea surrounding the mature seed. The seeds were soaked in water overnight before placing in soil to facilitate germination. Germinated seedlings were maintained in a growth chamber at 28/23C day/night temperature and 12 h photoperiod. The plantlets were moved to the greenhouse at the 6-le af stage and maintained at 28/23C day/night temperature and natural photoperiod.


66 RFLP marker analysis for apomixis RFLP analysis was followed as describe d by Martinez et al. (2003) with som e modifications. Following complete digestion with EcoR I, the samples were electrophoresed in a 1.5% w/v agarose gel/1xTAE overnight at 20 V. DNA was blotted onto nitr ocellulose membrane (Amersham Pharmacia Biotech, Piscataway, N. J. ) using 20x SSC following the neutral transfer procedure. The C1069 probe (Accession no. D15675) was amplified from rice ( Oryza sativa Japonica) genomic DNA using primers designed from the sequence information at the NCBI database. The PCR product was purified using th e Qiagen PCR purification kit (Qiagen Inc., Valencia, CA). Sequence analysis (MWG-Biotech Inc., High Point NC) was used to confirm the successful amplification of C1069. Following sequence confirmation, the C1069 probe was radio-labeled with [32P] dCTP using the Prime-a-gene labeling system (Promega U.S., Madison, WI). Overnight hybridiz ation with the hybridization bu ffer containing labeled probe and denatured herring sperm DNA (1 g l-1) was carried out at 65C Washing was performed at 65C and included the followi ng steps: quick wash using 2x SSC; 20 min low stringency wash using 2xSSC, 0.5% SDS; 20 min high string ency wash using 0.2x SSC, 0.1% SDS. The membrane was exposed to autoradiographic film (Kodak Biomax MS autoradiography) for 48 h at -80C. Results Transgenic Pollen Source Single copy bar gene integration in pollen donor line B9 was detected by Southern blot analysis (Fig. 4-2B). Dark red stained pollen indicated the production of viable pollen in the transgen ic line B9. Transgenic lin e B9 did not differ from wild-type in growth and seed set. This line produced viable seeds under open and self po llination. All 30 seed-derived progeny plants


67 were herbicide resistant to 0.3% glufosinate which is the recommende d rate for control of grassy weeds (data not shown). Anthesis and Synchrony of Flowering Weekly tagg ing of pollinating seedheads suppor ted monitoring of synchrony of flowering of transgenic and non-transgenic plants and iden tification of the peak flowering period. Seeds formed following this peak period were selected for seed progeny analysis. For each experiment, there were more than 30 inflorescences per genotype during this peak period. Pollen-mediated Gene Transfer from Transg enic Tetraploid to Diploid Bahiagrass The first greenhouse experim ent with a populatio n of diploid genotypes as pollen receptors showed 0.16% (13 herbicide resistant hybrids out of 8330 seedlings; Table 4-1) gene transfer from the transgenic apomictic pollen donor. The response of two clonal genotypes in the second and third experiment sets was different de pending on the genotype. Clone 1 showed a hybridization rate of 3.6% (12 herbicide resistant hybrids out of 332 seedlings) whereas clone 2 did not produce any hybrids (none out of 5209 seed lings; Table 4-1). It wa s also observed that clone 1 (1900 seeds) formed less seed as compared to clone 2 (13400 seeds). Greenhouse evaluation of field produced O.P. s eed from a population of diploid genotypes showed a hybridization frequency of 0 to 0.05% (Table 4-1). Out of 7102 seedlings from O.P. seeds produced in 2005, no herbicid e resistant hybrid plants we re detected. Five herbicide resistant hybrid plants were identified from 10,040 seedlings from O.P. seed produced in 2006 (0.05%). Pollen-mediated Gene Transfer from Transgen ic Tetraploid to Tetraploid Bahiagrass Gene transfer to three highly apom ictic te traploid genotypes was similar under greenhouse and field conditions (0.16% and 0.17 %, respectively). Evaluation of O.P. seed progeny from the fourth greenhouse experiment set yielded 10 he rbicide resistant hybrids out of 6297 seedlings.


68 Field evaluation of seed progeny from the same pollen receptors produced 11 herbicide resistant hybrids out of 6200 seedlings (Table 4-2). Seeds from the three different apomictic cultivars were separately harvested in the greenhouse. Am ongst the highly apomictic tetraploids, Paraguay 22 showed the highest hybridization frequency (0.32%), producing 5 herb icide resistant hybrids from 1559 seedlings analyzed. Argentine bahi agrass showed 0.11% hybridization frequency yielding 4 herbicide resistant hybrids out of 3669 seedlings analyzed; while Tifton 7 progeny produced one herbicide resistant hybrid out of 1062 seedlings (0.09% hybridization frequency). Paraguay 22 also produced the most seedheads, followed by Argentine, and Tifton 7 (data not shown). The facultative apomictic accession used as pollen receptor in the fifth greenhouse experiment set produced 15 herbic ide resistant hybrids out of 724 seedlings (2.7%; Table 4-2). Hybrids: Transgene Characterization All herb icide resistant hybrids plants which were detected by herbicide screening were confirmed to express the bar gene using the lateral membra ne flow immuno-chromatography assay (a representative image is shown in Fi g. 4-2A).The following herbicide resistant hybrid plants were selected for Southern blot analysis: a) R1, R2, R3, R4, R5, R6, and R7 resulting from hybridization of a population sexual diploid genotypes with the apom ictic transgenic tetraploid ; b) R8 and R20 resulting from hybridization of se xual diploid clonal plants with the apomictic transgenic tetraploid; and c) FR1, FR2 and FR3 obtained from gene transfer to population of sexual diploids under field conditions. All the hybrid plants analyzed showed a single functional copy of the bar gene identical to the pollen parent B9 (Fig. 4-2B). Hybrids: Cytogenetic Characterization The diploid and tetraploid peak values we re best optim ized at 99 and 316 respectively given the operating condition of the Partec Ploidy analyzer. Ploidy determination using the flow cytometer showed mean peak values intermedia te between the 4x and 2x controls. Hybrid R7


69 showed a peak value similar to the 2x control (Table 4-3). Root tip chromosome counts of representative plants were confirmed as follows: R1, 2n=30; R6, 2n=26 and R3, 2n=34(Fig. 4-3); R2, R4, R5, 2n=28; R7, 2n=20 and; FR3, 2n=30 (Table 4-3). Hybrids: Growth and Repro ductive Characteriz ation Embryo sacs of five hybrids which flowered during the first year (R1, R3, R4, R5, and R6) were characterized. A majority of the ovules in hybrids R1, R3, R5 and R6 showed multiple pairs of central cells and an or ganized sexual embryo sac (Table 44; Fig. 4-4C). A single sexual embryo was occasionally found in inverted orienta tion in some ovules of hybrids R1, R3, R4 and R5 (Table 4-4). The percent of aborted ovules was as high as 50% in hybrid R3. (Table 4-5). Flowering hybrids R1 and R3 had very low seed set as only ca. 1.0% of the ovules matured to form developed seeds. The seed set in hybrids R4, R5 and R6 was between 8.8-12.7%; at least one germinated seed was obtained fr om R4, R5 and R6 (Table 4-5). Differences in growth, habit and fertility of ten hybrids resulting from hybridization of sexual diploid population x apomictic transgenic te traploid are summarized in Table 4-5. Hybrid R1, R3, and R5 grew normally, while R2, R4, R6 R8, R9 and R10 showed slower growth as compared to the diploid parent (Table 4-5). Hybrid R2 displaye d a distinctive dwarf phenotype with fine leaves and slow growth. Hybrid R7 displayed a phenotype resembling diploid bahiagrass but had a very slow growth rate. Hybr ids R9 and R10 did not survive beyond the first year. Hybrids R2, R7, R8, R9, and R10 did not fl ower during an entire year of observation under natural photoperiod. Pollen stainability in hybri ds R1, R3, R4, R5 and R6 varied between 6075% (Fig. 4A-B; Table 4-5). When diploid pollen receptors were used th ere was segregation of the apospory locus in the F1 hybrids. The apospory-specific RFLP marker with a hybridizati on signal of 4.9 kb was detected in 15 out of 17 hybrids analyzed (Fig. 45A). When tetraploid cu ltivars were used as


70 pollen receptors the apospory-specific RFLP mark er with a hybridization signal of 4.9 kb was detected in all 12 tetraploid hybrids analyzed (Fig.4-5B). Discussion This study evaluates intraspecific pollen-m ediated transgene fl ow from apomictic tetraploid bahiagrass to diploid or tetraplo id bahiagrass under greenhouse and field conditions and characterization of hybrids. The potential for gene flow depends on a) the existence of synchronous flowering between the transgenic and the non-transgenic counterpart; b) compatibility between genotypes or the mating system; and c) mode of pollinati on. Among other factors affecting gene flow, the particular characteristics of the habitat wher e a species is grown is an important criteria especially for crop species grown in areas othe r than its center of di versity (Messeguer 2003). Risk assessment of transgenic crop species has to be addressed on a case by case basis due to the complexity of the nature of gene flow (Cha pman and Burke 2006). Cross pollinating perennial grasses impose a greater risk of gene transfer as compared to gr asses that are self-pollinating or reproduce asexually. Cro ss-pollinating transgenic perennial ryegrass ( Lolium perenne ) showed up to 2% gene transfer to nontransgenic ryegrass growing 14 4m away (Cunliffe et al. 2004). The frequency of gene transfer is highest at a short distance (0-1m) and decreases dramatically over distance (Messeguer et al. 2004; Cunliffe et al. 2004). The size of the pollen source plots also influences gene flow. A 162 ha transgenic cr eeping bentgrass field resu lted in hybridization rates of 0.03 to 2% at 21 km distance from the pollen source (Watrud et al. 2004). Kentucky bluegrass ( Poa pratensis L.) a facultative apomictic species has been reported to have a rate between zero to 16% gene transfer and an ove rall 0.5% hybridization frequency at 0 to 1m (Johnson et al. 2006). Proximal herbicide-tolerant and non-transgenic wild-type zoysiagrass ( Zoysia japonica Steud.) showed 6% cro ss-pollination, whereas the rate of hybridization was


71 reduced to 1.2% at 0.5m and 0.12% at 3m (Bae et al. 2008). Tall fescue ( Festuca arundinacea ) showed 5% hybridization frequency at 50 m, decreasing to 0.96% at 150 m and no gene flow was detectable at 200 m (Wang et al. 2004). The dr astic reduction in gene flow frequency with distance is related to a reduc tion in pollen density (Song et al. 2003; Song et al. 2004). Pollen-mediated gene flow in asexually reproducing species like P. notatum should therefore first be studied at a short distance since a very low gene flow frequency is expected and larger distances might not result in detectable gene flow. The frequency of gene transfer to a population of sexual diploid bahiagrass in both fi eld and greenhouse was lower as compared to other cross-pollinating perennial grasses. Tetraploid cultivars of bahiagrass such as Argentine are considered obligate apomicts. The differentiation of apomictic or meiotic embryos sacs has been used to char acterize the nature of seed production (Quarin and Hanna 1980; Quarin et al. 1982). Seed progeny from openpollinated transgenic tetraploids showed identi cal gene integration patterns as expected for apomictic inheritance (Sandhu and A ltpeter 2008). In pollination, timing is a critical factor in the expression of amphimixis in facultative apomictic P. notatum (Espinoza et al. 2002). In contrast to field studies, greenhouse experiments facilita te synchronization of fl owering of different genotypes. Deliberate synchronization of sexual di ploid genotypes and the transgenic apomictic tetraploid pollen donor could have resulted in a higher gene transf er to sexual diploid genotypes under greenhouse as compared to field conditions. Also, greater availability of wild-type sexual diploid pollen could be a plausibl e explanation for the lower gene transfer observed in the field as compared to the greenhouse. Since, transgen ic apomictic pollen donor and the non-transgenic highly apomictic tetraploid pollen receptors flow ered synchronously under field conditions, the


72 rate of gene transfer (0.16 and 0.17%) was very similar to that observed under greenhouse conditions. The use of self-incompatible clonal diploids as maternal parent and transgenic tetraploids as pollen source is a direct approach for enhancing the hybridi zation potential. In the current experiments, it also allowed differentiation of the degree of self-incompatibility that exists in the different sexual genotypes. This approach is similar to the use of male-sterile receptor plants designed to reduce pollen competitio n (Jia et al. 2007; Yuan et al 2007). Higher self-fertility in the second clone in contrast to the first clonal population was evident as the latter produced 7 times less seed, and 3.6% of those were hybrids. T hus, variation exists in the self-incompatibility of diploid genotypes as reported ear lier (Burton 1955; Acuna et al 2007) The use of male-sterile (ms) rice lines as pollen receptors produced ve ry high hybridization frequencies (3.1 to 36.1%; Jia et al. 2007). The relatively low hybridization frequencies observed in the present study could be due to the ploidy barrier be tween the pollen receptor and po llen donor. The results suggest that clonal diploids as pollen receptors overestim ates gene flow (0.16% vs 3.6%). These results are consistent with previous re ports on gene flow using male-ste rile plants as pollen receptors (Wang et al. 2006; Hoyle et al. 2007). The use of different pollen receptors in this study showed pollen competition as a significant factor affecting gene flow. A theoretical assessment has shown that even low hybridization rates of 0.1% could lead to the establishment of a modera tely advantageous (s=0.10) tr ansgene (Haygood et al. 2004). Hence, besides evaluating hybridization frequencie s, it is important to study the fate of such hybrids (Chapman and Burke 2006; Hails and Morley 2005). Earlier studies in perennial grasses did not include the phenotypic and reproductive characterization of intraspecific hybrids. Fate of hybrids is determined by their fitn ess criteria such as survival, fertility, mode of reproduction,


73 reproductive compatibility and transgene insertion (Chapman and Burke 2006; Jackson et al. 2004). Hybrid fitness is the best predictor of allelic spread (Chapman and Burke 2006). In P. limbatum a spontaneous hypotriploid hybrid from a diploid plant and an apomictic 4x male parent, was characterized as apomictic but seed sterile (Acuna et al. 2004). Apomictic triploid hybrids could release th e genetic variability in apomictic tetraploids and transfer the apomict trait from tetraploids to diploids (H anna and Burton 1986). However, spontaneous or manual production of triploids in different Paspalum species has resulted in a male sterile hybrid plant. Acuna et al. (2004) reported that the hypo triploid hybrid was 100% male sterile. Hanna and Burton (1986) also reporte d 0.02% viable pollen from a P. notatum apomictic triploid originating from an apomictic tetraploid plant an d a sexual diploid male parent. Our results are in contrast to earlier findings. The (near) triplo id hybrids produced in the present study from a sexual diploid plant and an apomictic tetraploid male parent produced apparent viable pollen although viable seed production was limited. High pollen stainability in triplo id plants is a rare occurrence. However, triploid F1 interspecific wheat hybrids have been detected with upto 94% pollen fertility based on aceto-carmi ne staining assay (Matsouka et al. 2007). A higher vigor of the triploid bahiagrass hybrid plant compared to both parents was repor ted by Hanna and Burton (1986). In contrast, most of the triploid hybrids in the current study showed reduced vigor compared to both parents, and some of them did not survive under greenhouse conditions. Seed set was reduced (1.5-12%) compared to an averag e seed set of 39% for O.P. diploids and 36% for O.P. apomictic tetraploids (Acuna 2006). Flow cytometry detected most hybrids as near triploids. The fact that peak values of tetraploids appeared reproducibly at more than d ouble the value of the dipl oid is an ununderstood anamoly. Karyotypic analysis using root-tip chro mosome counting further confirmed triploid or


74 near triploid nature with irregular chromosome numbers in hybrids obtained from sexual diploid maternal plants and tetraploid apomictic polle n donor plants. Triploid progeny produced from sexual diploid maternal and apomic tic tetraploid pollen donors of Taraxacum sect. Ruderalia are also characterized by irregular chromosome numbers (Martinf iova et al. 2007). Chromosome elimination during microsporogenesis was reported in P. subciliatum (Adamowski et al. 1998). Chromosome elimination has been widely observed in interspecific crosses or in allotetraploid genomes (Gernand et al. 2006; Adamowski et al 1998). Asynchronous cell cycle of two paternal species could cause meiotic instabilities a nd result in chromosome elimination. Lagging bivalents are characteristic cytological eviden ce for such meiotic irregularities leading to chromosome elimination. The tetraploid accessions of P. notatum originated from autopolyploidization and are known to show high frequency of meiotic irregularities (such as anaphase laggards, micronuclei formation) and abnormal tetrads leading to unbalanced gametes (Quarin 1992; Adamowski et al. 2005). Chromoso me irregularities observed in the triploid hybrids could be caused by such meiotic instabilities during microspore formation in tetraploid pollen donors. Interestingly, one hybrid (R7) with a complete chromosome complement of 20 chromosomes was detected from O.P sexual diploid genotypes. This hybrid (R7) did not resemble a typical diploid phenotype and was very slow growing. Further characterization of meiotic chromosome pairing in this hybrid (R7) was not possible as it did not flower. RFLP C1069 marker is considered a simple dose restriction fragment (Martinez et al. 2003) and is expected to segregate 1:1 for pr esence: absence in the F1 for a simplex (A---) condition (Wu et al. 1992). Earlie r reports have suggested ratios close to 3:1 expression of sexuality/apomixis in a segregating tetraploid hy brid population (Stein et al. 2004; Martinez et al. 2001). Such distortion towards sexuality has been explained by a lethal do minant effect of the


75 apospory gene with incomplete penetrance. Apospory in Bracharia spp., Panicum maximum and Pennisetum ciliare had been confirmed as a single domin ant gene with tetrasomic inheritance (Do Valle et al. 1994; Savidan 1981; Sherwood et al. 1994). Stein et al. (2004) suggested disomic inheritance based on AFLP markers link ed to the apoallele and preferential chromosome pairing in the a pospory specific genomic region (A SGR). In the present study openpollination of a sexual diploid female with transgenic, apomictic tetraploid male resulted in 13 apomictic and 2 sexual F1 individuals based on RF LP analysis. This ratio could suggest disomic inheritance of the apomixis locus. Cytogenetic observations of the embryo sac of five hybrids showed sexual or facultative apomixis in at least four of the five hybrids. These data may support Mogies hypothesis that there is a dosage requirement for expre ssion of apomixis. (Mogie 1988; Quarin et al. 2001). Martinez et al. (2001) discounted the diso mic model for inheritance of apomixis due to the lack of segregation for ap omixis in selfed F2 progeny from sexual F1s. However, it may be possible that female gametes have a lower transmission rate of the ASGR than the male gametes. Interestingly, all tetraploid hybrids evalua ted for the presence of apospory-specific RFLP marker tested positive. Linkage of the bar gene to the aposporous lo cus is an unlikely cause for this result, since segregation of the aposporous RF LP marker was detected in herbicide resistant triploid hybrids derived from the same pollen donor. Therefore, a low degree of amphimixis in the tetrapoid bahiagrass genotypes used as pollen receptor is the only plausible explanation for a lower frequency of pollen mediat ed gene transfer between apom ictic tetraploid genotypes. Thus, tetraploid apomictic bahiagrass does not provide complete transgene containment under unfavorable conditions. Howeve r, intra-specific gene transfer from apomictic bahiagrass is


76 drastically reduced compared to sexually reproducing perennial grasses and the majority of hybrids either did not produce seeds or resulting seedlings showed reduced vigor.


77 Table 4-1. Pollen-mediated gene transfer frequenc y from herbicide resistant apomictic tetraploid bahiagrass to diploid bahiagrass followi ng open pollination at 0.5 to 2.5m distance between the pollen donor and recipient. Location Pollen Recipient No. plants screened No. of hybrids (%) Greenhouse 1 Population of Pensacola genotypes 8300 13 (0.16%) Greenhouse 2 Single genotype of Pensacola clonally propagated (Clone 1) 332 12 (3.61%) Greenhouse 3 Single genotype of Pensacola clonally propagated (Clone 2) 5209 0 (0.00%) Field 2005 Population of Pensacola genotypes 7100 0 (0.00%) Field 2006 Population of Pensacola genotypes 10040 5 (0.05%) Table 4-2. Pollen-mediated gene transfer frequenc y from herbicide resistant apomictic tetraploid bahiagrass to tetraploid bahiagrass following open pollination at 0.5 to 2.5m distance between the pollen donor and recipient. Location Pollen Recipient No. of seedlings screened No. of herbicide resistant seedlings (%) Greenhouse 4 Highly apomictic genotypes 6297 10 (0.16%) Greenhouse 5 Facultative apomictic genotype 724 15 (2.07%) Field 2005 Highly apomictic genotypes 6200 11 (0.17%)


78 Table 4-3. DNA ploidy determination through flow cytometer and chromosome counting of F1 hybrids obtained from sexual diploid pollen r eceptors O.P. with transgenic apomictic tetraploid bahiagrass as pollen donor Sample Peak Chromosome counting (2n) 4x Known 316 40 2x Known 99 20 R1 182 30 R2 164 28 R3 220 34 R4 149 28 R5 149 28 R6 128 26 R7 110 20 R14 148 R17 213 R18 204 R19 135 R20 206 R21 130 *chromosome counting was not attempted in this sample Table 4-4. Embryo sac types in the F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass One ovule had single sexual embryo s ac with inverted orientation Two ovules had a single sexual embr yo sac with inverted orientation No. of ovules with Hybrid No. of ovules Apomictic Apomictic+Sexual Sexual Aborted R1 37 0 21 7 9 R3 40 0 17 3 20 R4 33 12 8 3 10 R5 33 4 20 5 4 R6 30 0 15 2 13


79Table 4-5. Growth, habit and fertility of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass Hybrid Growth rate Habit, phenotypic characteristics Pollen viability (%) Seed set (%) Viable seeds (%) R1 Normal Upright, wide leaves 70 13/850(1.5) 0/13 R2 Slow Upright, shorter internodes, fine leaves Did not flower in the first year R3 Normal Prostrate, wide leaves 75 7/550(1.3) 0/7 R4 Slow Upright, wide l eaves 60 33/260(12.7) 1/33 (3.0) R5 Normal Upright, fine leaves 60 26/260(9.6) 1/26 (3.8) R6 Slow Upright, fine leaves 60 43/488(8.8) 7/43 (16.3) R7 Very slow Upright, fine leaves Did not flower R8 Slow Upright, wide l eaves Did not flower R9 Slow Prostrate, wide leaves Did not flower and did not persist R10 Slow Prostrate, wide leaves Did not flower and did not persist Categorized for growth rate compared to a diploid plantstarting with very slow, slow or normal in ascending order Indicated as ratio of seeds /total number of seeds including seeds and empty seeds (empty seeds are those devoid of embryo, endosperm and nucellus)


80 Figure 4-1. Pollination experiments under greenhouse and field conditions and screening of O.P. seed. A and B) Greenhouse and field experi ment set up showing tetraploid pollen donor (middle) and tetraploid pollen recepto r plants during the peak flowering period. Newly emerged inflorescences were tagge d with colored tape and pollinated inflorescences were bagged in glassine bags. C and D) Six-week old seedlings derived from O.P. seed from the pollination experiments before and after herbicide application. A CD B


81 Figure 4-2. Transgene expre ssion and integration in F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass. A) Representative samples (R1 and R2) showing immuno-chromatographic determination of bar/PAT expression in the F1 hybrid plants using the Libertylink Sticx Test. The top band is common to all samples and indicated functionality of the stix assay. The lower PAT-specific band confirms the presence of PAT in the protein extract. B) Southern blot analysis showing bar gene integration of the B9 pollen parent and F1 hybrids (R-). Genomic DNA was digested with Bgl II, which excises a 5.0 kb band in pJFbar. The full length coding region of the bar gene was used as a probe. Wild-type bahiagrass was used as negative control (WT) and the transgenic pollen parent (B9) was used as positive control in both (A) an d (B); hybrids R1-R7 originating from a population of sexual diploid pollen receptors under greenhouse conditions; R8 and R20 originating from clonal diploid genotype under greenhouse conditions and; FR1 to FR3 originating fr om a population of sexual diploids under field conditions. WTB9R1R2A B WT B9 R1 R2 R3 R4 R5 R6 R7 R8 R20 FR1 FR2 FR3 B WT B9 R1 R2 R3 R4 R5 R6 R7 R8 R20 FR1 FR2 FR3


82 Figure 4-3. Karyotypi c analysis of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahi agrass as pollen donor.Root-tip chromosome preparations of herbicide resistant F1 hybrids showing R1 with 2n=30, R6 with 2n=26 and R3 with 2n=34. The root-tips were prepared by 4 h treatment with 8hydroxyquinoline and 24 h treatment with 95% ethanol: acetic acid, 3:1 v/v. Fixed roots were squashed using 45% acetic acid and stained with orcei n. The observations were made under a microscope with 40x magnification. Figure 4-4. Reproductive characterization of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic te traploid bahiagrass as pollen donor. A) and B) Pollen grains of hybrid R1 and R6. Dark red stained pollen indicates viable pollen, while light or transparent pollen are non-viable. C) An ovule from R6 hybrid plant bearing a sexual embryo sac characterized by antipodals, and aposporous embryo sac characterized by the second pair of polar nucle. References: a antipodals; e egg cell; p polar nuclei. R1R6 R3 AB Ca p p e


83 Figure 4-5. Molecular marker analysis of F1 hybrids using RFLP marker linked to apospory in tetraploid Paspalum notatum. A) RFLP analysis of F1 hybrids obtained from sexual diploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass as pollen donor. B) RFLP analysis of F1 hybrids obtained from apomictic tetraploid pollen receptors O.P. with transgenic apomictic tetraploid bahiagrass as pollen donor. Hybridization was carried with the C1069 ri ce probe following digestion of genomic DNA with EcoRI. Arrows indicate the apospor y specific 4900 bp (indicated by the arrow) restriction fragment. A= tetrap loid aposporous; S= diploid sexual; R-= herbicide resistant F1 hybrids; R1to R7 hybrids or iginating from a population of sexual diploid genotypes under greenhouse c onditions; R8 to R22 hybrids originating from clonal diploid genotype under gr eenhouse conditions; R23 to R45 hybrids originating from highly apomictic tetrap loid genotypes under greenhouse conditions and; R50 to R54 hybrids originating from facultative apomictic tetraploid genotypes. A S R1 R2 R3 R4 R5 R6 R7 R8 R12 R17 R18 R20 R21 R22 R14 A A S R1 R2 R3 R4 R5 R6 R7 R8 R12 R17 R18 R20 R21 R22 R14 A A S R23 R25 R26 R40 R41 R42 R43 R44 R45 R50R52 R54B A S R23 R25 R26 R40 R41 R42 R43 R44 R45 R50R52 R54B


84 CHAPTER 5 CONCLUSIONS Bahiagrass is a prim ary component of native grassland in southern Brazil, Paraguay, Uruguay and northeastern Argentina (Gates et al. 2004). Pseudogamous apomictic tetraploid races are the most common form of this specie s found near its center of genetic diversity. The sexual self-incompatible diploid cytotypes, commonly called Pen sacola inhabit only limited areas in its native habitat (Dau relio et al. 2004). Pensacola bahiagrass has become one of the major forage grasses in the southeastern Unit ed States since its in troduction in 1945 (Burton 1967; Gates et al. 2004). Genetic improvement of tetraploid bahiag rass through traditional breeding has been limited due to its asexual seed production (Blount et al. 2003). Gene tic transformation of apomictic cytotypes is thus an alternative approach to develop im proved cultivars. The introduction of a number of different transgenes in bahiagrass has resulted in plants with improved turf quality (Agharkar et al. 2007; Zhang et al. 2007), abiotic (James et al. 2008) and biotic (Luciani et al. 2007) st ress tolerance and herbicide resi stance (Sandhu et al. 2007). The transgenic lines are now being evaluated in the field. Concer ns about potential environmental risks associated with genetically modified tu rf or forage grasses currently slow their deregulation. Hybridization of transgenic plants with their wild relatives is one of the major environmental concerns. To address this poten tial problem, it is important to study both the pollen-mediated gene flow from the transgenic plants, as well the behavior and fate of transgenes. The production of uniform seed progeny wit hout segregation of genetic loci is a characteristic feature of apomic tic species such as Argentine bahiagrass. This feature supports transgene stability and the inhe ritance of complex transgene integration in the seed progeny


85 without segregation. It can thus allow stacking of multiple unlinked transgenes in the same transgenic line for pathway engineering or co-i ntroduction of multiple traits while maintaining uniform transgene expression in the seed progeny. It also reduces the risk of transgene dispersal through pollen. Stable and high co-expression of nptII and bar was confirmed in the apomictic seed progeny of herbicide re sistant transgenic bahiagra ss. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transform ed lines (95% co-expression frequency). Quantification of transgene co-expression revealed that several lines with complex integration patterns displayed a higher tran sgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplif ication and sequence analysis of transgene loci. However, the occurrence of complex transg ene loci was significantly reduced by lowering the amount of DNA during the microparticle coating. Transgenic bahiagrass plants were resistant to a field application of 0.6% glufosinate ammonium which is twice the recommended rate for weed control. They were mo rphologically identical to the non-transgenic w ild-type, and produced viable pollen. Under field conditions at clos e physical proximity (0.5 to 2.5m) with the non-transgenic bahiagrass, the average frequency of gene tran sfer from transgenic apomictic tetraploid bahiagrass to sexual diploid and apomictic tetraploid bahiagrass was 0.03% and 0.17% respectively. Under greenhouse conditions the fre quency of gene transfer from transgenic apomictic tetraploid bahiagrass to a non-transgenic population of sexual diploids or highly apomictic tetraploids was 0.16%. The majority of hybrids originating from sexua l diploid pollen receptors were either near triploids or triploids, although one diploid hybrid was detected. Chromosome variation in the


86 intraspecific hybrids is apparently a result of irregular meiosis during the formation of microspores in the pollen parent. The intra-sp ecific hybrids showed variation in habit and phenotype. Slow growth and reduced seed set compared to the diploid parent indicated compromised fitness of such hybrids. The increased proportion of apomictic intraspecific hybrids is in contrast to earlier reports. The data supports disomic inheritance of the apomixis gene or the presence of a low degree of amphimixis in cult ivar Argentine. Embryo sac analysis of five intraspecific hybrids showed the presence of meio tic and aposporous embryo sac, thus indicating facultative apomixis. Progeny analysis from the intra-specific hybrids wo uld thus be the most conclusive evidence to determine the facultative apomictic or sexual nature of such hybrids. Martinez et al. (2007) reported th at ploidy contribution of the male gametes from triploid hybrids depends upon the chromosome number of th e female gamete. Male gamete formation in the triploid and tetraplo id intraspecific hybrids obtained fr om this study would further enhance our understanding of transgene introg ression. Due to the unique feat ures of some hybrids (eg. R2 with a fine leaves and short in ternodes, non flowering), it would be interesting to study their performance, persistence and turf quality under field conditions. In summary, gene transfer between transg enic and non-transgenic bahiagrass can occur under field conditions, although the frequency of hybridization is low as compared to other transgenic cross-pollinating grasses and hybrids show reduced fitness.


87 APPENDIX LABORATORY PROTOCOLS USED FOR BIOLISTIC TRANSFORMATION, TISSUE CULT URE AND CHARACTERIZATION OF TRANSGENIC PARENT AND HYBRID BAHIAGRASS PLANTS Protocols for Molecular Cloning Glycerol Stocks 1. Initiate E. coli culture containing desired plasmid in 2 ml sterile LB broth containing the appropriate antibiotic (kanamycin for plasmid pJFbar). Incubate overnight at 37C on an orbital shaker with 220 rpm with 1 orbit. 2. Add 0.85 ml of the bacterial cult ure and 0.15 ml sterile glycerol into a sterile microfuge tube with a hole pierced in the lid. 3. Mix by vortexing and immediatel y freeze using liquid nitrogen. Store at -80C. Prepare several tubes for plasmid. Avoi d successive freeze-thaw cycles. Amplification and Purification of Plasmid DNA Using the QIAGEN Plasmid Midi Kit QIAGEN plasmid purification protocol as provided by the manufacturer All buffers (P1, P2, P3, QBT, QC, QF TE) are supplied with the QIAGEN kit 1. Inoculate a starter culture of 2-5 ml LB medium containing th e 1g/ml of the appropriate antibiotic. Frozen glycerol stocks or freshl y streaked selective plate can be used for inoculation. Incubate for ~8h at 37C with vigorous shaking (~220 rpm on an orbital shaker with 1 orbit). Use a tube or flask with a volume of at least 4 times the volume of the culture 2. Use the starter culture to initiate an overnigh t culture. Use 100l of the starter culture in 50 ml LB medium containing the appropriate an tibiotic. For low-copy plasmids, inoculate 100 ml medium. Incubate at 37C for 12-16 h on an orbital shaker. The vessel used should have a capacity atleast 4 times the volume of the culture 3. Harvest bacterial cells by centrifugation at 6000 g for 15 min at 4C. Remove all traces of supernatant by pipetting. 4. Resuspend the bacterial pellet in 4 ml buffe r 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. Incubate at room temperature for 5 min. Contents shou ld not be vortexed at this stage. 6. Add 4 ml of chilled buffer P3. Immediately mi x immediately by gently inverting the tubes 4-6 times. Incubate on ice for 15 to 20 min. Af ter 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 30 min at 4C. Re move supernatant containing plasmid DNA promptly. Before loading the centrifuge, the sample should be mixed again 8. Centrifuge the supernatant again at 20, 000g for 15 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 supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.


88 11. Wash the QIAGEN-tip with 2 times10 ml Buffer QC. 12. Elute DNA with 5 ml buffer QF. Coll ect the eluate in a 10 ml tube. 13. Precipitate DNA by adding 3.5 ml isopropanol to the eluted DNA. Mix and centrifuge immediately at 1500 g for 30 min at 4C. Care fully decant the supernatant. All solutions should be at room temperature in or der to minimize salt precipitation. 14. Wash DNA pellet with 2 ml 70% ethanol, and centrifuge at 1500 g for 10 min. Carefully decant the supernatant wit hout disturbing the pellet. 15. Air-dry the pellet for 5-10 min and redisso lve the DNA in a 200 l TE buffer. Allow the pellet to resuspend in TE buffer overnight at 4C before transferring to a sterile micorfuge tube. Preparation of Minimal Linear Expression Cassettes 1. Digest 100g of plasmid DNA usi ng restriction enzymes which excise the gene expression cassette (promoter, gene, poly A) and have no restriction site within the cassette. 2. Check for complete digestion by running a 1l a liquot of the digest on a 0.8% agarose gel. Complete digestion will be indicated by the exac t number of bands as expected. (eg. if 2 enzymes cutting one at each side of the expression cassette have single site in the plasmid, it will give 2 bands. The size of fragment of interest will depend on the relative size of the expression cassette to the whole plasmid. 3. Load 10-15 g/ well on a 0.8% agarose gel and electrophorese at 70V for 2 h to achieve good separation of bands. 4. Excise the band corresponding to the expression cassette using a UV-transilluminator. 5. Purify the excised band with the QIAquick ge l purification kit (Qiagen Inc., Valencia, CA) following the instructions manual. 6. Check quality by electrophoresing 2 l of the fragment DNA (expression cassette). 7. Quantify the fragment yield using na nodrop (ND-1000 spectrophotometer, Nanodrop Technologies, Wilmington, DE). Restriction Digestions to Prepa re Minimal Expression Cassettes pJFbar digestion Restriction Enzyme IsceI (Promega) pJFbar plasmid (100 g) 40 l Restriction Buffer 2 10X 10 l BSA 10X 10 l IsceI 10 l DdH2O 30 l Final volume 100 l Digest overnight at 37C


89 Gel Extraction Using QIAquick Gel Extraction Kit Prepare Buffer PE by adding 40 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 ma de fresh, when staining a gel make sure to use a fresh batch of ethidium bromide so as to not contaminate your plasmid with foreign DNA or DNase. 1. Visualize DNA bands under a UV transilluminator. 2. Excise the DNA fragment precisely from the ag arose gel using a clean sharp scalpel. Avoid excess agarose. Proceed quickly to avoid long UV exposure 3. Weigh the excised agarose gel and distribute approximately 400 mg per microfuge tube. 4. Add 3 volumes of Buffer QG to 1 volume of gel. 5. Incubate at 50C in a heating block for 10 min or until the gel has completely dissolvde. Vortex every 2 min during incubation to ensure complete dissolution of gel. After incubation check that the color of the mixture remains similar to Buffer QG (yellow), indicating that the pH has not changed. 6. Add 1 gel volume of isopropanol to the mixture and mix by inverting the tube several times. 7. Place a QIAquick spin column in a provided 2 ml collection tube and apply the sample to the column. 8. Centrifuge at 16,100g for 1 min us ing table top centrifuge. Repeat this step if the volume of the mixture is more than 800 l, the maximum capacity of the QIAquick column. 9. Discard the flow-through and place the co lumn in the same collection tube. 10. Wash the QIAquick column by adding 750 l Buff er 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 QIAquick column in a clean, sterile 1.5 ml microcentrifuge tube. 13. Add 30 l Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of the QIAquick membrane to elute DNA, let stand for 1 min and th en centrifuge for 1 min at 15,700g. 14. Store DNA at -20C. Protocol for Particle Bo mbardment using PDS-1000/He Preparation of Gold Stock (60mg ml-1) 1. Weigh 60mg of 1.0 m gold particles in a sterile 1.5ml microfuge tube. 2. Vortex for 3-5 min after adding 1ml of 70% ethanol. 3. Centrifuge briefly (5sec) to pellet the microparticles. 4. Discard the supernatant, follow by 3 washes with 1ml autoclaved ddH20. 5. Vortex for 1 min. 6. Centrifuge briefly (3-5sec) and again remove the supernatant. 7. Add 1ml sterile 50% v/v glycerol. 8. Store the gold stock at -20C.


90 Preparation of DNA Coat ed Microparticles 1. Transfer 30 l of the gold stock suspensi on after vortexing to a 1.5 ml sterile microfuge tube. 2. Add gene expression cassette (1-5g) and sterilized ddH2O to a final volume of 60l and vortex at low speed for 30 sec. 3. Place 20 l 0.1 M freshly prepared spermidine and 50 l 2.5 M CaCl2 on the lid of the eppendorf tube. 4. Mix all components by closing the lid and vortexing for 1 min. 5. Centrifuge briefly (3-5se c) to pellet the gold. 6. Discard the supernatant without disturbing the pellet and add 250 l absolute ethanol for washing. 7. Centrifuge briefly (3-5sec) and discard the supernatant. 8. Repeat the previous washing with ethanol once more. 9. Resuspend the pellet in 90 l absolute ethanol by sonication for 1 s. 10. Keep the DNA coated microparticles on ice. Biolistic Bombardment 1. Turn on PDS-1000/He Particle Delivery Syst em and vacuum pump; ensure the helium supply is at least 200 psi above the desired pressure optimum. 2. Place the rupture disk in the centre of the ruptur e disk holder and secure it properly inside the chamber. 3. Place macrocarriers into holders with forceps and push down with a sterile blunt object to secure it in holders. 4. Prior to use, resuspend the DNA-coated microparticles by briefly vortexing. 5. Apply 5 l of the suspension of DNA coated microparticles into the center (inner 5mm diameter) of the macrocarrier; allow for comp lete evaporation of ethanol before use. 6. Place stopping screen into the macrocarrier pl ate and insert the inverted macrocarrier assembly on top. Secure the lid on top of the shelf assembly. 7. Place macrocarrier plate containing the macro carrier at the highest level of the inner chamber. 8. Place tissue culture plate on shelf 2 below the macrocarrier plate (6 cm below). 9. Initiate a vacuum to 27.5 Hg; press and hold th e fire button until the disc ruptures at 1100psi. Eye protection must be worn. 10. Vent the vacuum and remove petri dish. 11. Dismantle the assembly and prepare for the next shot. Tissue Culture Induction, Maintenance, Selection and Plant Regeneration Seed Preparation for B ahiagrass Callus Induction 1. Weigh 2.0g seed in a vial and treat with conc entrated sulfuric acid for 12-16 min (increase the time for larger seed). Sulfuric acid is scarification treatment to break the dormancy. 2. Transfer the seeds to an empty beaker using 500 ml dH2O. Stir the mixture with a glass rod to mix. Decant the liquid and floating debris. 3. Add 500 ml dH2O to the seeds and strain them th rough two layers of cheesecloth.


91 4. Gently rub the seeds in the cl oth to remove the debris. 5. Wash the seeds with 500 ml dH2O and repeat the steps 3-5, two times. 6. After the third wash, leave the seeds in the cheesecloth and dry them for 15 min. 7. Transfer the seeds to a 9 cm Petridish. 8. For sterilization, mix 20 ml of 6% sodium hypochlorite solution a nd 10 ml glacial acetic acid. Place the resulting solution at the bottom of a glass dessi cator in the fume hood to avoid inhalation of fumes. 9. Place open Petridishes of seeds and lid in the dessicator for 1 h. Chlorine fumes from the mixture will sterilize the seeds and inside of the Petridishes. 10. Add enough autoclaved dH2O to submerge the seeds and leave to soak for at least 1 h before placing the seeds on the surface of on culture medium. 11. Place 20 seeds per Petridish. Tissue Culture of Seedling-derived Calli of Bahiagrass 1. Initiate cultures on a callus induction me dia (CIM; see media preparation below) and subcultured to a new medium of the same composition biweekly. 2. Maintain cultures under low light intensity (30 Em-2s-1 light) at 28C and 16 h photoperiod in an incubator. 3. Continue the callus induction phase for 2-4 we eks. Bahiagrass calli are bombarded 6-weeks after culture initiation. 4. Placed tissues on callus induction medium supplem ented with 0.4M sorb itol (osmoticum) 4-6 h prior to bombardment. Immediately after bombardment, or up to 16 h after bombardment,transferr tissues to callus induc tion medium (CIM) and maintain under 30 Em-2s-1 illumination at 28C and 16 h photoperiod for 7 days before initiation of selection. Selection and Regeneration Protocol for Bahiagrass using npt II/Paromomycin 1. Transfer calli onto selecti on medium 7 d after gene transfer and maintain at 30 Em-2sec-1 illumination, with a 16 h photoperiod at 25C. Th e selection medium consists of CIM with 50 mg L-1 paromomycin. Phytagel is replaced by agarose (Type I, Sigma). Track the identity of independent callus lines through selection and regenera tion procedure to recover independent transformants. 2. Subculture calli to new selection medium biweek ly until 28 d after initi ation of selection. 3. Transfer calli to regeneration selection medium containing all components of selection medium but with 1.1 mg L-1 BAP, replacing 0.1 mg L-1 BAP and no auxin. Maintain calli on regeneration selection medium for 14 d and increase the light intensity from 30 Em-2sec-1 to 150 Em-2sec-1 light. 4. Transfer calli with shoots to selection rooting media in deep Petri-dishes containing 50 mg L1 paromomycin without growth regulators. Incubate unde r high light intensity (150 Em-2s-1 light), 16 h photoperiod at 25C for 14 d. 5. Transplant elongated shoots with roots into compost and maintain in a growth chamber with 12 h photoperiod at 25C temperat ure. Keep the regenerated plan ts covered during the first 46 d with a transparent container to maintain humidity.


92 6. After 14 d of acclimatization in growth chambers move the transgenic plants to glasshouses maintained at 28C day and 22C night, under natural photoperiod. Molecular Techniques used in the Confirma tion of Putative Transgenic Plants Immunochromatographic bar /PAT QuickStix Test QuickStix Libertylink bar/PAT (EnviroLogix, Portland, Ma ine, US) kit for as provided by the manufacturer The QuickStix strips must be allowed to come to room temperature before use. 1. Harvest 3 leaf segments in a centrifuge tube 2. Add 150l of the extraction buffer supplied with the kit. 3. Grind the tissue until the buffer turns dark green 4. Centrifuge for 5 min 5. Remove the supernatant with a yellow pi pette without disturbi ng the leaf debris. 6. At this step, the total protein may be quantified or the Quick Stix may be directly used. (for protein estimation please see NPTII ELISA protocol). 7. Place the strip into the extraction tube. The samp le will travel up the strip. Use a rack to support multiple tubes if needed. 8. Allow the strip to develop for 10 min before making final assay interpretations. Positive sample results may become obvious much more quickly. Development of the control line within 10 min indicates that th e strip has functioned properly. The sample extract containing bar/PAT will develop a second line (test line) on the membrane strip between the control line and the protective tape, within 10 min of sample addition. NPTII ELISA Assay Stable Transformants Protein Extraction 1. Harvest 600mg of young fresh leaf tissue. Store samples on ice. 2. Add 10mg polyvinyl pyrrolidone (PVP) and 600 l 10x PEB1 buffer (supplied with the nptII Agdia ELISA kit) to each sample. 3. Grind the leaf samples using a micr opestle. Keep the samples on ice. 4. Centrifuge the samples at 20,800g at 4C for 15 min. 5. Transfer the supernatant to a new micr ocentrifuge tube and store on ice. Protein Estimation 6. Dilute Protein Determination Reagent (USB Corporation, product code 30098) 1:5 using sterile ddH2O. Prepare enough to use 1 ml per samp le including standards and blank. 7. Prepare a standard dilution series using BSA (0-20 g). 8. Add 1 ml diluted Protein Determination Reagen t to each cuvette. Add 5 l of sample to each cuvette and mix by vortexing. 9. Incubate at room-temperature while preparing the remaining samples. 10. Measure OD595 of each sample (ideally these should be between 0.2 and 0.8). 11. Plot a standard curve using BS A and use it to estimate total protein concentration of the samples. 12. Calculate the volume of each sample requi red for 15 g total protein per well.


93 Assay 13. Prepare the samples, including wild-type usi ng 15 g protein and the volume of buffer PEB1 required to make the total volume 110 l. 14. Prepare standards as follows: 110 l buffer PEB1 (negative control) and 110 l of the provided positive control. 15. Prepare a humid box by putting damp paper towel in a box with a lid. 16. Add 100 l of each sample and standard in the ELISA microplate provided with the kit. The order of samples should be noted at this time. 17. Place the plate in the humid box and incubate for 2 hours at room temperature. 18. Prepare the wash buffer PBST by dilu ting 5 ml to 100 ml (20x) with ddH2O. 19. Prepare the enzyme conjugate diluent by mi xing 1 part MRS-2 with 4 parts 1x buffer PBST. Make enough to add 100 l per well. 20. A few minutes before the incubation ends, add 10 l from bottle A and 10 l from bottle B per 1 ml of enzyme conjugate diluen t to prepare the enzyme conjugate. 21. When incubation is complete, remove plate from humid box and empty wells. 22. Fill all wells with 1x buffer PBST and then empty them again. Repeat 5x. 23. Ensure complete removal of wash solution by tapping the frame firmly upside down on paper towels. 24. Add 100 l of the prepared enzyme conjugate into each well and incubate the plate in the humid box for 2hrs at room temperature. 25. In the meanwhile, aliquot sufficient TMB substr ate (100 l per well) and allow it to warm to room temperature. 26. When the incubation is complete, wash th e plate with 1x buffer PBST as before. 27. Add 100 l of room temperature TMB substrat e solution to each well and place the plate back in the humid box for 15 min. A blue color will develop, the intensity of which will be directly proportional to the am ount of NPTII protein in the sa mple, while negative samples will remain white. 28. To stop the reaction, add 50 l 3M sulphuric acid to each well. The substrate color will change from blue to yellow. 29. The results must be recorded within 15 min af ter addition of the stop solution otherwise the reading will decline. 30. Color development can be visually scored or recorded with the help of an ELISA plate reader. DNA Extraction CTAB Method 1. Autoclave mortars, pestles and spatulas for 20 min and dry in an incubator oven at 60C. 2. Prepare enough CTAB buffer (5ml/gram leaf material) for use by adding mercaptoethanol fresh (200l -mercaptoethanol per 100ml buffer or 30 l in 15ml). Heat to 65C in a water bath. 3. Harvest 3g young leaf material and stor e on ice or freeze in liquid nitrogen. 4. Cool mortar and pestle by a dding liquid nitrogen. Chop fresh leaf into the liquid nitrogen using scissors or add leaf and then cut into small slices. Grind to a fine powder under nitrogen approximately 3 times. 5. Add frozen leaf powder to 15ml pre-heated buffer in a 50ml disposable polypropylene tube and mix well to remove lumps using a spatula or glass rod.


94 6. Incubate at 65C, 1 hour. Mix the conten ts in between as well (1-3 times). 7. Cool to room temperature. 8. Add equal volume (15ml) chloroform/isoamylalcohol (24:1) and mix gently to form an emulsion for approx 30 mins. Fist mix by hand and on an orbital shak er (Adams Nutator Mixer) for 15 min. Mix with hand by gently inverting the tubes and then again put it on the shaker for another 10-15 mins. 9. Spin, 4000rpm, 1 min. Remove lo wer layer using a 10ml pipette. 10. Spin, 4000rpm, 10 mins. Transfer top layer to a fresh 50ml tube. 11. Add 2/3 volumes isopropanol and mix gently by inverting. The DNA will precipitate. 12. Use a blue pipette tip to hold the DNA insi de the tube and pour away the liquid. 13. Transfer DNA to a clean 1.5ml eppendorf tube. 14. Wash 2 times in 70% ethanol by holding the DNA in the tube with the pipette tip. 15. Spin, max speed, 1 min. Remove supernatant. 16. Air-dry for 15 min in a clean bench and resuspend in 500l TE. 17. Add 2l RNaseA (30mg/ml) and incubate at 60C overnight. 18. Add 300l phenol/ chloroform (25:24:1 P:C:IAA) (lower layer in the bottle) and mix by inverting and tapping to form an emulsion. 19. Centrifuge for 7 min at 14000 rpm. Transfer supernatant to a clean tube without disturbing the interface. 20. Repeat phenol/ chloroform extraction. 21. Add 300l chloroform/isoamylalcohol (24: 1) and mix to form an emulsion. 22. Centrifuge for 7 min at 14000 rpm. Tran sfer supernatant to a clean tube. 23. Repeat chloroform extraction. 24. Add 1/10 volume (50l) sodium acetate and 2 volumes (1000l) 100% EtOH. 25. Centrifuge briefly and remove supernatant. 26. Wash two times using 70% ethanol. First wa sh, pour off the ethanol, and in second wash pipette the supernatant after a brief spin. 27. Dry the pellet for 15-25 mins in a vacuum rota tory evaporator (hea ting on) and resuspend in 200l autoclaved T1/10E. Allow the DNA pellet to re suspend overnight at 60C. 28. Prepare 10x dilution of the DNA stock. Use 1l to check concentrati on and quality using the Nanodrop spectrophotometer and by gel elec trophoresis of samples in a 0.8% agarose gel (80V, 60 min). 29. Store the dilutions and stocks at 4C. Southern Blotting Gel electrophoresis 1. Extract genomic DNA from transgenic lines as well as wild-type using the CTAB method as described above. 2. Digest 1g DNA from all samples with the re striction enzyme to be used for Southern blotting. Run the digested DNA on a 1% agar ose gel to ensure co mplete restriction digestion. 3. Use 15g DNA to set up restriction digests for Southern blotting. First, estimate the volume of each sample required to get 15 g DNA and pipet it into a sterile 1.5 ml microcentrifuge tube. Add steril e ddH2O to make up the volume to 30 l. 4. Set up the digestion as follows:


95 DNA (15 g DNA + sterile ddH2O) 30.0 l 10x BamHI buffer 6.0 l 100x BSA 0.6 l BamHI (1000U/l) 2.0 l Sterile ddH2O 21.4 l Final volume 60.0 l 5. Prepare a master mix for all samples that includes the buffer, BSA (if required for the enzyme chosen), enzyme and water. Mix by pipetting and add 30 l DNA. Mix well, centrifuge briefly and incubate in a waterbath at 37C over-night. 6. Run 2 l from each digest on a 1% agarose gel to ensure complete digestion. 7. Prepare a 1 cm thick 1% agarose gel for Southern blotting using 1x TAE. Concentrate all the digest s to reduce the volume by half (30 l) in a Speed-Vac. 8. Centrifuge the samples briefly and add 6 l 6x loading dye. Mix well by pipetting. Load samples on the gel. Also load a molecular weight marker (1 kb ladder, New England Biolabs) for estimating band size following hybridization. 9. Dilute the plasmid containing the tran sgene and load 50pg as a positive control. 10. Run the gel at 20V over-night (12-14 hours) in 1x TAE. 11. Remove the gel from the electrophoresis a nd cut excess gel using a scalpel. Measure the dimensions of the gel. 12. Stain the gel for one hour using freshly pr epared ethidium bromide stain (0.05g/ 13. Wash the gel five times using de-ionized water (DI H2O). 14. Visualize the gel on a gel-documentation sy stem to check complete digestion of all samples. Also measure and record the distance of the bands of the molecular weight marker from the top of the gel. DNA transfer blottingalkaline transfer procedure 15. Prepare 500 ml 0.25N hydr ochloric acid (HCl) and 3L 0.4N sodium hydroxide (NaOH) solution per blot. 16. Treat the gel with 0.25N HCl on a shak er with gentle shaking for 15 min for depurination. Wash the gel three times with DI H2O. 17. Cut three pieces of filter paper to match th e size of the gel and two pieces for the bridge (24x18 cm). Also cut a piece of the Hybond-N membrane (Amersham) to match the size of the gel. 18. Treat the gel with 0.4N NaOH on a sh aker with gentle shaking for 30 min. 19. Assemble the tray and the platform on which th e blot is to be set up. Place the filter paper bridge on top of each other on the platform and fold them so that it dips into the tray on both sides. 21. Pour 0.4N NaOH on the bridge to wet it co mpletely. Roll a glass rod over it several times to remove air bubbles. Pour more NaOH onto the bridge. 22. Place the gel in the center of the bridge, pour NaOH over it and remove air bubbles. 23. Mark the membrane to determine the orientation of the gel following blotting. 24. Wet the membrane using 0.4N NaOH, place the membrane on the gel; pour NaOH onto it and remove air bubbles. 25. Place three pieces of Whatmann filter pape r on the membrane; remove air bubbles after placing each piece. Pour more NaOH over the to p to avoid drying of the filter papers. 26. Place pieces of parafilm a ll around the gel to cover the br idge to ensure that the movement of the transfer buffer (0.4N NaOH) takes place only through the gel.


96 27. Fill the tray with the tran sfer buffer to the top and cover the tray with Saran wrap to prevent evaporation of the buffer during blotting. 28. Place a stack of absorbent paper towels on the gel and place another piece of plexi-glass on top. On this, place a small we ight ( ca. 750g) to ensure uniform blotting and leave over-night (16-18 hours). 29. Disassemble the blot. Wrap the membrane in a cling film and expose it to UV for 2 min to fix the DNA on the membrane. Store the membrane in a zip-loc bag at 4C. 30. Visualize the gel using a gel-documentati on system to make sure the transfer was complete. 31. Follow the hybridization protocol described below. For neutral transfer procedure, 20x SSC is used as transfer buffer in place of 0.4N NaOH. Treatment of the gel includes 10 min depurina tion treatment with 0.125N HCl, followed by 30 min treatment with denaturation buffer and 30 min treatment with neutralization buffer. All other steps are same as described above. Hybridization using the Prime-a-Gene Labeling System (Promega) 1. Check the radioactive working area for previous contamination before working. Thaw blocking DNA (sheared salmon sperm DNA), labeling buffer, dNTPs (dATP, dGTP, dTTP), BSA and probe on ice. Place [ -32P] dCTP behind the plexiglass shield to thaw. 2. Pre-heat the hybridization oven and water-bath to 65C. Place the hybridization buffer in the water-bath. 3. Roll the membrane and place it inside the hybrid ization tube. Add 50 ml 5x SSC to the tube and pre-wet the membrane for 5-10 min. Make sure that there are no leaks. 5. Estimate the volume of probe required to get 25 ng of the probe and pipette it into a clean microcentrifuge tube. Make up the volume to 30 l using the nuclease-free water provided with the labeling kit. 6. Close the tube tigh tly and boil the probe for 5 min. Al so boil 500 l of salmon sperm DNA in a separate tube. Place on ice fo r 5 min immediately after boiling. 7. Discard the 5x SSC into the sink and invert the hybridization tube s on a paper tissue to drain. 8. Prepare the dNTP mix by mixing equal parts of dTTP, dA TP and dGTP. Prepare enough to use 2 l per labeling reaction. 9. Set up the labeling reaction as follows: Add the following to the denatured probe5x labelling buffer 10 l Unlabeled dNTP mix 2 l BSA 2 l Klenow (5U/l) 1 l Total volume 45 l 10. Move the mixture behind th e plexiglass shield; add 5 l [ -32P] dCTP and mix well by pipetting. Final volume of the reaction is 50 l. 11. Incubate the mixture behind the plexigla ss shield at room-temperature for 4 hours.


97 12. For pre-hybridization, ad d 15 ml pre-heated (65C) hybridization buffer and 500l denatured salmon sperm DNA in the hybridization tube. 14. Place the tubes in the hybridization oven and incubate at 65C for 4h. 15. Add 500 l salmon sperm DNA to the labeled probe and boil for 5 min behind the plexiglass shield. 16. Discard the pre-hybridization solution into the sink and drai n the tube on a paper towel. 17. Just before the probe is ready, add 8 ml pre-heated hybridization buffer in the hybridization tube and move the tube behind the plexiglass shield. 18. Immediately after boiling, add the probe mi xture to the hybridizati on tube. Take care to avoid any spills. 19. Place the hybridization tube in the hybridi zation oven and incubate over-night at 65C (18 hours). 20. Prepare the wash solution (0.1x SSC + 0.1% SDS). Prepare enough to use 70 ml per wash for three washes per blot. 21. Heat the solution to 65 C using the water-bath. 22. Remove hybridization tubes from the oven and place behind the plexiglass shield. 23 Working behind the plexiglass shield, dispose the hybridization solutio n into the hazardous waste container using a funnel ta king care to avoid any spills. 24. Pour 70 ml pre-heated wash solution (65C) into the hybridization tube; replace the lid tightly and perform a quick wash by shaking the tube for a few seconds. 25. Dispose the wash solution into the hazardous waste contai ner and add another 70 ml preheated wash solution into the tube. 26. Place the tubes in the oven for 20 min. Working behind the plexiglass shield, dispose the wash solution into the hazardous waste contai ner and add 70 ml preheated wash solution into the tube for the final wash. 27 Place the tube in the oven for 20 min. Remove the tubes from the oven and place them behind the plexiglass shield. Dispose the wash solution into the hazardous waste container and wrap the membrane in Saran wrap. 28 Check for radioactivity on the membrane usi ng a Geiger counter. Also check the working area for any radioactive contamination. 29. Place the membrane with an X-ray film (Koda k) in an autoradiography cassette and allow 16-18 hours for exposure depending on the intensity of the signal from the Geiger counter. Place the cassette at -80C during exposure. Polymerase Chain Reaction Set up using the HotStarTaq DNA Polymerase (Qiagen) 1. Prepare a master-mix for all the sample s including the wild-type and a known positive control.as follows: 10x Buffer 2.50 l 5x Q solution 5.00 l 50x dNTP mix 0.50 l 50x MgCl2 0.50 l 10 M Forward primer 1.00 l 10 M Reverse primer 1.00 l HotStarTaq 0.15 l Sterile ddH2O 13.35 l


98 Final volume 24.00 l 3. Dispense 24 l of the master mix into each tube. 4. Add 1 l sample DNA for each sample tube (100-200 ng), 1 l sterile ddH2O to the negative control and 1 l plasmid (100pg/l) to the positive control. Mix well by pipetting, spin briefly and start the PCR program. Note: Remember to start the PCR program with 15 min at 95C to activate the HotStarTaq polymerase. Cytological Techniques used in the Charact erization of Herbicide Resistant Hybrids Embryo Sac Analysis 1. The flowering season of bahiagrass starts in May and continues until September. Maintain good plant growth by appropriate watering and biweekly application of fertilizer (e.g. Miracle Gro lawn food, The Scotts Company) (. Keep plants in greenhouse at 85F and natural photoperiod). 2. When the inflorescence has emerged and anthesis has started (generally the terminal portion of the raceme), remove the part of the raceme which flowered in the morning. 3. Next morning (between 7:00-9:00AM), rem ove the portion of the raceme which has flowering, (keeping the unflowered porti on intact) using a pa ir of scissors. 4. Fix the inflorescence in 70% ethanol: acetic ac id: methyl salicyclat e (3:1:1 v/v) for 2h. 5. Dissect the pistils carefully unde r a dissectoscope. Do not rem ove the two feathery stigmas (one on each side of the pistil). Prepare atleas t 25 pistils for each sample and place them in a clean microcentrifuge tube with the fixing solution. 6. Leave the pistils in the fixing solution overnight at room temperature. 7. Treat the pistils with a serious of solution as follows: Hydrogen peroxide 2 h 50% ethanol 45 min 75% ethanol 45 min 100% ethanol 45 min 50% ethanol: 50%met hyl salicyclate 45 min 25% ethanol: 75% met hyl salicyclate 45 min 100% methyl salicyclate overnight 8. Arrange the pistils on a clean si de. Add a drop of methyl salicyc late. Put the cover slip on it carefully. 9. Observe under a differential contrast microscope (DIC) at 20x magnification. Pollen Staining Procedure 1. Put a drop of orcein stain on a clean glass slide. 2. At the time anthesis between 7:00-9:00AM, collect the pollen by dusting it on the clean glass slide with a drop of the stain. 3. Gently cover it with a cover slip. 4. Observe the pollen grains under a microscope at 20x magnification.


99 Ploidy Determination using Flow Cytometer 1. Harvest the shoot meristem by excising atleast 5cm below the outermost leaf roll. Place it on a damp paper towel and put it in a zip-loc bag. Keep samples on ice. 2. Remove the outer layer of mature leaf. Exci se (1cm) the bottom part of the leaf roll. 3. Put 400l of CyStain A. Finely chop the meri stematic region usi ng a razor blade and a scalpel 4. Allow an incubation of 3 min at room temperature 5. Place the 1 micron nylon mesh filter on a clean disposable cuvette. Filter the material through the 1 micron mesh. 6. Add 800 l to the solution. Allow 3 min for staining 7. Place the cuvette in the sample holder on the Partec ploidy PA analyzer. 8. Read the diploid and tetraploid samples first, followed by the samples. Root-tip Preparations for Chromosome Counting 1. Harvest actively growing root s (5cm from the root tip). 2. Pre-treat the roots with 4hybdroxyquinoline for 4 h in the refrigerator. 3. Fix the material in 95% ethanol: acetic acid (3:1 v/v) overnight the refrigerator. 4. Store root samples in 70% ethanol. 5. Remove the root cap and place 5mm portion of the root-tip on a clean glass slide. 6. Put a drop of 45% acetic acid and macerate the sample using the a small round object (such as, rear end of a needle). 7. Put a drop of orcein stain. Allow the samples to stain for atleast 30 min. 8. Observe under a microscope, first at 20x and then at 40x. Buffers and Solutions Stock Solutions Kanamycin (10 mg/ml) Dissolve 100 mg kanamycin in 10 ml ddH2O. Filter sterilize and store in aliquots at 20C. Use 5 l per ml of LB broth. Paromomycin (50 mg/ml) Dissolve 0.5g paromomycin sulphate in 10 ml ddH2 O. Filter sterilize and store in aliquots at 20C. Use 1 ml/L media. CuSO4 (12.45mg/ml) 0.6225g CuSO4.5H2O dissolved in 50 ml ddH2O. Filter sterilize by usin g 1 micron filter fitted to a syringe. Store in aliquots at 20C. Use 100 l/L media. MS Vitamins (1000x) 5.156g powdered stock dissolved in 50ml ddH2O. Filter sterilize and store in aliquots at 20C. Use 1 ml/L media.

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100 BAP (1mg/ml) 0.025g powder dissolved in 500 l 1N NaOH. Make up to 25ml with ddH2O. Filter sterilize and store in aliquots at -20C. Use 1.1 ml/L media. Dicamba (2mg/ml) 0.1g dissolved in 1ml absolute EtOH (200 proof) by warming to 40C. Slowly add 49ml warm ddH2O (40C). Stir to dissolve. Filter sterilize and store in aliquots at -20C. Use 1.5 ml/L media. Media Callus Induction medium (IF) (400 ml) 1.72 g MS salts 40 l CuSO4 (12.45 mg/ml) 8 g Sucrose Dissolve in 350 ml ddH2O, adjust pH to 5.8 with 1N NaOH and make up the volume to 400 ml. Dispense into autoclavable media bottles and add 1.2 g phytagel. Autoclave for 20 min. Just before pouring in to Petri dishes add: 400 l MS Vitamins (1000x) 440 l BAP (1 mg/ml) 600 l Dicamba (2 mg/ml) Osmotic medium (0.4M IF) (400 ml) 1.72 g MS salts 40 l CuSO4 (12.45 mg/ml) 8 g Sucrose 29.12 g Sorbitol Dissolve in 350 ml ddH2O, adjust pH to 5.8 with 1N NaOH and make up the volume to 400 ml. Dispense into autoclavable media bottles and add 1.2 g phytagel. Autoclave for 20 min. Just before pouring in to Petri dishes add: 400 l MS Vitamins (1000x) 440 l BAP (1 mg/ml) 600 l Dicamba (2 mg/ml) Selection medium (IFP50) (400 ml) 1.72 g MS salts 40 l CuSO4 (12.45 mg/ml) 8 g Sucrose Dissolve in 350 ml ddH2O, adjust pH to 5.8 with 1N NaOH and make up the volume to 400 ml. Dispense into autoclavable media bo ttles and add 2.4 g agarose. Autoclave for 20 min. Just before pouring in to Petri dishes add: 400 l MS Vitamins (1000x) 440 l BAP (1 mg/ml) 600 l Dicamba (2 mg/ml)

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101 400 l Paromomycin (50 mg/ml) Regeneration medium (IFRP50) (400 ml) 1.72 g MS salts 40 l CuSO4 (12.45 mg/ml) 8 g Sucrose Dissolve in 350 ml ddH2O, adjust pH to 5.8 with 1N NaOH and make up the volume to 400 ml. Dispense into autoclavable media bo ttles and add 2.4 g agarose. Autoclave for 20 min. Just before pouring in to Petri dishes add: 400 l MS Vitamins (1000x) 40 l BAP (1 mg/ml) 400 l Paromomycin (50 mg/ml) Rooting medium (NHP50) (400 ml) 1.72 g MS salts 40 l CuSO4 (12.45 mg/ml) 8 g Sucrose Dissolve in 350 ml ddH2O, adjust pH to 5.8 with 1N NaOH and make up the volume to 400 ml. Dispense into autoclavable media bo ttles and add 2.4 g agarose. Autoclave for 20 min. Just before pouring in to Petri dishes add: 400 l MS Vitamins (1000x) 400 l Paromomycin (50 mg/ml) Buffers CTAB buffer (500 ml) 50 ml 1M Tris-HCl 20 ml 0.5M EDTA (disodium salt) 40.91 g NaCl 10g CTAB Make up the volume to 500 ml with ddH2O and autoclave for 20 min. 3M sodium acetate (50 ml) Dissolve 4.1 g sodium acetate in 50 ml ddH2O. Adjust pH to 5.2 with glacial acetic acid. 0.5M EDTA disodium salt, pH 8.0 (100 ml) Dissolve 18.61 g EDTA disodium salt in 100 ml ddH2O. 6x Loading dye (100 ml) 0.25% bromophenol blue 0.25% xylene cyanol FF 15% Ficoll Dissolve 15 g Ficoll in 60 ml ddH2O while stirri ng constantly. Add 20 ml ddH2O and warm the mixture. Add 0.25 g of both dyes, dissolve completely and make up volume to 100 ml with ddH2O. Autoclave for 20 min and store at room temperature.

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102 50x TAE (1L) 242 g Tris base 57.1 ml glacial acetic acid 100 ml 0.5M EDTA (pH 8.0) Combine all components in ddH2O, make the final volume to 1000 ml. Autoclave for 20 min. Use 1x TAE as running buffer. 5X TBE (1L) 54 g Tris base 27.5 g boric acid 20 ml 0.5M EDTA (pH 8.0) Combine all components in ddH2O, make the final volume to 1000 ml. Autoclave for 20 min. Use 0.5x TBE as running buffer. Hybridization Buffer (500 ml) 125 ml 1M Na2HPO4 (pH 7.4) 1 ml 0.5M EDTA (pH 8.0) 5 g BSA 175 ml 20% SDS Make up the volume to 500 ml with ddH2O. Autoclave for 20 min and store at 4C. 1M Na2HPO4, pH 7.4 (1L). Dissolve 142 g Na2HPO 4 in 800 ml ddH20. Adjust the pH to 7.4 with phosphoric acid and make up the volume to 1L. 20x SSC, pH 7.0 (1L) 175.3 g NaCl 88.2 g sodium citrate Dissolve in 800 ml ddH2O. Adjust pH to 7.0 w ith 1N HCl, make up the volume to 1L and autoclave for 20 min. 0.125N HCl 11ml conc HCl 898 ml dd H20 Denturation Buffer 87.66g NaCl 20g NaOH Dissolve in 800 ml ddH20 and make final volume to 1000 ml. Neutralization Buffer 87.66g NaCl 60.5g Trizma base Dissolve in 800 ml ddH20. Adjust pH to 7.5 with conc. HCl. Make final volume to 1000 ml with dd H20.

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103 Cytological Staining Solution Orcein Stain 0.4g Orcein 10 ml propinonic acid 10 ml lactic acid Disslove the mixture and filter it through Whatman filte r paper No. 1. Store it in the refrigerator at 4C.

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104 LIST OF REFERENCES Acuna CA (2006) Bahiagrass germ plasm reproduc tive characterization, and breeding at the tetraploid level. M.S. Thesis, University of Florida, Gainesville, FL Acuna CA, Blount AR, Quesenberry KH, Hanna WW, Kenworthy KE (2007) Reproductive characterization of bahiagrass germplasm. Crop Sci 47:1711-1717 Acuna CA, Martinez EJ, Quarin CL (2004) A pospory followed by sterility in a hypotriploid hybrid (2xX4x) of Paspalum. Caryologia 57:373-378 Adamowski E, Pagliarini MS, Batista LAR (1998) Chromosome elimination in Paspalum subciliatum (Notata group). Se x Plant Rep 11:272-276 Adamowski E, Pagliarini MS, Bonato ABM, Batista LAR, Valls JBM (2005) Chromosome numbers and meiotic behavior of some Paspalum accessions. Genet Mol Biol 28:773-780 Afolabi AS, Worland B, Snape JW, Vain P (2004) A large-scale study of rice plants transformed with different T-DNAs provides new insight s into locus composition and T-DNA linkage configurations. Theor Appl Genet 109:815-826 Agharkar M, Lom ba P, Altpeter F, Zhang H, Ke nworthy K, Lange T (2007) Stable expression of AtGA2ox1 in a low-input turfgrass (Paspalum notatum Flugge) reduces bioactive gibberellin levels and improve s turf quality in field conditions. Plant Biotechnol J 5:791801 Agrawal PK, Kohli A, Twyman RM, Christou P ( 2005) Transformation of plants with multiple cassettes generates simple transgene integr ation patterns and high expression levels. Mol Breeding 16:247-260 Aguado-Santacruz GA, Rascon-Cruz Q, Cabrera-Ponce JL, Martinez-Hernandez A, OlaldePortugal V, Herrera-Estrella L (2002) Transgenic plants of blue gram a grass, Bouteloua gracilis (HBK) Lag. ex Steud., from micr oprojectile bombardment of highly chlorophyllous embryogenic cells Theor Appl Genet 104:763-771 Altpeter F, Vasil 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, 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 and Visser R (2005) Particle bombardment and the genetic enhancem ent of crops: myths and realities. Mol Breeding 15:305-327 Altpeter F, James VA (2005) Genetic tr ansformation of turf-type bahiagrass (Paspalum notatum Flugge) by biolistic gene transfer. Intern Turfgrass Soc Res J 10:485-489

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108 Fu X, Duc LT, Fontana S, Bong BB, Tinjuangj un P, Sudhakar D, Twyman RM, Christou P, Kohli A (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic pl ants with simple integrati on patterns. Transgenic Res 9:11-19 Felber F, Kozlowski G, Arrigo N, Gaudagnualo R (2007) Genetic and ecological consequences of transgene flow to the wild flora. Green ge ne technology: Research in an area of social conflict. In: Advances in biochemical engineering/ bi otechnology 107:173-205 Gao CX, Jiang L, Folling M, Han LB, Nielse n KK (2006) Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer. Plant Cell Rep 25:19-25 Gao XR, Wang CK, Su Q, Wang Y, An LJ (2007) Phytase expre ssion in transgenic soybeans: stable transformation with a vector-less construct. Biotech Lett 29:1781-1787 Gates RN (2000) Response of incomplete Tifton 9 bahiagrass stands to renovation. J Range Manage 53:614-616 Gates RN, Mislevy P, Martin FG (2001) Herb age accumulation of three bahiagrass populations during the cool season. Agron J 93:112-117 Gates RN, Quarin CL and Pedreira CGS (2004) Bahiagrass. In: Warm-Season (C4) grasses, Agronomy monograph no 45s. Moser LE, Bu rson BL, Sollenberger LE (eds), ASA, CSSA, SSSA, Madison, WI Gernand D, Rutten T, Pickering R, Houben A (2006) Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatiniza tion. Cytogenet Genome Res 114:169-174 Goldman, JJ, Hanna WW, Flemi ng GH, Ozias-Akins P (2004) Ploidy variation among herbicideresistant bermudagrass plants of cv TifEagle transformed with the bar gene. Plant Cell Rep 22:553-560 Gondo T, Tsuruta S, Akashi R, Kawamura O, Hoffmann F (2005) Green, herbicide-resistant plants by particle inflow gun-mediated ge ne transfer to diploid bahiagrass (Paspalum notatum). J Plant Physiol 162:1367-1375 Gordon-Kamm WJ, Spencer TM, Ma ngano ML, Adams TR, Daines RJ, Start WG, OBrien JV, Chambers SA, Adams Jr. WR, Willets NG, Rice TB, Mackey CH, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603-618 Ha CD, Lemaux PG, Cho MJ (2001) Stable transf ormation of a recalcitrant Kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues. In Vitro Cell Dev Biol Plant 47:6-11

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109 Hadi MZ, McMullen MD, Finer JJ (1996) Transfor mation of 12 different plasmids into soybean via particle bombardment. Plant Cell Rep 15:500-505 Hanna WW, Burton GW (1986) Cytogenetics and breed ing behavior of an apomictic triploid in bahiagrass. J Heredity 77:457-459 Hanna WW (1995) Use of apomixis in cu ltivar development. Adv Agron 54:333-350 Hails RS, Morley K (2005) Genes invading new populations: a risk assessment perspective. Trends Ecol Evol 20:245-252 Hare PD, Chua NH (2002) Excision of selectable marker genes from transgenic plants Nat Biotechnol 20:575-580 Hartman CL, Lee L, Day PR, Tumer NE (1994) Herbicide-resi stant turfgrass (Agrostis palustris Huds) by biolistic transformation. Bio-Tech 12:919-923 Haygood R, Ives AR, Andow DA (2004) Population genetics of transgene containment. Ecol Lett 7:213-220 Hobbs SLA, Kpodar P, Delong CMO (1990) Th e effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transf ormants. Plant Mol Biol 15:851-864 Hoyle M, Hayter K, Cresswell JE (2007) Effect of pollinator abundance on self-fertilization and gene flow: application to GM canola. Ecological Adaptations 17:2123-2135 Iyer LM, Kumpatla SP, Chandrashekharan MB Hall TC (2000) Transgene silencing in monocots. Plant Mol Biol 43:323-346 Jackson MW, Stinchcombe JR, Korves TM, Schmitt J (2004) Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana. Mol Ecol 13:3609-3615 Jakowitsch J, Papp I, Moscone EA, van der Winden J, Matzke M, Matzke AJM (1999) Molecular and cytogenetic char acterization of a transgene lo cus that induces silencing and m ethylation of homologous promot ers in trans. Plant J 17:131-140 James VA, Avart C, Worland B, Snape JW Vain P (2002) The relationship between homozygous and hemizygous transgene expressi on levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:53-661 James VA, Neibaur I, Altpeter F (20 08) Stress inducible expression of DREB1A transcription factor from xeric Hordeum spontaneum L. in turf and forage grass (Paspalum notatum Flugge) enhances abiotic stress to lerance. Transgenic Res 17:93-104 Jia S, Wang F, Shi L, Yuan Q, Liu W, Liao Y, Li S, Jin W, Peng H (2007) Transgene flow to hybrid rice and its male-sterile lines. Transgenic Res 16:491-501

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110 Johnson PG, Larson SR, Anderton AL, Patterson JT, Cattani DJ, Nelson EK (2006) Pollenmediated gene flow from Kentucky bluegrass under cultivated field conditions. Crop Sci 46:1990-1997 Joersbo M, Donaldson I, Kreiberg J, Petersen S, Brunstedt J, Okkels FT (1998) Analysis of mannose selection used for transformati on of sugar beet. Mol Breeding 4:111-117 Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, Christou P (1999a) Molecu lar characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J 17:591-601 Kohli A, Gahakwa D, Vain P, Laurie DA, Christou P (1999b) Transgene expression in rice engineered through particle bombardmen t: molecular factors controlling stable expression and transgene silencing. Planta 208:88-97 Kononov ME, Bassuner B, Gelvin SB (1997) Inte gration of T-DNA binary vector 'backbone' sequences into the tobacco genome: Ev idence for multiple complex patterns of integration. Plant J 11:945-957 Kron P, Suda J, Husband BC (2007) Applications of flow cytometry to evolutionary and population biology. Annu Rev Ec ol Evol Syst 38:847-876 Kumar J, Shukla SM, Bhat V, Gupta S, Gupta MG (2005) In vitro plant regeneration and genetic transformation of Dichanthium annulatum. DNA Cell Biol 24:670-679 Lange M, Vincze E, Moller MG, Holm PB (2006) Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation. Plant Cell Rep 25:815-820 Li L, Li R, Fei S, Qu R (2005) Agrobacterium-mediated transformation of common bermudagrass (Cynodon dactylon). Plant Cell Tissue Organ Cult 83:223-229 Loc NT, Tinjuangjun P, Gatehouse AMR, Christou P, Gatehouse JA (2002) Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins confe rring insect resistance. Mol Breeding 9:231244 Luciani G, Altpeter F, Yacta yo-Chang J, Zhang H, Gallo M, Meagher R, Wofford D (2007) Expression of cry1Fa in bahiagrass enhances resist ance to fall armyworm. Crop Sci 47:2430-2436 Luo, H, Hu Q, Nelson H, Longo C, Kausch AP, Chandlee JM, Wipff JK, Fricker CR (2004) Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothr icin selection results in a high frequency of single-copy transgene integration. Plant Cell Rep 22:645-652

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117 BIOGRAPHICAL SKETCH Sukhpreet S andhu was born in Jammu, India, to parents Col. Manjit. S. Sandhu and Jaswant Sandhu. Although originally from Punjab, she spent her childhood in different parts of India including, Jammu and Kash mir, Darjeeling, and the desert s of Rajasthan. She graduated from BCM Model Senior Secondary School, Ludhi ana in 1996, and pursued a B.S. (Science) from Government College for Women, Ludhiana. She was awarded the Meri t Certificate for an outstanding academic achievement. She joined the Punjab Agricultural University (PAU), Ludhiana, and graduated with an M.S. (Biochemistry) in 2002. Im mediately after graduation, she joined as a Research Fellow, at the Department of Agronomy, PAU. She continued to work until she came to US to pursue a Ph.D at the Univ ersity of Florida (UF). The Department of Agronomy, UF awarded her the Fred Hull Award for excellence in academics and research. She graduated with a Ph.D. (Agronomy) from UF in 2008.