Effects of Gibberellin 2-Oxidase, Phytochrome B1, and Bas1

Home , Gibberellin, Phytochrome

EFFECTS OF GIBBERELLIN 2-OXIDASE, PHYTOCHROME B1, AND BAS1

GENE TRANSFORMATION ON CREEPING BENTGRASS

PHOTOMORPHOGENESIS UNDER VARIOUS LIGHT CONDITIONS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Jia Yan, M.S.

*****

The Ohio State University

2007

Dissertation Committee: Approved by Dr. Karl Danneberger, Advisor

Dr. David Gardner

Dr. Lisa Lee

Dr. Jim Metzger Advisor Graduate Program in Dr. Guo-liang Wang Horticulture and Crop Science

ABSTRACT

Sunlight is the primary energy source to drive photosynthesis, as well as the signal to stimulate series of developmental events ranging from germination to flowering in plants. Light can induce leaf formation, leaf expansion, and chloroplast differentiation, and inhibit stem elongation. Light has great impact on turfgrass quality and turfgrass performs best with a minimum of four to six hours of full sun per day. However, under certain circumstances, a grassed area may be shaded for most or all of the day, making it a problem for turfgrass to obtain enough light energy. Modern stadiums create a shaded environment that requires turfgrass for sports usage, while some area of a semi-enclosed arena receives little or no direct sunlight especially during winter season. Moreover, when sunlight penetrates the canopies and reaches the surface of the turf, both light quantity and quality (red/far-red ratio) drop dramatically. Shade stress causes succession of harmful physiological and morphological changes in plants, such as thinner, more delicate leaves, reduced tillering, poor shoot density, altered pigment concentration, and affected carbohydrate reserve. Moreover, it is difficult for turfgrass to recover from wear under shade.

Vertical growth remains a critical problem in turfgrass management. Practical methods can be used to control the plant growth. For example, application of chemical regulators can inhibit gibberellin (GA) biosynthesis and reduce the stem

growth. Alternatively, improving the characteristics of cultivars by biotechnology is a long-term option. GA 2-oxidase (GA2ox) genes are important in control of GA levels.

Overexpression of OsGA2ox causes a dwarf phenotype and delay in reproductive development in transgenic rice. Moreover, genetic engineering of phytochrome genes has provided a potential means to control vegetative growth and reproductive development. Phytochrome represents a family of red-light-absorbing photoreceptors that exist in the physiologically inactive Pr form and the active Pfr form.

Transformation and overexpression of the PHYB gene in Arabisopsis and tobacco resulted in a dwarf phenotype. Additionally, BAS1 is a gene regulating brassinosteroid

(BR) levels and light responsiveness in Arabidopsis. Overexpression of the BAS1 gene leads to decreased BR levels and a BR-deficient dwarf phenotype.

The objectives of our studies were: 1) develop creeping bentgrass plants transformed with GA2ox gene from runner bean (Phaseolus coccineus), BAS1 gene from Arabidopsis, and PHYB1 gene from tobacco (Nicotiana tabacum), respectively;

2) evaluate growth responses of transgenic plants under various light conditions and

reveal possible interactions between hormones and photo-receptors.

Our results showed that vertical growth, internode extension, and leaf growth of

transgenic creeping bentgrass plants were inhibited by GA2ox transformation under

reduced low light conditions in the field. True GA2ox gene transformants showed

increase in overall quality, shoot density, or stolon density compared with control

plants. Moreover, GA2ox transgenic lines tended to keep horizontal growth habit, possibly due to the increased GA metabolism by GA2ox overexpression, or the collaborated work of GA and light receptors in signal transduction. Greenhouse studies revealed the similar results to field studies. Strong transformants, such as

iii

GA6548 and GA6549, displayed dwarfism and superior quality under all light

conditions. RT-PCR results confirmed that mRNA level of foreign GA2ox was

correlated with the selection indices ranking of creeping bentgrass plants both in the

greenhouse and the field.

Furthermore, reduction in R:FR increased vertical growth and erectness of both

PHB1 gene transformants and control plants. As group, transgenic plants exhibited delayed vertical shoot growth and more horizontal shoot architecture, but did not demonstrate significant change in leaf growth or visual quality. PB0701 exhibited highest visual quality and lowest leaf growth rate among all plants. Leaf growth was observed to be affected mainly by the change of PPF rather than R:FR. Visual quality rating increased with the rise of both PPF and R:FR. RT-PCR results revealed the transcription of PHYB1 gene in all creeping bentgrass lines, with PHYB1 transformant PB0701 displayed highest transcription level. It may also be associated with its high quality and alleviation of shade response under low light conditions.

Finally, reduction in R:FR increased vertical growth and erectness of both BAS1 gene transformants and control plants, however, overexpression of BAS1 gene induced dwarfism in transgenic creeping bentgrass by delaying vertical shoot growth and altering shoot architecture. True BAS1 transformants (BS1305, BS1307, BS1701) had the best performance in most traits even under reduced PPF and R:FR.

To my parents

Benxiu Yan and Aihua Zhang

my husband

Hailu Meng

Thank you for the support and dedications

v ACKNOWLEDGMENTS

My dream may never come true without the support of many intelligent people. I would like to extend my sincere appreciation to my major advisor Dr. Karl

Danneberger for his support, guidance, encouragement, and friendship. It has been wonderful experience working with Karl for the past five years. I am also indebted to

Dr. David Gardner, Dr. Lisa Lee, Dr. Jim Metzger, and Dr. Guoliang Wang for serving on my committee and providing continuous and generous support.

Special thanks to Dr. Bo Zhou and other members in Dr. Guoliang Wang’s lab, to

Craig Yenkrek, Adri McKelvey, and other members in Dr. Jim Metzger’s lab, to Pam

Sherratt, Jill Taylor, and other turf group members for their support, assistance, and friendship. I am also grateful to Dr. Bob Harriman, Dr. Eric Nelson and other scientists in Scotts Company for their training and support.

To Mom and Dad: I want to thank both of you for teaching me the value of diligence and devotion, and for supporting me constantly.

To Hailu: Thank you for your consideration and encouragement during my most stressful time. You are a great companion during the long and tough journey.

VITA

February 24, 1978...... Born – Maanshan, Anhui Province, China

2001………………………...M.S. Biology, Nanjing University, Nanjing, China

2001-Present……………...... Graduate Research Associate, The Ohio State University

PUBLICATIONS

Referred Journal Articles

Yan, J., J. Li, Y. Xu, and J. Zhang. 2001. Studies on situations, causes and

controlling strategies of ecological pollution in Nanjing City. Urban

Environment & Urban Ecology 14:36-38.

FIELDS OF STUDY

Major field: Horticulture and Crop Science

Specialization: Turfgrass Physiology and Ecology

Turfgrass Molecular Breeding

vii

TABLE OF CONTENTS

Page

Abstract ………………………………………………………………………. ii

Dedication ……………………………………………………………………. v

Acknowledgements …………………………………………………………. vi

Vita …………………………………………………………………………. vii

List of Tables ………………………………………………………………… x

List of Figures ………………………………………………………………... xi

Chapters

1. Literature review…………………………………………………………. 1

Creeping Bentgrass……………………………………………………… 1

Genetic Transformation of Creeping Bentgrass………………………….. 2

Shade Studies Review…………………………………………………… 4

Phytochrome B Review…………………………………………………… 8

Gibberellin Review………………………………………………………. 13

Brassinosteroid Review…………………………………………………… 21

References…………………………………………………………………. 27

2 Field and greenhouse evaluation of transgenic creeping bentgrasses transformed with runner bean GA2ox gene………………………………… 43

viii

Abstract…………………………………………………………………….…. 43

Introduction……………………………………………………………….…. 44

Materials and Methods………………………..……………………………… 47

Results………………………………………………………………………... 54

Discussion…………………………………………………….……………… 60

References…………………………………………………………………… 79

3 Development and characterization of transgenic creeping bentgrass

transformed with tobacco PHYB1 gene……………………………………… 83

Abstract…………………………………………………………………….…. 83

Introduction………………………………………………………………….. 84

Materials and Methods……………………………………………………….. 86

Results…………………….………………………………………………….. 90

Discussion……………………………………………………………………. 94

References……………………………………………………………………. 108

4 Development and characterization of transgenic creeping bentgrass

transformed with Arabidopsis BAS1 gene……………………………………. 113

Abstract…………………………………………………………………….…. 113

Introduction………………………………………………………………….. 114

Materials and Methods……………………………………………………….. 115

Results……………….……………………………………………………….. 120

Discussion……………………………………………………………………. 122

References……………………………………………………………………. 132

ix LIST OF TABLES

Table Page 2.1 Vertical growth rate, internode length, and leaf length of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in 62 the field.…………………………………………..……………………..

2.2 Visual quality, shoot density, and stolon density of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in 64 the field ……………………...…………………………………………..

2.3 Color and erectness of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in the 66 field………………………….... ………………………………………...

2.4 Base index rankings of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in the 68 field...……………………………………………………………...

2.5 Instant photosynthetic photon flux and red:far-red ratio of the three light treatments………………………………………………………... 70

2.6 Visual rating, erectness rating, leaf growth rate, vertical growth rate, base index of 17 creeping bentgrass lines in the 71 greenhouse ………... ……………………………………………..

3.1 Instant photosynthetic photon flux and red:far-red ratio of the four light treatments ……………………………………………………………….. 97

3.2 Effects of full sun, neutral shade, canopy shade, and far-red light absorbing filter treatments on overall quality, erectness, leaf growth rate, and vertical growth rate of 4 PHYB1 transgenic, 1 NSC control, and 2 98 wild type control cultivars in 2005………………………………..

4.1 Instant photosynthetic photon flux and red:far-red ratio of the three light treatments ………………....…………………………………………….. 125

4.2 Visual rating and erectness of 9 creeping bentgrass cultivars in the greenhouse.………………………………………………………………. 127

LIST OF FIGURES

Figure Page

2.1 Schematic map of GA2ox expression cassette. Final construct also contained CP4-EPSPS and Kan genes as selective markers ………. 72

2.2A-C Treatments established in open sun light (A), under a shade cloth covered Quonset structure (B), and under mature deciduous 73 hardwoods (C)……………………………………………………….

2.3A-C Wild type control LS44, glyphosate-resistant control RR6260, and GA2ox gene transformed creeping bentgrass plants GA6549, GA6476, GA6547, and GA6548 (left to rigth) grown under clear filter(A); LS44, RR6260, GA6547, GA6476, GA6549, and GA6548 74 (left to right) under canopy shade(B); LS44, RR6260, GA6476, GA6549, GA6547, and GA6548 (left to right) under black shade cloth(C)………………………………………………………………. 2.4 Comparison of wild type plant LS44 and transgenic lines Ax6547 and Ax6548 (left to right) under full sun in the greenhouse…………. 75

2.5 Vertical growth rate(mm/day) of glyphosate-resistant cultivars(RR6260 and RR6261), positive GA2ox transformants (GA6435,GA6548, and GA6549), negative GA2ox transformant 76 (GA6487), and wild type controls (Crenshaw and LS44) in the greenhouse…………………………………………………………….

2.6 Erectness ratings of glyphosate-resistant cultivars(RR6260 and RR6261), positive GA2ox transformants (GA6435,GA6548, and GA6549), negative GA2ox transformant (GA6487), and wild type 77 controls (Crenshaw and LS44) in the greenhouse…………………….

2.7 Reverse-transcription (RT-PCR) of RNA samples from putative GA2ox transformants (GA6465, GA6465, GA6476, GA6548, GA6549, GA6555, and GA6487), conventional control plants (Crenshaw and LS44), and glyphosate-resistant control plants 78 (RR6237 and RR6260). Arrows specify the expected GA2ox and Actin bands……………………………………………………………

3.1 Schematic map of PHYB1 final construct……………………………. 99

3.2A-H Production of transgenic creeping bentgrass (Agrostis palustris) plants using biolistic bombardment of embryogenic callus cells…….. 100

3.3A-C LS44 (A), no shot control (B), and PB0701 (C) under canopy shade, neutral shade, full sun, and far red light (AFR) filter (left to right)…… 102

3.4A-D Visual quality ratings of wild type cultivars (Crenshaw and LS44), no shot control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) under A 103 Neutral shade, B Canopy shade, C Full sun, and D far-red light absorbing (AFR) filter…………………………………………………

3.5A-D Erectness ratings of wild type cultivars (Crenshaw and LS44), no shot control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) under A Neutral 104 shade, B Canopy shade, C Full sun, and D far-red light absorbing (AFR) filter……………………………………………………………. 3.6A-D Leaf growth rate(mm/day) of wild type cultivars (Crenshaw and LS44), no shot control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) 105 under A Neutral shade, B Canopy shade, C Full sun, and D far-red light absorbing (AFR) filter……………………………………………

3.7A-D Vertical growth rate(mm/day) of wild type cultivars (Crenshaw and LS44), no shot control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) 106 under A Neutral shade, B Canopy shade, C Full sun, and D far-red light absorbing (AFR) filter……………………………………………

3.8 Reverse-transcription (RT-PCR) of RNA samples from putative PHYB1 transformants (PB1101, PB1102, PB0701, PB0501), 107 conventional control plants (Crenshaw and LS44), and tissue culture control plant (NSC)…………………………………………………... 4.1 Schematic map of BAS1 gene expression cassette. Final construct also contained CP4-EPSPS gene as the selective marker……………. 128

4.2A-D Production of transgenic creeping bentgrass (Agrostis palustris) plants using biolistic bombardment of embryogenic callus cells…….. 129

4.3 Reverse-transcription (RT-PCR) of RNA samples from transgenic glyphosate resistant plants, conventional control plants (Crenshaw 130 and LS44), and tissue culture control plants (NSC). Arrows specify the expected BAS1 and Actin bands…………………………………. 4.4 Vertical growth rate(mm/day) of transgenic lines (BS1305, BS1307, BS1701, BS0101), conventional cultivars (Crenshaw and LS44), 131 tissue culture control (NSC) plants of creeping bentgrass (Agrostis palustris) under neutral shade, canopy shade, and full sun.

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CHAPTER 1

LITERATURE REVIEW

CREEPING BENTGRASS

Creeping Bentgrass Taxonomy and Characteristics

Within the family Gramineae (Poaceae) and subfamily Pooideae, the genus

Agrostis (bentgrass) belongs to the supertribe Poeae and tribe Aveneae (Turgeon, 1991;

Warnke, 2003). The taxonomy within the genus Agrostis is difficult and sophisticated

(Warnke, 2003). Agrostis consists of approximately 150-220 species which are mainly native to Eurasia (Christians, 1998; Harlan, 1992; Hitchcock, 1950; Turgeon, 1991). It was reported that about 40 bentgrass species are nearly equally distributed in eastern and western United States (Gould, 1968). Five species have been utilized as turfgrass including Agrostis stolonifera-creeping bentgrass (2n=4x=28), Agrostis canina

L.-velvet bentgrass (2n=2x=14), Agrostis capillaries L.-colonial bentgrass

(2n=4x=28), Agrostis castellana L.-dryland bentgrass (2n=4x=28), and Agrostis gigantean L.-redtop bentgrass (2n=6x=42) (Christians, 1998; Warnke, 2003).

Research proved that creeping bentgrass is generally a strict allotetraploid with a genome designation of A2A2A3A3(Warnke, 2003).

Since its introduction from Eurasia during the colonial period, creeping bentgrass

has been naturalized in all states of US, except warmer southern part of states in the

Southeast (Gould and Shaw, 1983; Xu and Huang, 2001). It is named after its

vigorous creeping stolons that develop at the surface of the ground and initiate new

roots and shoots from the nodes (Beard, 1973). The optimum temperature for its shoot

growth is between 16 and 24°C, while between 10 and 19°C for root growth (Beard,

1973). Creeping bentgrass tolerates severe coldness, while its heat tolerance is fair

(Christians, 1998). It can survive in a variety of soil types, but grows best in moist, fertile, fine-textured, slightly acidic soil (Beard, 1973).

Use and Culture

Creeping bentgrass is best known for its fine texture and tolerance of mowing

heights as low as 3.2 mm (Emmons, 2000). It forms a dense, uniform, fine-textured

turf when closely mowed, thus it has been used extensively on golf greens and

fairways (Bell and Danneberger, 1999b). Besides, creeping bentgrass can be

propagated both vegetatively and by seeds. Crenshaw and LS44 are both synthetic

seeded cultivars. Crenshaw is a six-clone bentgrass released in 1994 by the Texas

Agricultural Experimental Station.(Yamamoto and Engelke, 1998). LS44 is Dr.

Virginia Lehman’s fourth generation of bentgrass released in 2003 (Overbeck, 2003).

GENETIC TRANSFORMATION OF CREEPING BENTGRASS

Agrostis Species and Traits of Interest

Gene transformation has been successfully performed in several Agrostis species, including Agrostis alba (Asano and Ugaki, 1994), Agrostis stolonifera (Asano et al.,

1998; Chai et al., 2002; Dai et al., 2003; Dalton et al., 1998; Fu et al., 2005; Guo et al.,

2003; Han et al., 2005; Hartman et al., 1994; Lee et al., 1996; Luo et al., 2004; Wang

and Ge, 2005a; Xiao and Ha, 1997; Yu et al., 2000; Zhong et al., 1994), and Agrostis

tenuis (Chai et al., 2004). Genetic engineering has been utilized to develop traits of

interest in bentgrass such as disease resistance (Chai et al., 2002; Dai et al., 2003; Fu

et al., 2005; Guo et al., 2003) and herbicide resistance (Gardner et al., 2004; Gardner

et al., 2003).

Transformation Method

Various lab methods have been developed to deliver gene of interest into plant genome. Transgenic bentgrasses were generated by direct gene transfer mediated by polyethylene glycol treatment (Lee and Day, 1998; Lee et al., 1996), silicon carbide fibers or whiskers (Asano et al., 1991; Dalton et al., 1998), or electroporation (Asano and Ugaki, 1994; Asano et al., 1991; Asano et al., 1998; Sugiura et al., 1998).

Currently, transgenic bentgrass has been mainly generated with protoplast-independent methods, including microprojectile bombardment (Lee and

Day, 1998) and Agrobacterium-mediated transformation(Chai et al., 2004; Han et al.,

2005; Luo et al., 2004; Yu et al., 2000).

Tissue Culture and Gene Transformation

The establishment of a tissue culture system to regenerate plants is essential for

genetic manipulation of monocot plants (Wang et al., 2001). Callus culture has been the basis in bentgrass transformation. Embryogenic calli generated from seeds are often used as direct targets for both microprojectile bombardment and Agrobaterium infection. Instead, embryos are used for transformation and calli are induced from

them afterwards. However, the process of callus induction and plant regeneration

from the induced callus are lengthy and difficult. It may also bring about somaclonal

variation (Wang and Ge, 2006). Alternatively, a direct, callus-free transformation

system using stolon nodes was recently developed for three grass species, including

creeping bentgrass. The new method only takes one-third of the time required for

other reported transformation systems (Ge et al., 2006; Wang and Ge, 2005a; Wang

and Ge, 2005b).

SHADE STUDIES REVIEW

Solar energy is the ultimate energy source for life on earth. Green plants harvest

solar energy by photosynthesis and pass it on to other forms of life. Competition for light is of vital importance for plants to survive and thrive. Quantity, quality, direction,

and duration of light all have great impact on photosynthesis and other growth and

developmental events in plants (Franklin and Whitelam, 2005). Light has great impact

on turfgrass quality and turfgrass performs best in full sun with a minimum of four to

six hours of full sun per day (Reicher and Throssell, 1998).

Over the long period of evolution, plants, the photosynthetic and sessile

organisms, have developed adaptation mechanisms in response to changes of the

natural environment (Fernández et al., 2005). Light is not only the primary energy

source for plants, but also as a stimulus to trigger photomorphogensis, series of

developmental events including germination, deetiolation, leaf expansion, stem and

petiole elongation, circadian clock, and transition from vegetative to reproductive

growth (Nagy and Schäfer, 2002; Quail, 2002)

Light Intensity

Solar energy has the traits of both wave and particle. Photosynthetically active

radiation (PAR) is defined as solar radiance between 400- to 700-nm of the light

spectrum (Bell et al., 2000; Parks et al., 1996). Plant pigments, including chlorophyll,

carotein, and zeaxanxin, absorb PAR best at particular wavelength. PAR is termed

photosynthetic photon flux (PPF) when it is expressed on a quantum basis (Taiz and

Zeiger, 2002c). PPF is measured as irradiance per unit area per unit time at

400-700nm wavelengths (Wherley, 2003). PPF can decline to 1-5% of sunlight in the

shade of deciduous canopy (Shirley, 1945).

Perform of turfgrasses varies differently under low PPF. Warm-season turfgrass

plants (C4) are generally less shade tolerant than cool-season grasses (C3). The latter

usually have a low light compensation point and maximize photosynthesis in low light

conditions (Danneberger, 1993). Light compensation point is the point at which CO2 uptake in photosynthesis equals CO2 release in photorespiration in plant leaves. When light intensity drops below the compensation point, turf plants utilize or even deplete carbohydrate reserve for respiration. Turfgrass quality, vigor, and ability to recover from wear will decrease eventually.

Creeping bentgrass usually has good shade tolerance compared to other turfgrasses. However, when grown on golf courses, creeping bentgrass is mowed as low as 13 mm. Intensive mowing removes most of leaf surface area and reduces turfgrass photosynthesis and recuperative ability under low light conditions (Bell and

Danneberger, 1999b). Besides, reduction of air flow under trees usually increases humidity and occurrence of turfgrass disease (Bell and Danneberger, 1999a).

Different sources including trees and buildings could pose shade on turfgrass

surface. Modern stadium design creates a shaded environment that substantially

increases the stress on turfgrass for sports usage through its usage of semi and fully

enclosed structures (Tegg and Lane, 2004). PPF drops dramatically when the sunlight

penetrates the canopies and reaches the surface of the turf. Shade stress causes increase of vertical growth and succession of harmful physiological and

morphological changes in plants, such as thinner, more delicate leaves, reduced

tillering, poor shoot density, altered pigment concentration and affected carbohydrate

reserve (Bell and Danneberger, 1999b).

Light Quality

Light quality is described as the ratio of photon irradiance in red light (R, photon

irradiance between 655 and 665 nm) to that in far-red light (FR, photon irradiance

between 725 and 735 nm). The R:FR ratio is usually constant all year round,

averaging about 1.15. However, R:FR ratio could drop to as low as 0.7 at dusk, which

is referred to as end-of-day far-red light (Smith, 1982). Moreover, R:FR ratio under

vegetation canopy falls in the range 0.05-0.7 (Smith, 1982). The decrease in light

quality was reported to be greater under deciduous than coniferous tree shade (Beard,

1973).

Phytochromes are the photosensors of light quality change in the red and far red

region of the spectrum. Phytochromes are usually in the dynamic equilibrium of Pr

and Pfr forms. Decrease in R:FR triggers conformational change towards biologically

inactive Pr form and affects downstream reactions leading to shade avoidance

response (Franklin and Whitelam, 2005). Shade avoidance syndrome usually include

stem and petiole elongation, reduction in chlorophyll content and leaf thickness, elevation in leaf angle (erectness), reduced tillering in grasses (Casal et al., 1986).

Low light quality may also promote flowering and seed setting (Dorn et al., 2000;

Halliday et al., 1994; Smith and Whitelam, 1997). Plants having shade avoidance often result in lodging damage or mechanical injury and lost of compatibility in a long run (Casal and Smith, 1989).

Management Practice

Practical management program can be conducted to maintain turfgrass quality in shade. The methods include: pruning and tree removal which improve sunlight penetration through the tree canopy and allow more air circulation under the tree; raising mowing height and leaving maximum leaf area for photosynthesis; deep and infrequent irrigation which helps minimize disease pressure on turf; reduction in traffic which allows damaged turf to recuperate; decrease of Nitrogen application which helps control shoot growth and preserve carbohydrate in turf; pest management which lowers the competition pressure; overseeding which improves turf density in shaded areas; selection of turf species and cultivars adequate for shaded areas; application of plant growth regulators for stature control.

Plant Growth Regulators

Plant growth regulators (PGRs) are synthetic chemicals which are exogenously applied on plants to promote or retard elongation (Rademacher, 2002). The PGR, trinexapac-ethyl (TE; Primo), was introduced for use on turf in 1991 (Watschke and

DiPaola, 1995). TE interferes with the action of GA by inhibiting the conversion of

GA20 to GA1, which is the bioactive GA in plants (Adams et al., 1992). TE application

has been proven successful in suppression of shoot growth of many turfgrass species

(Fagerness and Penner, 1998; Johnson, 1992; King et al., 1997; Qian and Engelke,

1999). TE application also can improve turfgrass quality and color under unfavorable

light conditions (Goss et al., 2002; Qian and Engelke, 1999). However, PGRs need to

be applied repeatedly to achieve effective control of plant height. Cost, effectiveness,

possible undesired environmental consequences, and public concerns are all important

issues to consider at time of PGR application (Busov et al., 2003).

PHYTOCHROME B REVIEW

Plant Photoreceptors

Plant growth and development are regulated by environmental light signals, including changes in light quality, quantity, and daily light/dark cycles (Fernández et

al., 2005; Jiao et al., 2005). Photoreceptors absorb sunlight in the wavelength range

from ultraviolet (Neff et al., 1999) to near infrared. UVB photoreceptor(s) have been

characterized, but not yet isolated (Beggs and Wellman, 1994). Conversely, other

photoreceptors have been isolated, including cryptochromes cry1 and cry2 (Cashmore

et al., 1999), phototropins phot1 and phot2 (Sakai et al., 2001), and phytochromes

phyA–E (Clack et al., 1994). Cryptochromes are blue light and UV-A sensors which

control de-etiolation, circadian rhythms, and time of flowering (Cashmore et al., 1999;

Lin, 2002). Phototropins perceive blue light and regulate phototropic curving, stomata

opening, chloroplast movement, and other physiological responses (Liscum et al.,

2003; Wada et al., 2003). Phytochromes sense the light quality change in the red and

far-red region of the spectrum (Franklin and Whitelam, 2005).

Phytochrome was first successfully purified from etiolated oats (Avena sativa)

in 1964 (Siegelman and Firer, 1964). However, completely intact, native phytochrome

was not isolated until 1983 (Vierstra and Quail, 1983). Phytochromes in higher plants are encoded by a small multi-gene family (Quail, 1994) and can be divided into two distinctive groups, type I, ‘light-labile’ and type II, ‘light-stable’ phytochromes. Type I phytochromes are involved in the very low fluence rate response (VLFR) and the far-red high irradiance response (HIR). On the contrary, type II phytochromes control the red/far-red reversible (low fluence rate, LFR) light response (Fernández et al.,

2005). Angiosperms contain three chief phytochromes, phyA, phyB and phyC, apoprotein of which are encoded by the genes PHYA, PHYB, and PHYC. The other two phytochromes, phyD, and phyE, were found in dicotyledonous plants (Mathews and Sharrock, 1997). PHYA–PHYE genes have been sequenced and identified in model plants Arabidopsis thaliana (Clack et al., 1994; Sharrock and Quail, 1989).

Arabidopsis phyA belongs to the ‘light-labile’ and phyB–phyE to the ‘light-stable’ groups (Fernández et al., 2005). Interestingly, light-labile phyB was found in a photoperiod-insensitive barley line (Hanumappa et al., 1999). PHYB, PHYD, and

PHYE genes are more closely related to each other than to either of PHYA and PHYC.

Therefore, it is proposed that PHYB-PHYE genes constitute an individual subgroup within the Arabidopsis PHY gene family (Goosey et al., 1997).

Phytochrome Structures

Phytochromes are dimers made up of two identical subunits called holoproteins.

Each holoprotein includes a polypeptide chain (apoprotein) linked to a light-absorbing,

linear tetrapyrrole pigment molecule (chromophore) via a covalent bond (Taiz and

Zeiger, 2002a). Phytochromes have two photointerconvertible forms, inactive Pr form

absorbing maximally in the red region (660 nm) and bioactive Pfr form with highest absorption in the far-red region (730 nm). Pr protein obtains activity through conversion to Pfr form, triggered by red light; similarly, far-red light promotes the conversion of Pfr to Pr. Normally phytochromes are mixtures of both forms. The concentration of active Pfr form of phytochrome reflects the relative amount of R and

FR in ambient light (Franklin and Whitelam, 2005; Mathews, 2005). Slight decrease in R:FR ratio may result in substantial reduction in ratio of Pfr to Ptotal (Smith,

1982).

Shade Avoidance and Phytochromes

Phytochromes play an important role in a wide range of growth and development events, including seed germination, seedling de-etiolation, gene expression, chloroplast differentiation, floral induction or suppression, and senescence (Chory,

1997). Besides, phytochromes involve in gravitropism (Parks et al., 1996), shade avoidance response (Smith, 1995), and circadian mechanism of plants (Kreps and Kay,

1997). In this review shade avoidance will be discussed in further details.

Creation of single, double, or triple mutants of phytochrome genes facilitates the study of function of each individual phytochrome, as well as their interaction

(Franklin and Whitelam, 2005). phyA controls plant development under far-red light while phyB acts as the chief phytochrome regulating plant development under continuous red light irradiation (Somers et al., 1991). PhyC has similar function as phyB and mediates photomorphogenesis during the whole life span of plants the plant.

Nonetheless, the phenotype of phyC mutant is phyB-dependent (Monte et al., 2003).

The only function of phyD in inhibiting hypocotyl growth is so weak and therefore is probably negligible in some natural environments (Aukerman et al., 1997). At last, the

lack of shade avoidance response of phyE single mutant revealed that phyE is essentially functional in adult plants (Devlin et al., 1998).

Phytochromes function as sensors of shading by the surrounding vegetation (Taiz and Zeiger, 2002a). Plants quickly initiate escape response in response to decrease in

R:FR ratio of light by allocating more resource to vertical growth rather than storage organ development. Other characteristics of plants in shade include low chlorophyll content, show reduced leaf thickness, elevated leaf angle, and enhanced apical dominance leading to reduced branching in dicots and tillering in monocots, respectively (Casal et al., 1986). In general, plants exhibit shade-avoidance syndrome which facilitate them to grow above the canopy and obtain more unfiltered and photosynthetically active light (Taiz and Zeiger, 2002a). However, in an extended unfavorable growth environment where R:FR ratio remains low, plants favor reproductive success instead by promoting flowering and seed set (Dorn et al., 2000;

Halliday et al., 1994; Smith and Whitelam, 1997). Plants may benefit from shade avoidance in a short term; however, they also become more susceptible to lodging damage or mechanical injury and eventually lose compatibility (Casal and Smith,

1989).

Leading role of phyB in perceiving R:FR ratio signal was revealed by

‘constitutive shade avoidance’ of phyB mutants of various plant species grown in white light (Devlin et al., 1992; Lo´pez-Juez et al., 1992; Reed et al., 1993; Somers

DE, 1991). In light grown plants, phyB is the major sensor of red light (Neff et al.,

2000) and regulates cell expansion or elongation. Hypothetically Arabidopsis phyB

controls cell size by regulating nuclear endoreduplication in hypocotyls (Gendreau et

al., 1998). Arabidopsis PHYD gene overexpression can restore the red light

stimulated response of hypocotyl elongation in tobacco hgl2 mutant, whereas it hardly

works in phyB-9 Arabidopsis mutant (Fernández et al., 2005). Moreover, the function

redundancy of phytochromes has been confirmed in controlling shade avoidance,

especially those of phyB, phyD, and phyE (Franklin et al., 2003; Goosey et al., 1997;

Mathews and Sharrock, 1997).

Massive accumulation of phyA only occurs in etiolated seedlings due to fast degradation of phyA in red light (Quail, 1994). phyA mainly regulates the FR high irradiance-induced development of de-etiolating seedlings (Smith and Whitelam,

1990). Studies on phyA, phyAphyB mutants, as well as PHYA transformants, have

suggested that phyA suppresses phyB-regulated hypocotyl elongation in shade

avoidance responses (Johnson et al., 1994; Robson et al., 1996; Yanovsky et al.,

1995).

Phytochrome Signal Transduction

Light-induced conformational change of phytochromes between Pr and Pfr forms

affects the subcellular localization, stability (only phyA), and the protein kinase

activity of phytochromes (Neff et al., 2000).

PhyA and phyB have been proved to have partially overlapped early signaling

pathways. Both pathways contain positively and negatively acting factors. The

pathways eventually join together downstream in the regulation of

photomorphogenesis and the circadian clock (Quail, 2002).

In general, there may be at least two separate light signal transduction pathways

from phytochrome photoreceptors to photoresponsive genes. In the rapid pathway,

light signals regulate gene transcription by directly targeting to the promoters of

primary response genes; while in the slower and more indirect pathway, regulation of

the transcription of target genes involves post-translational, proteolytic modulation of

transcription-factor levels (Quail, 2002).

GIBBERELLIN REVIEW

Discovery of Gibberellins

Gibberellins (GAs) are defined as a family of naturally occurring tetracyclic

diterpenoid acids based on the ent-gibberellane carbon skeleton (Sponsel and Hedden,

2004). Some GAs have complete 20 carbons while others only have 19. GAs were

first discovered in Japan in the 1930s. They were named after the fungus Gibberella

fujikuroi from which they were first isolated. GAs secreted by the fungus caused the

“foolish seedling” disease, extensive overgrowth and little seed production of the

infected rice plants (Taiz and Zeiger, 2002b). GAs were mainly identified in vascular

plants and also found in bacteria and fungi. They were numbered in chronicle order of

their identification. To date 136 GAs have been completely characterized and

assigned GA1 through GA136. Nevertheless, hardly any GAs are intrinsically bioactive.

Only GA1, GA3, GA4, GA5, GA6, and GA7 meet the requirement for bioactive GAs,

which are “C19-GAs with a carboxylic acid group on C-6,” “possess a 3β-hydroxyl

group or some other functionalization at C-3, such as a 2,3-double bond.” GA1 was first identified from runner bean (Phaseolus coccineus) and has been detected in 86 plants. It is believed to be the major active GA influencing stem elongation. However,

GA4 has been found to co-exist with GA1 in many species and lead a major role of GA

activities in some species. Besides, GA3 (gibberellic acid), which was discovered in G.

fujikuroi and 45 plants, is also physiologically active (Sponsel and Hedden, 2004).

Biosynthesis of GAs

GAs biosynthesis starts from the geranylgeranyl diphosphate via isopentenyl

diphosphate (IPP), the 5-carbon building material for all terpenoid/isoprenoid compounds. There was substantial indirect evidence to propose that IPP is formed solely via the mevalonate(MVA)-independent methylerythritol phosphate (MEP) pathway in plants, however, it is yet to be confirmed whether there is any cross-over

of intermediates from the MVA-dependent pathway (Hedden and Kamiya, 1997;

Lange, 1998). GA biosynthesis in higher plants usually comprises of three parts. The

first part occurs in proplastid and leads to biosynthesis of the tetracyclic hydrocarbon

ent-kaurene. In the second part, ent-kaurene is eventually oxidized to GA12 and its

13-hydroxylated analog GA53 in the endoplasmic reticulum. Finally, in the third

which takes place in the cytosol, GA12 and GA53 are further oxidized to other C20-GAs

and C19-GAs (Sponsel and Hedden, 2004).

Major genes for GA-biosynthesis enzymes have been cloned and characterized

(Hedden and Kamiya, 1997; Olszewski et al., 2002). Terpene cyclases including

diphosphate synthase (CPS) and ent-kaurene synthase (KS) catalyze the two-step

conversion of GGDP to ent-kaurene. In the second stage of GA biosynthesis,

membrane-bound monooxygenases utilizing cytochrome P450 catalyze the oxidization of highly hydrophobic ent-kaurene to GA12-aldehyde. Subsequently,

mono-oxygenases catalyze the following C7-oxidation of GA12-aldehyde to yield

GA12 and the 13-hydroxylation of GA12 to GA53. Soluble 2-oxoglutarate-dependant

dioxygenases involve in the third stage of the pathway (Hedden and Kamiya, 1997).

GA 20-oxidase (GA20ox) can catalyze the conversion of GA12 and GA53 to GA9 and

GA20, respectively, by successive oxidation of C-20 from a methyl group, through the

alcohol and aldehyde, followed by the lost of this C atom as CO2. GA9 and GA20 are

further converted to physiologically active GAs, GA4 and GA1, respectively, by the

3β-hydroxylation catalyzed by GA 3-oxidase (GA3ox). In some species, further

oxidation of GA9 and GA20 catalyzed by GA3ox results in the formation of GA7 and

GA3, respectively, via 2, 3-didehydro-GA9 and GA5. A third class of dioxygenase, GA

2-oxidase (GA2ox), is responsible for irreversible deactivation of GAs by

2-hydroxylation and, in several species, additional oxidation to 2-keto derivatives

(Hedden and Kamiya, 1997).

Regulation of GA Levels

Developmental regulation and sites of GA biosynthesis

Growing seeds and developing fruits are sites of GA biosynthesis and contain the

highest level of GA. Nonetheless, GA level normally reaches the maximum in early

stage of seed and fruit growth, and drops to zero in mature seeds (Sponsel and Hedden,

2004; Taiz and Zeiger, 2002d).

GA biosynthesis sites also include quickly growing sections of the plants, such as young, actively growing buds, leaves, and upper internodes (Elliott et al., 2001). For

example, instead of mature chloroplasts, ent-kaurene is synthesized in immature

plastids in actively-growing plant parts (Aach et al., 1997). Besides, it was suggested

that GA biosynthesis and action take place in the same tissues or even cells.

GA homeostasis

GAs maintain their own homeostasis in plants by limiting GA biosynthesis and

promoting GA degradation (Taiz and Zeiger, 2002b). For instance, GA application

brings about a down-regulation of GA20ox and GA3ox genes (negative feedback

regulation) and an up-regulation of GA2ox gene (positive feed-forward regulation)

(Sponsel and Hedden, 2004; Taiz and Zeiger, 2002b). The self-regulation mechanism

facilitates the change of active GA level responding to developmental and

environmental cues, and may also help restore GA concentration to normal levels,

especially after dramatic alteration (Vidal et al., 2003).

Regulation by light

The interaction between GA and light will be discussed in details later in this

review.

Regulation by temperature

Low temperatures are crucial for the germination of some seeds (stratification)

and flowering in some species (vernalization). GAs engage in both processes and can

induce the same effect in the absence of cold treatment. ent-kaurenoic acid builds up to high quantity in the shoot tip, the perception site of the cold stimulus, without cold induction. After vernalization and a following return to high temperature, the level of the ent-kaurenoic acid drops gradually (Hazebroek et al., 1993). Recent study on

Arabidopsis thaliana seeds has displayed the up-regulation of a subset of GA biosynthesis genes by low temperature. It has also shown an increased level of bioactive GAs and transcript abundance of GA-inducible genes (Yamauchi et al.,

2004).

Hormonal regulation

Levels of other hormones can have a substantial impact on GA biosynthesis. It

has been exhibited that auxin significantly influences GA biosynthesis and

metabolism. A particular auxin, 4-chloroindole-3-acetic acid, up-regulates GA20ox

gene expression in growing pods (Huizen et al., 1997). It was also reported that auxin

from the apical bud maintained the internode GA1 level by promoting GA1 biosynthesis, especially the step from GA20 to GA1, and by preventing GA1

deactivation (O'Neill and Ross, 2002; Ross et al., 2000). In addition, brassinosteroid

(BR) appears to up-regulate GA20ox gene expression independent of GA.

GA Signal Transduction

GA signaling in cereal aleurone

GA signaling pathways connect GA perception to downstream GA-induced

responses (Bethke and Jones, 1998). Throughout seed germination and early stage of

seedling growth, growing embryo obtains soluble food resource by the digestion of

the food reserves of the endosperm. Breakdown of the starch and protein storage

materials is catalyzed by numerous hydrolytic enzymes in aleurone cells, which

include α-amylase, proteases, and cell wall–degrading enzyme. After being

synthesized by the embryo and transported into the endosperm, GAs stimulate gene

expression of the hydrolytic enzymes (Olszewski et al., 2002).

Overall GA response

GA metabolism has been shown to closely interact with GA response pathways.

However, it remains unclear how the GA metabolic pathway is regulated both by

developmental programs and the GA response pathway (Olszewski et al., 2002).

Physiological Functions of GA

GAs involve in many growth and developmental processes. The major

GA-regulated events are listed as follows (Taiz and Zeiger, 2002d):

GAs stimulate stem elongation by promoting cell elongation and cell division.

Cell elongation may involve GA-induced activity of the enzyme xyloglucan endotransglycosylase (XET) and expansins, which are cell wall proteins facilitating wall loosening by breaking hydrogen bonds between wall polysaccharides in acidic conditions (Taiz and Zeiger, 2002d).

GAs can cause bolting and stem elongation in long day plants when grown in long day conditions (Sponsel and Hedden, 2004).

GA application promotes formation of staminate (male) flowers in dicots such as cucumber, hemp, and spinach, whereas it stimulates transition to pistillate (female) flowers in maize (Taiz and Zeiger, 2002d) .

GA application breaks seed dormancy and induces seed germination mainly by promoting gene transcription of hydrolases which can catalyze the breakdown of storage materials in aleurone labyers (Taiz and Zeiger, 2002d).

GAs can also be applied as a commercial spray or dip to stimulate the stalk growth of parthenocarpic (seedless) grapes. Besides, GAs can delay senescence in citrus fruits and extend the market period (Taiz and Zeiger, 2002d).

Interaction of GA with Light Signals

Seed germination

Red light promotes seed germination by stimulating GA biosynthesis (Yamauchi

et al., 2004) or tissue responsiveness to GA (Hilhorst and Karssen, 1998). It is

suggested that red light induces 3β-hydroxlation of GA and this effect is controlled by

phytochromes due to red/far-red photoreversibility (Olszewski et al., 2002).

De-etiolation

The de-etiolation process, including inhibition of stem elongation, form change of

apex, and leaf and chloroplast development (Wei and Deng, 1996), is controlled by

phytochromes. It is still questionable whether light signal can lead to decrease of GA

content. Recent studies showed that GA content dropped initially within 2 hrs of light

exposure, and then went up after de-etiolation. It was proved that decline of GA level

resulted from up-regulation of gene of GA metabolism enzyme, GA2ox (O'Neill and

Ross, 2002).

Stem elongation

Reduction of light intensity results in inversely proportional increment of stem

elongation. It is suggested that low irradiance cause the increase of bioactive GA by

promoting GA1 synthesis and reducing GA1 metabolism (García-Martinez and Gil,

2002).

Photoperiod

Day length (in fact night length) involves in stem elongation and bolting, bud

dormancy, flowering, tuber formation, and other developmental events. Photoperiod

seems to influence steps throughout the GA-biosynthesis pathway (García-Martinez

and Gil, 2002). GAs was documented to induce bolting of long day (LD) rosette

plants, such as spinach. LD treatment raised GA20 content rapidly, and built up GA1 level gradually (Zeevaart et al., 1993). It was also observed that when Arabidopsis plants were moved from short day (SD) to LD, the level of bioactive GAs, GA1 and

GA4, both went up in the rosette leaves. The increase of stem elongation may be mediated only by GA20ox genes (Xu et al., 1997). Moreover, in some woody plants, bioactive GA content increases when the plants are transferred to LD. LD may affect

GA biosynthesis at the step of GA 3-oxidation. The overexpression of PHYA seemed to decrease stem growth and diminish the LD effect (Olsen et al., 1997).

Daily changes of GA content and metabolism

Diurnal changes of content of some GAs have been observed in Sorghum bicolor, a SD plant (Foster and Morgan, 1995). It has been suggested that phyB regulates the daily change of GA20 biosynthesis (García-Martinez and Gil, 2002). Besides, GA20ox

gene transcription appeared to change daily in other plants, however, it is doubtful whether the cycling results from circadian rhythm (Carrera et al., 1999; Jackson et al.,

2000).

Light quality

Regardless of inconsistent results, lots of research have supported that phytochrome regulates stem elongation by affecting GA biosynthesis and signaling, in response to the change of light quality (Kamiya and Garcia-Martinez, 1999).

Phytochrome controls the GA1 level and vertical height of some plants (Jackson et al.,

1996; Olsen et al., 1997). Sometimes it also changes the diurnal rhythm of GA1

content (Foster and Morgan, 1995). However, it has no effect on GA content in some occasions (Lopez-Juez et al., 1995; Reed et al., 1993; Weller et al., 2001).

The decrease of R: FR ratio causes shade avoidance syndrome of plants growing underneath other plants. It is suggested phyB is implicated in shade avoidance response and GA controls effect of FR (García-Martinez and Gil, 2002).

Moreover, it is hypothesized that GA4 and phytochrome mediate stem elongation via different pathways, however, they may interact with each other when approaching the final response step (Lopez-Juez et al., 1995).

Furthermore, phytochrome may regulate cell elongation of light-grown seedlings by stimulating GA1 catabolism by GA2ox (Martínez-García et al., 2000).

BRASSINOSTEROID REVIEW

Brassinosteroids (BRs) represent a sixth class of plant hormones in addition to abscisic acid (ABA), auxins, cytokinins, ethylene, and GAs. In general, BRs are defined as naturally-occurring 5α cholestane steroids that influence plant growth and development in nano- or micromolar concentrations (Clouse and Sasse, 1998).

Primarily, young growing tissues contain higher level of BRs than mature tissues.

Pollen is the original source of brassinolide and immature seeds are the richest sources. Various reviews have covered different aspects of BR research including BR structure, synthesis, metabolism, transport, signaling, and response (Altmann, 1998a;

Altmann, 1998b; Bishop and Yokota, 2001; Bishop and Koncz, 2002; Castle et al.,

2003; Clouse and Sasse, 1998; Hooley, 1996; Li and Chory, 1999; Pereira-netto et al.,

2003; Yokota, 1997). This review highlighted research on BR biosynthesis, regulation,

signal transduction, physiological function, and interaction with light.

Research History of BRs

Independent groups of scientists in Japan and USA initiated BR research from

1968 and 1970, respectively (Marumo et al., 1968; Mitchell et al., 1970). Marumo et

al.(1968) concluded that a new type of plant hormone was found in active fractions

isolated from leaves of Distylium racemosum Sieb et Zucc, an evergreen tree in Japan.

Mitchell et al. (Mitchell et al.) named the pollen extract of rape (Brassica napus)

Brassin, which promoted stem elongation in the bean second internode test.

Brassinolide, the most bioactive form of BR, was isolated and characterized as a plant

steroid compound promoting cell division and plant growth (Grove et al., 1979).

Specific bioassay methods were used to determine BL biological activity. It was found that BL at very low concentrations promoted the lamina inclination of rice plants

(Wada et al., 1981). Besides, BL application induced epicotyls elongation and epinastic curvature of the epicotyl and petioles of young mung beans(Gregory and

Mandava, 1982). Furthermore, it was observed that BRs inhibited root elongation

(Clouse et al., 1993; Guan and Roddick, 1988a; Guan and Roddick, 1988b).

Field trials demonstrated that BR treatment on seeds did not enhance yield, while

BR spraying on plants improved yield of many crops (Ikekawa and Zhao, 1991;

Steffens, 1991). Nevertheless, results showed that BL had higher impact on crop yield under more stressful conditions (Ikekawa and Zhao, 1991).

Later on, BR mutants became the most effective tools to disclose the mechanisms of BR biosynthesis, metabolism, signaling, and physiological actions. More details will be covered in the following sections.

Biosynthesis of BRs

Since the isolation of the first BR, Brassinolide (Olszewski et al., 2002), more

than 40 natural BRs have been found in different organs of plant species from several families (Pereira-netto et al., 2003). BRs belong to a group of molecules called

triterpenoids and are a cluster of modified sterols. Brassinolide (BL) biosynthetic

pathways consist of two major parts: sterol specific pathway starting from squalene to campesterol (Rommens et al., 2004) and BR-specific pathway from campesterol to

BL. Mevalonic acid (MVA) serves as the starting molecules of terpernoid pathway and is condensed and modified to from squalene (Choe, 2004). The final product BL is synthesized from CR via either early or late C-6 oxidation routes.

BR biosynthesis mutants exhibit dwarf and de-etiolated phenotype of typical BR

deficiency (Bishop and Yokota, 2001). Several BR biosynthesis genes were found to

encode cytochrome P450 monooxygenases (P450s or CYPs) in molecular analysis of

BR biosynthesis mutants (Bishop and Yokota, 2001).

Regulation of BR Levels

BR homeostasis is maintained through the balance between the rates of

biosynthesis and metabolism or inactivation. Studies of BR biosynthesis gene,

constitutive photomorphogenesis and dwarfism (cpd), indicated a mechanism for autoregulation of BR levels (Bishop and Yokota, 2001). Besides, cellular BR levels are also regulated by the catabolism of BL and/or its precursors. Activation-tagged

Arabidopsis bas1-D (phyB activation-tagged suppressor1-D) mutant exhibited dwarf phenotype, contained no detectable BL, and accumulate biologically inactive

26-hydroxybrassinolide in feeding experiment (Neff et al., 1999).

BR Signal Transduction

It was suggested that plant steroid played a major function in altering plant gene

transcription and regulating growth and development. BR signal transduction

appeared to be similar to the non-genomic steroid pathways in animals (Bishop and

Koncz, 2002). Exogenously applied BL insensitive mutants were identified and

utilized in studies on BR signal transduction. Mutant studies revealed that the plasma

membrane localized receptor kinase BRI1 recognized and transduced signals, whilst

DET3, BIN2, BRS1BR and other unknown genes may act downstream from BRI1 to

promote gene expression and cell expansion (Friedrichsen and Chory, 2001).

Physiological Functions of BR

Plant growth is defined as “an irreversible increase in volume” which involves cell division and cell elongation (Taiz and Zeiger, 2002b). Mature walls are rigid and highly cross-linked by covalent bonds. The key steps in cell elongations include wall relaxation and loosening, as well as integration of new polymers into the expanding cell wall (Clouse and Sasse, 1998). Expansins may be the major enzymes involved in cell wall relaxation, while glucanases, other wall hydrolases, and xyloglucan endotransglycosylases (XET) affect the expansin activity by changing the viscosity of

the matrix (Cosgrove, 1997).

Cell elongation is regulated both by light and plant hormones (Azpiroz et al.,

1998). BR can stimulate cell elongation at extremely low concentration (Clouse and

Sasse, 1998). BR is implicated in cell elongation through regulation of genes encoding expansin and XET (Bishop and Koncz, 2002; Clouse and Sasse, 1998). It

was hypothesized that BL modified the biophysical properties of cell walls and

regulated cell elongation via a microtubule independent process following a

microtubule dependent pathway (Mayumi and Shibaoka, 1995). Besides, BR-induced

cell expansion is associated with cell wall acidification and loosening caused by

activity of H+ pump on cell membrane in response to BR (Dahse et al., 1990;

Schumacher et al., 1999).

BRs play a role in cell division and expansion of embryonic and post embryonic

development of plants (Jang et al., 2000). BRs also involve in induction of pollen tube

elongation in both light and dark conditions (Hewitt et al., 1985). BRs have been

shown to induce germination of GA biosynthetic mutants and GA perception mutants

(Steber and McCourt, 2001). BRs are also implicated in cell division and

differentiation of vascular tissues at the stage prior to secondary cell wall formation

and cell death (Fukuda, 1997; Yamamoto et al., 1997). Furthermore, BRs may play a

crucial function in accelerating senescence in normal plants (Choe et al., 2000).

Additionally, preliminary studies exhibited that the application of BRs increased crop tolerance to unfavorable environmental conditions including heat, chilling, and

salt stress, as well as pathogen attack. Recent studies showed that BL directly induced

the translation of heat shock proteins which enabled the BL-treated plant to endure

heat stress (Dhaubhadel et al., 1999; Dhaubhadel et al., 2002).

Interaction of BRs with Light Signals

Early studies displayed that BR counteracted the effect of light on stem

elongation and morphogenesis (Krizek and Mandava, 1983). Many brassinosteroid

mutants carried out de-etiolation in the absence of a light cue. When grown in the

dark, mutant seedlings showed light grown phenotype of short hypocotyls with

developing cotyledons, while adults exhibited dwarfism with dark green epinastic leaves, short stems and petioles, and delayed senescence (Li and Chory, 1999).

However, it is undetermined how quality and quantity of light are linked to BR biosynthesis and signaling pathway through photoreceptors (Bishop and Yokota,

2001).

Light seems to play no role in the regulation of the activity of brassinosteroid biosynthetic genes or the hypothetical receptor. Endogenous BR levels in wild type light-grown pea seedlings were observed to be higher than dark-grown ones (Symons et al., 2002). What's more, genetic analysis discovered an interaction between multiple photoreceptors and brassinosteroid catabolism (Neff et al., 1999).

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CHAPTER 2

FIELD ANDGREENHOUSE EVALUATION OF TRANSGENIC CREEPING

BENTGRASSES TRANSFORMED WITH RUNNER BEAN GA2OX GENE

ABSTRACT

Development of creeping bentgrass (Agrostis palustris) plants that constantly exhibits dwarfism and superior quality is important for turfgrass management under extended shade conditions. Transgenic creeping bentgrass plants were previously generated by transformation with runner bean (Phaseolus coccineus) GA 2-oxidase gene in particle bombardment. GA2ox transformed lines, glyphosate-resistant control, and wild type control lines were subjected to neutral shade (NS), canopy shade (CS), and full sun (FS) treatments both in field and greenhouse studies. Our results showed that vertical growth, internode extension, and leaf growth of transgenic creeping bentgrass plants were inhibited by GA2ox transformation under reduced low light conditions in the field. As a group, GA2ox trangenic creeping bentgrass plants did not

show increase in overall quality, shoot density, or stolon density compared with wild type plants. Moreover, GA2ox transgenic lines tended to keep horizontal growth habit,

possibly due to the increased GA metabolism by GA2ox overexpression, or the

collaborated work of GA and light receptors in signal transduction. Greenhouse

studies revealed the similar results to field studies. Strong transformants, such as

GA6548, displayed dwarfism and superior quality under all light conditions. RT-PCR

results confirmed that mRNA level of foreign GA2ox was associated with the slection indices ranking of creeping bentgrass plants both in the greenhouse and the field.

INTRODUCTION

Creeping bentgrass (Agrostis stolonifera) is commonly used on golf course greens, tees and fairways throughout the temperate regions of the world. Light plays a critical role in creeping bentgrass growth and development. Creeping bentgrass plants utilize light both as energy source to drive photosynthesis and as a stimulus to trigger series of developmental events ranging from germination to flowering (Kendrick and

Kronenberg, 1986). Usually, turfgrass performs best in full sun with a minimum of four to six hours of full sun per day (Reicher and Throssell, 1998). However, creeping bentgrass is often subjected to low light or shaded conditions for extended duration.

Both photosynthetic photon flux (PPF) and red/far red light ratio (R/FR) drop dramatically when the sunlight penetrates the canopies and reach the surface of the turf. Reduction in photosynthetic irradiance causes increase of vertical growth and succession of harmful physiological and morphological changes in turf plants, such as thinner, more delicate leaves, reduced tillering, poor shoot density, altered pigment concentration and affected carbohydrate reserve (Bell and Danneberger, 1999). It was also reported that tall fescue plants exhibited reduced tillering, leaf blade width and thickness when R:FR dropped under low light intensity (Wherley et al., 2005).

Given that creeping bentgrass is often subjected to low mowing heights, less than 3 mm on putting greens, slight changes in growth habit specifically shoot elongation and reduced tillering, would have great impact on turfgrass quality. In response to shade, increased shoot elongation results in more tissue removal by 44

mowing and may cause scalping. Reduced tillering is associated with slower turf

recovery from injury and decreased wear tolerance (Danneberger, 1993).

Turf loss in shade can be reduced through management practices such as pruning

and removing trees, raising mowing height, decreasing Nitrogen application, and

overseeding. Furthermore, creeping bentgrass plants that have dwarfism characteristics could reduce the probability of shoot elongation in shade and potentially enhance turf quality. Dwarfism is commonly associated with deficiencies in Gibberellic acid (GA) levels or signaling (Peng et al., 1999). The gibberellins F form a large group of tetracyclic diterpenoid carboxylic acids, certain members of which function as natural regulators of growth and development throughout the life cycle of higher plants (Hooley, 1994). One of the outstanding characteristics of the gibberellins is their ability to promote cell elongation and shoot vertical growth (Taiz

and Zeiger, 2002a).

Plant growth regulators (PGRs) have been proven successful in suppressing shoot

growth and enhancing quality of many turfgrass species, even under unfavorable light

conditions (Fagerness and Penner, 1998; Goss et al., 2002; Johnson, 1992; King et al.,

1997; Qian and Engelke, 1999). The PGR, trinexapac-ethyl (TE; Primo), was

introduced for use on turf in 1991 (Watschke and DiPaola, 1995). TE interferes with

the action of GA by inhibiting the conversion of GA20 to GA1, which is the bioactive

GA in plants (Adams et al., 1992). Single applications of TE have proven successful for significantly reducing shoot growth of numerous turfgrass species (Fagerness and

Penner, 1998; Johnson, 1992; King et al., 1997). However, stature control through

"anti-GA" plant growth retardants requires repeated application of synthetic chemicals that is costly, variable in effectiveness, and can have undesired environmental consequences or public perceptions (Busov et al., 2003). 45

Biotechnological manipulation of GA levels provides an alternative approach and can be achieved through various means, including up- or down-regulating genes encoding enzymes involved in GA biosynthesis and catabolism (Hedden and Phillips,

2000). Endogenous GA level is regulated by the sequential action of cyclases, membrane-associated monooxygenases, and soluble 2-oxoglutarate-dependent dioxygenases (Hedden and Kamiya, 1997). The dioxygenases catalyze the later steps in the biosynthesis pathway, including the removal of C-20 by GA 20-oxidase (Philips et al., 1995; Xu et al., 1995) and the introduction of the 3-hydroxyl group by GA

3-hydroxylase (Chiang et al., 1995). A third dioxygenase introduces a 2-hydroxyl group, resulting in biologically inactive products that cannot be converted to active forms (Thomas et al., 1999). It was shown that ectopic expression of

GA2oxidase(GA2ox) gene caused a dwarf phenotype in rice (Sakamoto et al., 2001).

Biolistic bombardment was applied to generate creeping bentgrass plants transformed with runner bean (Phaseolus coccineus) GA2ox gene.

The purposes of this study were to explore the phenotypic changes of transgenic creeping bentgrass plants transformed with GA2ox gene and to evaluate the performance of 30 lines of putative creeping bentgrass GA2ox transformants under altered PPF and R/FR.

MATERIALS AND METHODS

Creeping bentgrass transformation and regeneration

Induction and maintenance of embryogenic callus cells were described previously

(Busov et al., 2003). Mature seeds (caryopses) of creeping bentgrass cultivar,

‘Crenshaw’, were surface sterilized in 70% ethanol for 1 min, followed by 30-min

treatment of 50% commercial bleach and 0.1% Tween-20 under vacuum. Surface

sterilized seeds were rinsed three times with sterile distilled water and then cultured

on MSI medium (Lee et al., 1996; Zhong et al., 1991), MS basal medium

supplemented with 500 mg L-1 Casein enzymatic hydrolysate (Sigma), 3% sucrose, 30

μM 3,6-dichloro-o-anisic acid (dicamba), and 2.2 μM 6-benzylaminopurine (BA), solidified with Gel-GroTM (MP biomedical). After 4-5 wk at 25 ºC in the dark, calli

were selected and transferred to new MSI media. Embryogenic calli were subcultured

onto fresh media every 4-6 wk.

Structure of GA2ox expression cassette

The 2.4kb NotI/NotI GA2ox expression cassette included a 1kb BamHI/EcoRI

fragment of a full length GA2ox cDNA (Thomas et al., 1999), a CaMV35S promoter, a ract1 intron, and a nos terminator. The final construct also contained CP4-EPSPS gene as selective markers (Fig. 2.1).

Transformation and regeneration

Embryogenic calli were placed on 9-cm filter disks (0.3g tissue per disk) in plates of MSI medium containing 0.3 M mannitol and 0.3 M sorbitol at least 4 hr prior to bombardment (Kern et al., 1993). Plasmid DNA containing GA2ox gene construct was delivered into embryogenic creeping bentgrass callus cultures using the biolistic transformation system (Harriman et al., 2002; Lee et al., 2002; Vain et al., 1993). 47

After 6-8 wk on selective MSI media containing 2mM glyphosate, embryogenic

callus was regenerated on MSS medium, MS medium including 3mg L-1 BA and 0.2

mM glyphosate. Appearing shoots were transferred to MSR medium, MS medium

containing 0.2 mM glyphosate for rooting. Finely rooted plants were grown in

metro-mix 350 (Scotts Miracle-Gro Co., Marysville, OH). Simultaneously, control

plants were recovered from embryogenic calli bombarded with plasmid DNA containing CP4 EPSPS after glyphosate selection. Herbicide resistance of

transformants was confirmed by spraying plants with 0.9g/m2 Roundup® herbicide

(Scotts Miracle-Gro Co., Marysville, OH).

Field Study

Plot design and root zone preparation:

The study was conducted at the Ohio Turfgrass Foundation (OTF) Research and

Education Facility, Columbus, OH (39°59’N 83°01’W; elevation 243.8m) from July

2004 through August 2005. Experimental design was similar to the one described by

(Wherley et al., 2005). Plots were constructed by digging to a 20-cm root zone depth and refilling with a mix of 90% sand and 10% peat (v/v) covering the native clay soil.

Randomized complete block design was utilized at 3 different locations: full sun, deciduous tree shade under mature hardwoods, mainly Acer. spp. and Fraxinus spp.

that were 20-25 m tall, and neutral shade under a 3-meter-high Quonset hut covered

with black shade cloth (Fig 2.2). Full sun plots received sunlight of highest PPF and

Red:Far-red (R:FR) ratio. PPF dropped dramatically both in deciduous shade (DS) and neutral shade (NS) plots (11.8% and 10.5% of full sun, respectively). R:FR ratio decreased differently in neutral shade and deciduous shade (Fig 2.2).

Light measurement

Continuous measurement of PPF was taken every 5 min from 29 June 2004 through 15 Oct. 2004 and from 10 May 2005 through 30 August 2005. One cosine corrected PAR light sensor (Spectrum Technologies, Plainfield, IL) was attached to a plastic pole 30 cm above the ground and positioned at the edge of every treatment plot.

Accumulative PPF data were stored in a Spectrum Technology data logger and average daily PPF (mol·m-2d-1) was determined by Specware software (Spectrum

Technologies). Red: far-red (R:FR) values are calculated from readings taken in June and August 2004 and 2005. R and FR photon flux were measured at 660±5nm and

730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience,

Lincoln, NE).

Plant transfer and propagation

All transgenic creeping bentgrass plants were previously developed by Scotts Miracle-Gro

Company (Marysville, OH) using the abovementioned procedures. Thirty-eight creeping bentgrass (Agrostis palustris) lines were used in the study, including 8 glyphosate-resistant control lines (Ar plants), events of primary transformants containing CP4 EPSPS gene and 32 glyphosate-resistant experimental lines (GA plants), events transformed with CP4 EPSPS and runner bean (Phaseolus coccineus)GA2ox genes (Table 2.1). Transgenic creeping bentgrass plants were developed from the callus of cultivar ‘Crenshaw’ using biolistic bombardment procedure similar to the one described by Hartman et al. (1994) Two conventional creeping bentgrass cultivars, ‘LS44’ and ‘Crenshaw’, were also included as wild type control. All transgenic and non-transgenic plants were transferred from Marysville,

OH in May 2004 and propagated in metro-mix 350 (Scotts. Co., Marysville, OH) in

10-cm pots. Fertilizer (20N-10P2O5-20K2O) was applied at 100ppm weekly; 49

pesticide was applied monthly to prevent disease and insects; all plants were watered daily to prevent wilting and trimmed to 2.0 cm once a week.

Plot Maintenance

Plants were manually watered every two days, or once a day on hot summer days to avoid moisture stress. Slow release fertilizer (18N-4P2O5-18K2O) was applied at

4.9g/ m2 4 times a year. Roundup was sprayed at 0.9g/m2 to eliminate weeds. All plants were mowed to 1.60 cm every three wk, starting from the third week after transplanting.

Measurement and Evaluation

Monthly growth measurements were made, including vertical growth, leaf length, and internode length. Quality and growth habit were evaluated including visual quality, shoot density, genetic color, stolon density, and erectness. Visual quality ratings were based on 1-9 scale; 1 was poor density and uniformity, dormant/dead turf or bare soil, and coarse texture, 9 was ideal turf, dark green color, excellent density and uniformity, and fine texture; acceptable turf quality≥6. Shoot density ratings were based on 1-9 scale with 9 equaling maximum density. Stolon density ratings were based on 1-0 scale with 9 being maximum stolon density. Color ratings were based on

1-9 scale with 1 being light green and 9 being dark green. Erectness was visual rating of angles between turf stand and the ground. It was based on 1-9 scale: 9= 90°; 1=0°.

All evaluations were conducted following NTEP turfgrass evaluation guidelines.

Experimental design and data analysis

A randomized complete block design was employed under three different light conditions. Data were analyzed with a PROC GLM procedure of SAS (PC version 9.1, the SAS Institute, Cary, NC). A base index was developed to facilitate simultaneous trait selection as previously described. The only revision was that all phenotypic 50

values were standardized using SAS PROC STDIZE. A base index weights each trait

based on its economic importance, and is calculated by the following equation: I= a1P1+a2P2+…+anPn, with a representing the economic weight and P representing

the standized phenotypic value (Baker, 1986; Hanks et al., 2005). Economic weights were -3 for vertical growth rate, -1 for internode length, -1 for leaf length, 3 for visual

quality rating, 3 for shoot density rating, 1 for stolon density rating, 3 for color rating,

and -3 for erectness rating. Least significance difference (LSD) mean separation and

CONTRAST procedures were performed to test hypothesis of external GA2ox gene function.

Greenhouse Pot Trial

Plant material transfer and propagation

Plant material for the study was established from stolon nodes of 12 primary

transgenic GA2ox events, 3 glyphosate-resistant control lines, and 2 conventional lines (Table 2.6) in 7-cm square plastic pots filled with metro-mix 350 (Scotts

Miracle-Gro Co., Marysville, OH). Throughout the trial, plants were kept at 21°C

/16°C (day/night) under a 14/10-hr (day/night) photoperiod. Pots were weekly

fertilized at a rate of 100ppm N with a 20N-10P2O5-20K2O soluble fertilizer (Scotts

Miracle-Gro Co., Marysville, OH). Turf was irrigated twice daily (0900 and 1300 hr) with automatic irrigation system. Fungicide and insecticide were applied monthly to

prevent disease and insect problems. All plants were trimmed weekly to the height of

2.5 cm with clippings autoclaved and discarded.

Experimental design

A randomized split-plot study with 3 blocks was performed in the greenhouse at

The Ohio State University, Columbus, OH between May 30 and July 11, 2005. Three

light treatments were assigned at random to the main plots within each block, including non-photoselective polyethylene film (full sun control), mature soybean plants that were 60-70 cm in height (canopy shade), and black shade cloth (neutral shade). Creeping bentgrass lines were assigned random to the subplots within each main plot. PPF was averaged over weekly readings taken during the study using a quantum light meter (Spectrum Technologies, Plainfield, IL). Red:far-red (R:FR) values were calculated from the weekly measurements at 660±5nm and 730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience, Lincoln, NE).

Average PPF dropped to 16% and 26% of the value in full sun when measuring canopy and neutral shade, respectively. Average R:FR remained same when measured under neutral shade and full sun, while reading under canopy shade was only 53% of the value in full sun (Table 2.5).

Data collection

Overall quality ratings, erectness ratings, leaf extension rates, and vertical growth rates were obtained weekly, starting from wk 3. Visual quality ratings were based on

1-9 scale; 1 was poor density and uniformity, dormant/dead turf or bare soil, and coarse texture, 9 was ideal turf, dark green color, excellent density and uniformity, and fine texture; acceptable turf quality≥6. Erectness was visual rating of angles between turf stand and the ground. It was based on 1-9 scale: 9= 90°; 1=0°. All evaluations were conducted following NTEP turfgrass evaluation guidelines. Leaf extension rate was defined as the difference between leaf lengths measured 24 and 36 hours after trimming. Vertical growth rate was determined by the difference of plant height (height from media level to plant apex) right before clipping event and plant height right after the previous clipping (usually 2.5 cm), divided by day numbers between the two clipping events. 52

RNA isolation and RT-PCR analysis

Two-wk-old leaf tissue of transgenic and non-transgenic plants growing in the greenhouse was collected fresh and snap frozen in liquid nitrogen. Total RNA was isolated from approximately 1.5g frozen tissue using TRIzol® reagent (Invitrogen,

Carlsbad, CA) and further treated with DNaseI (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. First strand cDNA was obtained through reverse transcription of an aliquot of 1µg RNA from each sample using

Oligo-dT primers with the 2-step Reverse Transcription System (Promega, Madison,

WI). An aliquot of 1µl cDNA out of each 20µl sample was analyzed by PCR using

Taq DNA polymerase (New England Biolabs, Ipswich, MA) and gene-specific primers. Primers for GA2ox were GA2ox-F

5'-CCGATGCCAAGAATCTCATAGTG-3' and GA2ox-R

5'-ATTATCTGTGGGTCTGTGTGCTC-3' which generated a 526bp DNA fragment.

PCR reaction was conducted under the following conditions: 94℃ for 5 min for one cycle, followed by 94℃ for 30 sec, 55℃ for 40 sec, and 72℃ for 1 min for 30 cycles, and a final incubation at 72℃for 5 min. PCR product from GA6548 was isolated on 0.8% agarose gel, purified with Qiagen MinElute purification kit (USA —

QIAGEN Inc., Valencia, CA), and sequenced using GA2ox-F primer. Furthermore, primers actin-5 (5'-GAGAAGATGACCCAGATCATGTTTG-3') and actin-3

(5'-TCCTAATATCCACGTCGCACTTCAT-3') were used to amplify the actin gene

(Wang and Luthe, 2003).

Data analysis

Data were analyzed with a PROC MIXED procedure of SAS (PC version 9.1, the

SAS Institute, Cary, NC). A base index was developed to facilitate simultaneous trait

selection as previously described. The only revision was that all phenotypic values

were standardized using SAS PROC STDIZE. A base index weights each trait based

on its economic importance, and is calculated by the following equation: I=

a1P1+a2P2+…+anPn, with a representing the economic weight and P representing

the standized phenotypic value (Baker, 1986; Hanks et al., 2005). Economic weights were 3 for visual quality rating, -1 for vertical growth rate, -1 for erectness rating, and

-1 for leaf growth rate. LSMEANS and CONTRAST procedures were performed to

test hypothesis of gene transformation, light effect, and their interaction.

RESULTS

Field studies

Vertical height varied significantly among putative GA2ox transgenic lines

under all 3 light conditions. It ranged from 4.2 cm (GA6548) to 9.1 cm (GA6480) in

neutral shade, from 3.2cm (GA6548) to 8.3cm (GA6511) in canopy shade, and 2.9 cm

(GA6548) to 5.4 cm (GA6476) in full sun. As group, vertical height of putative

GA2ox transgenic plants was significantly lower than wild type control plants, but not

significantly different from glyphosate-resistant control plants under neutral shade.

Putative GA2ox transfomants had significantly lower vertical height than both wild

type and glyphosate-resistant control plants under canopy shade. Under full sun

conditions, vertical height of putative GA2ox transformants were not significantly

different from wild type plants, but significantly lower than glyphosate-resistant

control plants. Particularly, GA6548 and GA6549 grew significantly lower than 54

control plants under all light conditions (Table 2.1).

There was significant difference of internode length among putative GA2ox

transformants under all light conditions. It ranged from 0.8 cm (GA6539) to 3.1cm

(GA6462) in neutral shade, from 0.9 (GA6548) cm to 2.6 cm (GA6480) in canopy

shade, and 0.5 cm (GA6562) to 2.3 cm (GA6480) in full sun. Besides, on average,

internode length of GA2ox plants was not significantly different from glyphosate-resistant plants, but significantly shorter than wild type plants under any light condition (Table 2.1).

As a group, GA2ox plants had significantly shorter leaves than

glyphosate-resistant and wild type control plants under all light conditions, except that

no significance was observed between the leaf lengths of putative GA2ox

transformants and glyphosate-resistant controls under neutral shade (Table 2.1).

Significant variation of leaf length among GA2ox plants (Table 2.1) appeared only

when they were grown under deciduous trees ranging from 3.4cm (GA6476) to 6.5cm

(GA6509).

There was significant difference between average quality ratings of wild type,

glyphosate-resistant, and GA2ox transgenic plants. As group, visual ratings was

highest for wild type and lowest for glyphosate-resistant plants under all light treatments. However, putative GA2ox line GA6548, GA6547, GA6509, GA6549, and

GA6465 were visually rated top 20% among all lines under neutral and canopy shade

(Table 2.2).

Average shoot density ratings were significantly different between each two

groups of wild type, glyphosate-resistant, and putative GA2ox transgenic plants. On

average, wild type plants had the highest shoot density rating while

glyphosate-resistant plants had the lowest one despite the changes in PPF and R:FR.

However, putative GA2ox lines were rated significantly different in shoot density under both canopy shade and full sun. Particularly, GA2ox line GA6548, GA6509,

GA6547, and GA6465 had the highest density ratings among all experimental lines under canopy shade, while GA6548 and GA6549 had the highest density under neutral shade (Table 2.2).

Average stolon density of wild type plants was significantly higher than putative

GA2ox transgenic plants under all light conditions. The latter were rated significantly better in stolon density than glyphosate-resistant plants under both shade conditions, however no significant difference was observed between the two groups under full sun (Table 2.2).

Putative GA2ox transformants varied significantly in color ratings under both neutral shade and full sun. On average, color rating of putative GA2ox transgenic

plants was significantly lower than wild type plants and higher than

glyphosate-resistant plants under all light conditions. Nonetheless, top 20% greenest

lines under neutral shade were all GA2ox transgenic lines including GA6548, GA6569,

GA6567, GA6437, GA6547, GA6480, and GA6549; similarly, all top 20% greenest

lines under canopy shade belonged to putative GA2ox group, including GA6548,

GA6549, GA6567, GA6569, GA6480, GA6558, and GA6562.

When compared as a group, putative GA2ox trangenic plants grew significantly

more horizontal than glyphosate-resistant and wild type control plants under all light

conditions, except that wild type plants had the most horizontal growth habit among

the three groups under full sun. The difference of erectness ratings varied significantly

among GA2ox transgenic plants grown under full sun, ranging from 1.0 (GA6548).to

7.5 (GA6535). GA2ox transgenic line GA6548 had the most horizontal growth habit 56

among all plants regardless of light condition change. Furthermore, erectness ratings

of putative GA2ox transformants GA6548, GA6555, GA6509, GA6517, GA6487,

GA6549, and GA6569 were the bottom 20% among all line under low PPF, while

GA6548, GA6539, GA6555, GA6509, GA6487, GA6549, and GA6562 had the

lowest erectness ratings under low PPF and R:FR (Table 2.3).

Base index ranking results revealed that top 20% best lines under neutral shade were GA6548, GA6549, GA6539, GA6547, GA6509, GA6569, GA6562, and

GA6557; top 20% best lines under canopy shade were GA6548, GA6549, GA6555,

GA6569, GA6517, GA6509, GA6547, and GA6568; top 20% best lines in full sun were GA6548, Crenshaw, LS44, GA6465, GA6517, GA6549, GA6564, and GA6509.

In summary, top 20% best lines were GA6548, GA6549, GA6509, GA6517, GA6547,

GA6569, GA6557, and GA6465. In contrary, RR6249, the best line among glyphosate-resistant control plants, was ranked number 12 on the list, while wild type control plants Crenshaw and LS were ranked number 15 and 18 respectively.

Greenhouse pot trial

PPF had significant impact on visual quality of creeping bentgrass plants. Visual quality of plants grown in full sun was rated significantly higher than those grown under shade conditions. However, R:FR change under low light conditions did not have significant impact on plants visual quality (Table 2.6). Furthermore, as group, average visual quality of putative GA2ox transformed plants significantly higher than

glyphosate-resistant control plants, but not significantly different from wild type control plants (Table 2.6). However, visual quality varied significantly among GA2OX transgenic creeping bentgrass plants with GA6548, GA6549, and GA6547 having the highest quality rating among all lines, while GA6487 and GA6531 the lowest (Table

2.6; Fig 2.3A-C). Specifically, GA6548 and GA6547 were much greener and denser 57

than other transgenic and wild type control plants (Fig 2.4).

Light treatments had significant effect on leaf orientation of creeping bentgrass plants, both transgenic and no transgenic. Growth habit of plants grown in shade was significantly more upright than those grown under full sun. Reduction in R:FR did not significantly change the leaf orientation of the plants under low PPF (Table 2.6, Fig

2.3A-C). On average, GA2ox transgenic plants grew significantly more horizontally than wild type and glyphosate-resistant control plants. Besides, growth habit varied significantly among GA2ox transgenic plants ranging from GA6548, GA6549,

GA6547, and GA6465 having the most horizontal growth habit to GA6539, GA6476, and GA6487 the most upright (Table 2.6, Fig 2.6). Particularly, under full conditions, erectness ratings of RR6260 and RR6261 were 2 to 3 times as GA6548 and 1.5 to 2 times as GA6549, while LS44 and Crenshaw were almost 2 times as GA6548 and 1.5 times as GA6549. When PPF and R:FR dropped, ratings of GA6548 and GA6549 were less effected than either wild type or glyphosate-resistant plants (Fig 2.6).

Leaf growth rates of plants grown in full sun were significantly lower than those grown under shade conditions. There were no significant difference between leaf growth rates of plants under neutral shade and those in canopy shade. Average leaf growth rate did not vary significantly among putative GA2ox transgenic lines. As group, putative GA2ox transformants had significantly lower leaf growth rates than glyphosate-resistant control and wild type control plants (Table 2.6). GA6548,

GA6435, GA6549, and GA6465 had the lowest leaf growth rates among all lines under all light conditions (Table 2.6).

Changes in PPF and R:FR significantly affected vertical growth rate. Plants grown in shade had significantly higher vertical growth rates than those grown under full sun. Decrease in R:FR further stimulated plant vertical growth rates under low 58

light conditions. Average of vertical growth rate of putative GA2ox transformants creeping bentgrass was significantly lower than wild type and no shot control plants

(Table 2.6). There was also significant difference of vertical growth rate among

GA2ox transgenic plants with GA6548, GA6465, GA6547, and GA6549 having the lowest vertical growth rates and GA6539, GA6487, and GA6568 the highest among all lines under all light conditions (Table 2.6; Fig 2.3A-C, Fig 2.5). In addition, less than 30% increase in vertical growth of GA6548 and GA6549 was observed when

PPF and R:FR dropped, in compare with the up to 90-110% increase of glyphosate-resistant and wild type control plants (Fig 2.5).

Cultivars that were ranked top 40% on the base index ranking list all belonged to putative GA2ox group. Specifically, the top 4 cultivars were GA6548, GA6547,

GA6549, and GA6465. GA6487, GA6531, and GA6539 were the 3 worst cultivars among putative GA2ox transformant group. Additionally, glyphosate-resistant control plants were ranked lowest on the list (Table 2.6).

RT-PCR analysis

Transcription of the GA2ox gene in creeping bentgrass was studied by RT-PCR analysis of RNA samples isolated from putative GA2ox transformants, glyphosate-resistant control, and wild type control plants. Results indicated that the expected GA2ox band of the right size (526bp) appeared in glyphosate-resistant lines

GA6435, GA6465, GA6476, GA6548, GA6549, and GA6555, but not in GA6487, glyphosate-resistant control line (RR6237 and RR6260), or the 2 conventional lines

Crenshaw and LS44 (Fig 2.7). GA6548 and GA6549 exhibited highest transcription level of GA2ox gene, which was at least 10 fold higher than weak transformants

GA6435 and GA6476, and 4-5 fold higher than GA6465 and GA6555. Sequencing result confirmed that the RT-PCR product from GA6548 was 100% identical to part of 59

Phaseolus coccineus GA2ox gene sequence from gene bank. Primers for actin were used as a positive control and its transcript was present in all samples (Fig 2.7).

DISCUSSION

This paper introduced an experimental system to compare the morphological traits of transgenic creeping bentgrass plants under various light conditions. It also demonstrated the potential for creating new phenotypes of creeping bentgrass plants using biolistic bombardment of GA2ox gene.

Field studies

Our results showed that vertical growth, internode extension, and leaf growth of transgenic creeping bentgrass plants were inhibited by GA2ox transformation under reduced low light conditions. This might be due to the metabolism of bioactive GA in absence of light cue.

Although as a group, GA2ox trangenic creeping bentgrass plants did not show improvement when compared with wild type plants., individual lines, such as

GA6548, were superior to control plants in visual quality and shoot density ratings,

Reduction in chlorophyll content and leaf thickness is one of the shade avoidance syndromes of plants under shade (Casal et al., 1986). Although GA2ox transformed lines did not have darker green color than wild type control plants as a group, extremely dwarf lines, such as GA6548, exhibited the darkest green leaf color among all plants. It was in agreement with the studies on rice (Oryza sativa) plants transformed with rice GA2ox gene (Sakamoto et al., 2001).

GA2ox gene transformation also altered growth habit of transgenic creeping

bentgrass lines. Plant leaves maximize absorption of sunlight when the leaf surface is

perpendicular to the incident light (Taiz and Zeiger, 2002b). Transgenic and

non-transgenic plants all exhibited more horizontal growth habit in full sun, compared to shade conditions. Glyphosate-resistant and wild type plants grew more upright under shaded conditions as a shade avoidance response. GA2ox transgenic lines tended to keep horizontal growth habit, possibly due to the increased GA metabolism by GA2ox overexpression, or the collaborated work of GA and light receptors in signal transduction.

Greenhouse pot trial

Similar results were obtained in greenhouse studies to those from field work.

GA2ox gene transformation led to significant reduction of vertical growth rate and

leaf growth rate, as well as alteration of growth habit of transgenic plants, compared

to control plants, most possibly resulting from the inactivation of bioactive GA by

GA2ox.

Transcription level of representatives from putative GA transformants were

analyzed with RT-PCR. The results reveal that the ranking of mRNA level of GA2ox

matches the ranking of individual traits and selection indices of creeping bentgrass

plants in the greenhouse and the field. It shows that GA2ox transformation is

associated with the phenotypic changes in creeping bentgrass and as a result, strong

transformants exhibited strong shade tolerance and would be the candidates for

further breeding program.

Vertical height Internode length Leaf length Cultivar (cm)† (cm)‡ (cm)§

NS¶ CS# FS†† NS CS FS NS CS FS Glyphosate-resistant control (RR)‡‡ RR6234 6.5 4.9 3.6 2.4 1.5 1.7 4.4 3.2 1.7 RR6235 6.5 7.0 3.9 2.3 2.0 1.8 6.3 5.8 2.6 RR6237 4.6 4.5 3.3 2.0 1.3 1.0 5.9 5.4 3.9 RR6249 6.5 7.3 4.6 1.3 1.8 1.1 3.6 4.2 2.5 RR6253 8.2 7.7 4.8 1.1 1.1 0.8 6.7 5.6 4.0 RR6260 5.8 6.8 4.1 2.5 1.5 1.2 9.4 8.5 4.6 RR6261 6.1 6.0 3.4 2.2 1.1 1.8 4.9 3.7 2.3 RR6263 7.0 6.3 3.8 1.3 1.2 1.1 5.2 5.1 2.7 Putative GA2ox transformants (GA)§§ GA6435 5.8 4.7 3.8 2.2 1.5 1.8 4.7 3.6 2.4 GA6437 8 6.8 4.4 2.8 2.5 2.2 6.1 5.2 2.8 GA6441 7.1 5.4 3.6 2.2 1.7 2.0 6.8 4.3 2.5 GA6448 6.0 5.9 3.6 1.6 1.0 1.0 5.4 4.4 2.3 GA6462 8.8 8 4.5 3.1 1.8 1.5 8.0 6.4 2.8 GA6465 8.0 6.5 3.7 2.6 1.5 1.2 5.9 5.2 2.8 GA6476 6.6 5.1 5.4 2.1 1.2 1.8 5.1 3.4 2.4 GA6480 9.1 7.8 4.4 3.1 2.6 2.3 7.3 6.2 3.5 GA6487 7.8 7.4 5.1 2.2 1.7 1.7 3.8 4.1 2.4 GA6509 6.2 6 3.8 1.9 1.4 1.2 7.4 6.5 4.1 GA6511 7.8 8.3 4.6 2.1 2.1 1.5 4.1 4.8 2.2 GA6517 4.8 4.4 3.1 2.3 1.3 0.9 3.2 4.9 2.6 GA6525 6.2 6.3 4 0.8 1.0 0.9 6.0 5.0 2.6 GA6531 5.6 5 3.6 1.4 1.3 0.8 8.2 3.9 2.7 GA6535 5.2 5.9 3.1 2.3 1.9 1.7 4.3 4.5 2.5 GA6536 5.5 6 3.6 1.4 1.3 1.4 4.6 3.8 3.0 GA6539 4.3 4.3 3 0.8 1.0 0.7 4.6 3.8 2.2 GA6546 6.3 6.3 3.5 1.1 1.3 1.1 3.9 3.9 2.5 GA6547 7.2 4.8 3.2 2.6 1.2 1.5 7.1 5.6 2.4 GA6548 4.2 3.2 2.9 2.0 0.9 0.6 7.4 3.6 2.6 GA6549 4.9 3.9 3.8 1.2 1.2 0.8 6.8 5.6 3.2 GA6555 5 4.1 3.4 1.9 1.0 1.0 4.7 4.2 2.4 GA6557 4.7 4.8 3.4 1.3 2.1 0.6 5.3 4.0 2.9 GA6558 7.1 6.8 3.6 2.0 1.4 1.4 6.9 5.1 3.4 GA6562 4.8 5.6 3 1.2 1.0 0.5 4.8 4.8 2.2

Table 2.1: Vertical growth rate, internode length, and leaf length of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in the field. Continued on next page

Table 2.1 Continued.

Cultivar Vertical height (cm) Internode length(cm) Leaf length(cm)

NS CS FS NS CS FS NS CS FS Putative GA2ox transformants (GA) GA6564 5.9 5.9 3.7 1.6 1.4 0.8 7.0 3.7 3.9 GA6567 8.1 8.0 4.0 2.4 1.8 1.2 6.9 5.7 3.2 GA6568 4.9 5.7 3.7 0.8 1.0 0.7 4.8 4.8 2.5 GA6569 6.2 4.9 3.7 2.0 1.0 0.7 7.5 5.1 3.3 GA6577 6.2 6.5 3.6 1.0 1.1 1.2 4.8 4.8 2.6 Wild type control (WT)¶¶ Crenshaw 8.4 7.8 3.8 2.8 2.0 2.3 9.0 6.4 3.4 LS44 8.6 7.6 3.6 2.2 1.8 1.2 8.4 6.6 2.9 LSD (0.05) 1.0 0.8 0.5 0.8 0.5 0.5 1.9 1.4 0.7

Source of P>F P>F P>F P>F P>F P>F P>F P>F P>F variance

Cultivar *** *** *** *** *** *** *** *** *** RR vs.GA NS *** * NS NS NS NS * * GA vs. WT *** *** NS ** ** *** *** *** * Among GA *** *** *** *** *** *** NS * NS †Values are means of 18 numbers; 2 replicates averaged across 9 dates (23 July 2004, 11 Aug. 2004, 31 Aug. 2004, 21 Sep. 2004, 13 Oct. 2004, 16 May 2005, 16 June 2005, 18 Jul. 2005, and 25 Aug. 2005). ‡ Values are means of 8 numbers; 2 replicates averaged across 4 dates (13 Aug. 2004, 21 Sep. 2004, 17 June 2005, and 19 July 2005). §Values are means of 4 numbers; 2 replicates averaged across 2 dates (21 Sep. 2004 and 20 June 2005). ¶Neutral shade (NS) #Canopy shade (CS) ††Full sun (FS) ‡‡ Glyphosate-resistant controls are transgenic creeping bentgrass containing CP4 EPSPS gene. §§Putative GA2ox plants are glyphosate-resistant creeping bentgrass regenerated from callus transformed with CP4 EPSPS and runner bean GA2ox genes. ¶¶Wild type controls are non-transgenic creeping bentgrass. *, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively. NS, not significant at P<0.05.

Cultivar Visual quality† Shoot density‡ Stolon density§

NS¶ CS# FS†† NS CS FS NS CS FS Glyphosate-resistant control (WT)‡‡ RR6234 3.8 5.0 2.4 4.0 5.0 2.3 8.5 8.0 6.5 RR6235 4.6 5.0 5.8 4.7 4.3 6.0 6.0 7.0 8.0 RR6237 5.0 4.8 6.7 4.9 4.4 6.4 6.5 7.0 6.0 RR6249 3.0 2.5 4.0 2.5 3.0 4.8 3.0 6.0 4.0 RR6253 2.3 2.3 6.4 3.5 3.1 6.0 1.0 1.5 5.5 RR6260 4.3 5.5 6.3 5.0 5.3 5.8 7.5 6.0 6.0 RR6261 4.1 5.8 4.8 4.5 5.5 4.9 7.5 7.5 7.5 RR6263 3.8 3.5 5.3 4.4 3.6 5.8 4.0 4.5 6.5 Putative GA2ox transformants (GA)§§ GA6435 4.3 5.0 4.6 4.8 5.3 4.5 7.0 6.5 7.0 GA6437 5.4 4.8 5.8 5.9 5.3 5.5 9.0 8.5 9.0 GA6441 5.4 5.5 6.0 5.5 5.3 6.0 6.5 7.5 7.5 GA6448 4.5 4.3 5.8 4.3 4.4 6.3 6.5 5.0 6.0 GA6462 3.8 3.5 5.5 4.3 3.8 5.3 8.0 7.0 6.5 GA6465 5.5 6.3 7.4 5.8 6.1 7.8 7.5 7.0 8.0 GA6476 5.3 4.0 6.4 5.8 4.3 6.8 6.5 7.0 6.0 GA6480 4.5 5.0 7.0 4.8 4.7 6.3 8.5 9.0 9.0 GA6487 3.6 3.3 4.8 4.3 4.6 5.3 6.5 7.0 7.0 GA6509 5.8 6.0 7.1 5.8 6.3 7.5 8.0 6.0 5.5 GA6511 3.0 4.8 4.9 3.3 4.1 5.3 5.0 6.5 7.5 GA6517 5.3 5.3 6.0 4.6 5.3 7.1 7.5 6.0 6.0 GA6525 2.3 4.8 3.9 4.0 4.7 4.5 3.5 6.0 6.0 GA6531 4.9 4.0 6.1 5.3 4.2 6.3 6.5 5.0 4.0 GA6535 4.4 4.8 4.8 5.1 5.0 4.8 6.5 7.0 6.0 GA6536 3.8 2.8 4.8 3.3 3.4 4.3 4.0 4.0 4.0 GA6539 4.0 5.3 4.8 5.1 5.4 4.5 4.5 4.0 1.5 GA6546 4.3 4.3 5.0 4.5 5.2 5.3 6.0 6.0 6.0 GA6547 6.8 6.3 5.0 6.9 6.3 5.5 8.5 8.0 6.0 GA6548 7.5 7.3 8.7 7.8 7.5 8.3 8.0 8.0 6.0 GA6549 5.8 5.8 6.8 5.9 5.3 6.4 6.5 7.5 6.5 GA6555 5.2 4.5 5.7 5.5 4.3 6.3 7.5 6.0 6.0 GA6557 4.3 5.5 6.4 4.7 5.5 6.1 6.5 7.0 4.0 GA6558 4.5 4.5 6.5 4.5 4.8 6.0 6.5 4.5 6.5 GA6562 2.9 5.3 5.3 3.3 5.5 4.7 6.0 7.5 2.0

Table 2.2: Visual quality, shoot density, and stolon density of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in the field. Continued on next page

Table 2.2 Continued.

Cultivar Visual quality Shoot density Stolon density

NS CS FS NS CS FS NS CS FS Putative GA2ox transformants (GA) GA6564 2.8 3.3 6.6 3.2 4.3 6.4 6.5 6.0 6.0 GA6567 4.8 4.8 6.0 4.8 4.8 6.3 8.0 6.0 7.5 GA6568 4.8 5.8 6.3 5.4 5.8 6.8 5.5 6.0 2.5 GA6569 5.8 5.0 6.8 5.3 5.0 5.5 7.5 5.5 4.0 GA6577 3.3 4.3 5.4 4.0 4.8 5.8 4.0 6.0 6.0 Wild type control (WT)¶¶ Crenshaw 5.4 5.5 8.5 6.0 6.0 8.4 8.5 7.5 8.5 LS44 4.9 5.3 7.8 6.1 5.8 7.8 7.5 7.0 6.5 LSD (0.05) 0.7 0.9 0.6 0.7 0.8 0.7 1.6 1.4 1.6

Source of P>F P>F P>F P>F P>F P>F P>F P>F P>F variance

Cultivar *** *** *** *** *** *** *** *** *** RR vs. GA *** *** *** *** *** *** *** * NS GA vs. WT ** * *** *** *** *** ** * *** Among GA NS NS NS NS * ** * *** *** †Visual quality ratings are based on 1-9 scale; 1 is poor density and uniformity, dormant/dead turf or bare soil, and coarse texture, 9 is ideal turf, dark green color, excellent density and uniformity, and fine texture; acceptable turf quality≥6. Values are means of 12 numbers; 2 replicates averaged across 6 dates (24 Sep. 2004, 13 Oct. 2004, 15 May 2005, 15 June 2005, 14 July 05, and 26 Aug. 2005). ‡Shoot density ratings are based on 1-9 scale with 9 equaling maximum density. Values are means of 12 numbers; 2 replicates averaged across 6 dates (24 Sep. 2004, 13 Oct. 2004, 15 May 2005, 15 June 2005, 14 July 05, and 26 Aug. 2005). §Stolon density ratings are based on 1-0 scale with 9 being maximum stolon density. Values are means of 4 numbers, 2 replicate across 2 dates (24 Sep. 2004 and 15 June 2005). ¶Neutral shade (NS) #Canopy shade (CS) ††Full sun (FS) ‡‡Glyphosate-resistant controls are transgenic creeping bentgrass containing CP4 EPSPS gene. §§Putative GA2ox transformants are glyphosate-resistant creeping bentgrass plants regenerated from callus bombarded with CP4 EPSPS and runner bean GA2ox genes. ¶¶Wild type controls are non-transgenic creeping bentgrass. *, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively. NS, not significant at P<0.05.

Cultivar Color† Erectness‡

NS§ CS¶ FS# NS CS FS Glyphosate-resistant control(WT)†† RR6234 4.3 4.5 2.0 8.0 7.0 7.5 RR6235 4.8 5.0 3.5 7.0 6.5 7.5 RR6237 4.8 4.8 6.3 6.0 5.5 4.5 RR6249 4.5 4.8 5.3 7.5 7.0 7.0 RR6253 5.3 3.8 6.8 7.5 8.0 7.5 RR6260 6.3 6.0 6.5 6.5 7.0 5.5 RR6261 5.0 5.3 3.8 8.0 6.5 8.0 RR6263 4.8 4.0 6.5 8.0 8.5 7.5 Putative GA2ox transformants (GA)‡‡ GA6435 5.0 5.3 5.3 4.0 5.5 4.5 GA6437 6.8 4.8 6.5 8.0 5.0 4.5 GA6441 5.5 5.5 6.0 6.0 4.5 5.0 GA6448 5.8 4.8 6.3 4.0 6.5 3.0 GA6462 5.8 5.8 7.3 5.0 7.0 4.5 GA6465 6.3 5.5 6.8 6.0 5.5 3.0 GA6476 5.8 3.8 4.3 5.5 4.0 6.5 GA6480 6.5 6.3 6.5 7.0 5.5 3.0 GA6487 3.3 4.8 4.3 3.5 3.5 6.0 GA6509 5.5 5.8 7.0 2.5 3.0 3.5 GA6511 3.5 5.8 4.0 8.0 6.5 6.0 GA6517 6.0 5.5 6.3 3.0 5.0 2.5 GA6525 4.5 4.5 4.0 6.0 4.0 5.0 GA6531 5.5 5.5 5.5 6.0 6.0 3.5 GA6535 4.5 4.3 4.5 5.5 4.0 7.5 GA6536 5.3 3.8 5.8 6.5 8.0 6.5 GA6539 5.3 5.5 4.8 5.5 2.0 6.0 GA6546 4.5 3.3 5.3 6.0 6.0 3.5 GA6547 6.8 5.8 6.3 6.5 5.5 2.5 GA6548 8.8 7.8 9.0 2.0 1.5 1.0 GA6549 6.5 7.3 7.5 3.5 3.5 3.0 GA6555 5.8 4.0 5.8 2.0 2.5 4.5 GA6557 6.0 6.0 5.5 5.5 4.5 4.5 GA6558 6.3 6.3 7.5 5.5 7.0 5.5 GA6562 5.5 6.3 6.5 4.5 3.5 5.0

Table 2.3: Color and erectness of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in the field. Continued on next page

Table 2.3 Continued.

Cultivar Color Erectness

NS CS FS NS CS FS Putative GA2ox transformants (GA) GA6564 4.8 5.0 7.5 5.0 5.5 2.5 GA6567 7.3 6.8 6.8 7.5 6.5 3.5 GA6568 5.3 5.8 5.3 6.0 6.0 6.5 GA6569 7.5 6.8 7.3 3.5 3.5 6.0 GA6577 4.3 3.0 4.8 6.5 6.0 5.5 Wild type control(WT)§§ Crenshaw 6.3 5.8 7.8 7.5 6.0 2.0 LS44 6.0 6.3 7.0 7.5 7.5 2.5 LSD (0.05) 0.6 0.7 0.7 1.3 1.1 1.2

Source of P>F P>F P>F P>F P>F P>F variance

Cultivar *** *** *** *** *** *** RR vs. GA *** *** *** *** *** *** GA vs. WT ** ** *** *** *** *** Among GA *** NS *** NS NS ** †Color ratings are based on 1-9 scale with 1 being light green and 9 being dark green. Values are means of 12 numbers; 2 replicates averaged across 6 dates (24 Sep. 2004, 13 Oct. 2004, 15 May 2005, 15 June 2005, 14 July 05, and 26 Aug. 2005). ‡Erectness is visual rating of angles between turf stand and the ground. It is based on 1-9 scale: 9, 90°; 1=0°. Values are means of 6 numbers; 2 replicates averaged across 3 dates (13 Oct. 2004, 15 May 2005, and 14 July 05). §Neutral shade (NS) ¶Canopy shade (CS) #Full sun (FS) ††Glyphosate-resistant controls are transgenic creeping bentgrass containing CP4 EPSPS gene. ‡‡ Putative GA2ox transformants are glyphosate-resistant creeping bentgrass regenerated from callus bombarded with CP4 EPSPS and runner bean GA2ox genes. §§Wild type controls are non-transgenic creeping bentgrass. *, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively. NS, not significant at P<0.05.

Cultivar Ranking of base index† NS‡ CS§ FS¶ Sum# Glyphosate-resistant control (RR) †† RR6234 17 37 40 29 RR6235 33 28 33 32 RR6237 16 14 10 33 RR6249 39 38 38 12 RR6253 40 39 29 40 RR6260 24 22 24 38 RR6261 15 29 36 24 RR6263 37 33 28 36 Putative GA2ox transformants(GA)‡‡ GA6435 13 15 30 19 GA6437 26 19 26 25 GA6441 11 17 21 16 GA6448 28 12 9 17 GA6462 36 36 23 34 GA6465 12 16 4 8 GA6476 22 10 34 22 GA6480 29 31 19 28 GA6487 31 32 39 37 GA6509 5 6 8 3 GA6511 35 40 37 39 GA6517 9 5 5 4 GA6525 18 35 35 30 GA6531 21 13 11 13 GA6535 19 18 32 23 GA6536 38 27 31 35 GA6539 3 9 27 11 GA6546 32 23 22 27 GA6547 4 7 12 5 GA6548 1 1 1 1 GA6549 2 2 6 2 GA6555 14 3 15 9 GA6557 8 11 13 7 GA6558 25 20 16 20 GA6562 7 21 18 14

Table 2.4: Base index rankings of 40 creeping bentgrass cultivars under neutral shade, canopy shade, and full sun in the field. Continued on next page

Table 2.4 Continued. Cultivar Ranking of base index NS CS FS NS Putative GA2ox transformants GA6564 30 34 7 26 GA6567 27 24 14 21 GA6568 10 8 20 10 GA6569 6 4 17 6 GA6577 34 30 25 31 Wild type control(WT)§§ Crenshaw 20 25 2 15 LS44 23 26 3 18 † Base index was calculated from the standardized values of the following traits assigned with different economic weight: vertical growth rate(-3), internode length(-1), leaf length(-1), visual quality(3), shoot density(-3), stolon density(1), color rating(3), and erectness(-3). All values were ranked from high to low. ‡Neutral shade (NS) §Canopy shade (CS) ¶Full sun (FS) #Sum of base indices under all three conditions was from high to low. ††Glyphosate-resistant controls are transgenic creeping bentgrass containing CP4 EPSPS gene ‡‡Putative GA2ox transformants are glyphosate-resistant creeping bentgrass regenerated from callus bombarded with CP4 EPSPS and runner bean GA2ox genes. §§Wild type controls are non-transgenic creeping bentgrass.

Percentage of FS Percentage of FS Treatment (instant PPF) † (R:FR) ‡

Non-selective cover (full sun control) 100 100

Canopy shade (CS) 16 53

Neutral shade (NS) 26 99 †Instant PPF(mol m-2s-1) was averaged over weekly readings taken using a quantum light meter (Spectrum Technologies, Plainfield, IL) around 2pm during the study from May 30 through July 11, 2005. ‡Red:far-red (R:FR) values were calculated from the weekly measurements at 660±5nm and 730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience, Lincoln, NE) around 2pm during the study from May 30 through July 11, 2005.

Table 2.5: Instant photosynthetic photon flux and red:far-red ratio of the three light treatments.

Visual Leaf growth rate Vertical growth Erectness‡ Base index# Rank rating† (mm/day)§ rate(mm/day)¶ Cultivar Glyphosate-resistant control(RR)†† RR6237 4.4 6.8 3.3 2.7 -3.2 14 RR6260 4.2 7.2 4.2 3.2 -5.6 16 RR6261 3.7 8.4 3.2 3.3 -6.9 17 Putative GA2ox transformants(GA)‡‡ GA6435 5.4 6.8 2.5 2.3 0.8 6 GA6465 6.0 5.1 2.6 1.9 4.4 3 GA6476 5.7 7.6 3.1 2.8 -0.2 9 GA6487 4.7 8.2 3.0 3.0 -3.5 15 GA6531 4.9 6.7 3.5 2.7 -2.0 12 GA6539 5.1 7.6 3.6 2.9 -2.3 13 GA6547 6.2 4.4 2.8 2.1 3.8 4 GA6548 7.3 3.4 2.0 1.6 9.4 1 GA6549 6.3 4.3 2.6 2.1 4.9 2 GA6555 5.1 5.1 2.7 2.5 0.5 7 GA6564 5.4 5.3 3.3 2.5 0.8 5 GA6568 5.7 7.5 3.2 2.8 -0.3 10 Wild type control(WT)§§ Crenshaw 6.0 7.0 3.5 2.9 0.5 8 LS44 5.8 7.1 3.8 3.2 -0.8 11 Light treatment Neutral shade(NS) 5.3 6.8 3.2 2.8 -1.1 2 Canopy shade(CS) 5.2 6.9 3.5 3.1 -1.8 3 Full sun(FS) 5.8 5.5 2.6 1.9 2.9 1 Source of variance Cultivar *** *** ** *** *** Among GA *** *** NS *** *** GA vs. RR *** *** ** *** *** GA vs. WT NS *** ** *** * Treatment *** *** *** *** *** NS vs. CS NS NS NS *** NS Shade(NS+CS) vs. FS *** *** *** *** *** Cultivar×Treatment NS * NS *** NS †Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05). ‡Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05). §Values are means of 15 numbers; 3 replicates averaged across 5 dates (6 June 2005, 21 June 2005, 30 June 2005, 8 July 2005, and 14 July 2005). ¶Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, and 13 July 2005). #Base index was calculated from the standardized values of the following traits assigned with different economic weight: visual rating (3), erectness (-1), leaf growth rate (-1), and vertical growth rate(-1). ††Glyphosate-resistant controls (RR) are glyphosate-resistant creeping bentgrass regenerated from calli bombarded with CP4 EPSPS gene. ‡‡Glyphosate-resistant GA2ox (GA) plants are glyphosate-resistant creeping bentgrass regenerated from calli bombarded with CP4 EPSPS and runner bean GA2ox genes. §§Wild type controls are non-transgenic conventional creeping bentgrass plants from seeds. *, **, ***Significant at 0.05, 0.01, and 0.001 probability levels, respectively. Table 2.6 Visual rating, erectness rating, leaf growth rate, vertical growth rate, base index of 17 creeping bentgrass lines in the greenhouse 71

P35S Ract-Intron GA2ox nos

5’ 3’

1.0 kb

Figure 2.1: Schematic map of GA2ox expression cassette. Final construct also contained CP4-EPSPS and Kan genes as selective markers 72

A B C

73 Fig.2.2A-C: Treatments established in open sun light (A), under a shade cloth covered Quonset structure(B), and under mature deciduous hardwoods(C). Photosynthetic photon flux averaged at 28.3 mol·m-2d-1 in full sun, 3.0 mol·m-2d-1 in neutral shade, and 3.3 mol·m-2d-1 in deciduous shade, from 29 June 2004 through 15 Oct. 2004 and from 10 May 2005 through 30 August 2005. R: FR was measured in June and August of both years and averaged at 1.101 in full sun, 0.954 under neutral shade, and 0.395 under deciduous shade.

Figure 2.3A-C: Wild type control LS44, glyphosate-resistant control RR6260, and GA2ox gene transformed creeping bentgrass plants GA6549, GA6476, GA6547, and GA6548 (left to rigth) grown under clear filter(A); LS44, RR6260, GA6547, GA6476, GA6549, and GA6548 (left to right) under canopy shade(B); LS44, RR6260, GA6476, GA6549, GA6547, and GA6548 (left to right) under black shade cloth(C). Yellow represents the vertical height of creeping bentgrass plants.

Figure 2.4: Comparison of wild type plant LS44 and transgenic lines GA6547 and GA6548 (left to right) under full sun in the greenhouse.

Neutral shade Canopy shade 4 Full sun

Vertical growth rate(mm/day) Vertical 1

0 RR6260 RR6261 GA6435 GA6487 GA6548 GA6549Crenshaw LS44

Figure 2.5 Vertical growth rate(mm/day) of glyphosate-resistant cultivars(RR6260 and RR6261), positive GA2ox transformants (GA6435,GA6548, and GA6549), negative GA2ox transformant (GA6487), and wild type controls (Crenshaw and LS44) in the greenhouse.

Neutral shade Canopy shade Full sun 8

4 Erectness rating

0 RR6260 RR6261 GA6435 GA6487 GA6548 GA6549Crenshaw LS44

Figure 2.6 Erectness ratings of glyphosate-resistant cultivars(RR6260 and RR6261), positive GA2ox transformants (GA6435,GA6548, and GA6549), negative GA2ox transformant (GA6487), and wild type controls (Crenshaw and LS44) in the greenhouse.

w 5 5 6 8 9 5 7 a 7 0 46 43 47 54 54 55 48 nsh 4 23 26 x6 x6 x6 x6 x6 x6 x6 re S4 r6 r6 A A A A A A A C L A A kb 0.5 GA2ox

0.5 Actin

Figure 2.7: Reverse-transcription (RT-PCR) of RNA samples from putative GA2ox transformants (GA6465, GA6465, GA6476, GA6548, GA6549, GA6555, and GA6487), conventional control plants (Crenshaw and LS44), and glyphosate-resistant control plants (RR6237 and RR6260). Arrows specify the expected GA2ox and Actin bands.

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CHAPTER 3

DEVELOPMENT AND CHARACTERIZATION OF TRANSGENIC CREEPING

BENTGRASS TRANSFORMED WITH TOBACCO PHYB1 GENE

ABSTRACT

Reduction in photosynthetic photon flux (PPF) and light quality (red:far-red light ratio) adversely affected turfgrass growth in shade. Overexpression of phytochrome genes provides a fast and convenient way to control plant vegetative growth. Tobacco

(Nicotiana plumbaginifolia Viv.) PHYB1 gene was inserted into creeping bentgrass

(Agrostis palustris) by biolistic bombardment, with CP4EPSPS gene as the selectable marker. Four transgenic plants were recovered following transformation and glyphosate selection. Tissue culture control (NSC) was regenerated from non-bombarded callus at the same time. Together with 2 wild type cultivars, LS44 and

Crenshaw, transgenic and NSC plants were multiplied and subjected to 4 light treatments: full sun (FS), canopy shade (CS), neutral shade (NS), and far-red light absorbing filter (AFR). Our results showed reduction in R:FR increased vertical growth and erectness of both transgenic and control plants. As group, transgenic plants exhibited delayed vertical shoot growth and more horizontal shoot architecture, but did not demonstrate significant change in leaf growth or visual quality. PB0701 exhibited highest visual quality and lowest leaf growth rate among all plants. Leaf

growth was observed to be affected mainly by the change of PPF rather than R:FR.

Visual quality rating increased with the rise of both PPF and R:FR. RT-PCR results

revealed the transcription of PHYB1 gene in all creeping bentgrass lines, with

PHYB1 transformant PB0701 displayed highest transcription level. It may also be

associated with its high quality and alleviation of shade response under low light

conditions.

INTRODUCTION

Creeping bentgrass (Agrostis palustris) has been used extensively on golf greens

and fairways throughout the United States (Bell and Danneberger, 1999b; Emmons,

2000). Its growth and development are influenced by changes in light quality, quantity,

and duration (Franklin and Whitelam, 2005). Bentgrass performs best with a

minimum of four to six hours of full sun per day (Reicher and Throssell, 1998). Both photosynthetic photon flux (PPF) and red/far red ratio (R/FR) decline dramatically when sunlight penetrates the canopy and reaches the surface of the turf (Shirley, 1945;

Smith, 1982). Shade stress causes increase of vertical growth and succession of harmful physiological and morphological changes in plants, such as thinner, more delicate leaves, reduced tillering, poor shoot density, altered pigment concentration and affected carbohydrate reserve (Bell and Danneberger, 1999b). Furthermore, intensive mowing on golf course removes most of leaf surface area, reduces bentgrass photosynthesis and recuperative ability, and increases occurrence of bentgrass disease under low light conditions (Bell and Danneberger, 1999a; Bell and Danneberger,

1999b).

Phytochromes are photoreceptors sensing the light quality change in the red and far-red region of the spectrum (Franklin and Whitelam, 2005). They are usually in the dynamic equilibrium of Pr and Pfr forms. Decrease in R:FR triggers conformational change towards biologically inactive Pr form and affects downstream reactions leading to shade avoidance response (Franklin and Whitelam, 2005). Shade avoidance syndrome usually include stem and petiole elongation, reduction in chlorophyll content and leaf thickness, elevation in leaf angle (erectness), reduced tillering in grasses (Casal et al., 1986). Shade avoidance often results in lodging damage or mechanical injury and lost of compatibility in a long run (Casal and Smith, 1989).

Phytochromes are encoded by a small multi-gene family in higher plants (Quail,

1994) and can be divided into two distinctive groups, type I, ‘light-labile’ and type II,

‘light-stable’ phytochromes. Type I phytochromes are involved in the very low fluence rate response (VLFR) and the far-red high irradiance response (HIR). On the contrary, type II phytochromes control the red/far-red reversible (low fluence rate,

LFR) light response (Fernández et al., 2005). PHYA–PHYE genes have been sequenced and identified in model plants Arabidopsis thaliana (Clack et al., 1994;

Sharrock and Quail, 1989). Arabidopsis phyA belongs to the ‘light-labile’ and phyB–phyE to the ‘light-stable’ groups (Fernández et al., 2005). Leading role of phyB in perceiving R:FR ratio signal was revealed by ‘constitutive shade avoidance’ of phyB mutants of various plant species grown in white light (Devlin et al., 1992;

Lo´pez-Juez et al., 1992; Reed et al., 1993; Somers DE, 1991). In light grown plants, phyB is the major sensor of red light (Neff et al., 2000) and regulates cell expansion or elongation. Hypothetically Arabidopsis phyB controls cell size by regulating nuclear endoreduplication in hypocotyls (Gendreau et al., 1998).

Vertical height control remains a key problem in turfgrass management under

shade. Dwarfism could improve turfgrass quality and reduce management cost.

Genetic engineering of phytochrome genes provides a fast and convenient way to

control plant vegetative growth. Arabidopsis or rice (Oryza stivum L.) PHYB gene

transformation led to short hypocotyls in transgenic Arabidopsis seedlings

(McCormac et al., 1993; Wagner et al., 1991). Besides, reduction of stem elongation of mature tobacco (Nicotiana tabacum L.) and potato (Solanum tuberosum L.) plants

resulted from overexpression of Arabidopsis PHYB gene (Halliday et al., 1997; Thiele

et al., 1999). Furthermore, decrease of vertical growth and increase of branching

angles were observed in transgenic chrysanthemum (Dendranthema ×grandiflora K.)

plants transformed with tobacco (Nicotiana plumbaginifolia Viv.) PHYB1 gene

(Zheng et al., 2001). We hypothesized that transformation of tobacco PHYB1 gene would enhance the sensitivity of transgenic creeping bentgrass to red light and promote the downstream R-regulated reaction, which would lead to reduction of vertical growth and leaf growth rate, alteration of plant architecture and visual quality.

MATERIALS AND METHODS

Construction of PHYB1 expression cassettes

A 4kb BamHI/SacI fragment of a full length Nt-PHY-B1 cDNA (Murashige et al.,

1962)was cloned into BamHI/SacI sites of pCAMBIA1305.2, forming pCB. pMON vector was cut by NotI, and a 2.4kb fragment containing a CaMV35S promoter, ract1 intron, and a nos terminator was cloned in the sense direction into the NotI site in pGEM-T Easy vector, resulting in the vector pGM. pKN vector was developed by cloning a complete EcoRI/NotI nos fragment from pGM into EcoRI/NotI sites of pKS vector. A fragment of BamHI/SalI PHYB1 from pCB was ligated with a BamHI/SacI

fragment pGM containing a CaMV35S promoter and a ract1 intron, and a SalI/SacI nos fragment from pKN. The resulting pGB was digested with NotI and a 5.3 kb fragment containing the Nt-PHY-B1, 35 S promoter and terminator was ligated in the sense direction with the NotI fragment of pMON which includes a CP4-EPSPS gene.

The 5.3 kb NotI/NotI PHYB1 expression cassette included a 4kb BamHI/SalI fragment of a full length PHYB1 cDNA, a CaMV35S promoter, a ract1 intron, and a nos terminator. The final construct also contained CP4-EPSPS gene as selective markers (Fig. 3.2).

Callus induction and maintenance

Induction and maintenance of embryogenic callus were described previously

(Busov et al., 2003). Mature seeds (caryopses) of creeping bentgrass cultivar,

‘Crenshaw’, were surface sterilized in 70% ethanol for 1 min, followed by 30-min treatment of 50% commercial bleach and 0.1% Tween-20 under vacuum. Surface sterilized seeds were rinsed three times with sterile distilled water, cultured on MSI medium, MS basal medium (Lee et al., 1996; Zhong et al., 1991) supplemented with

500 mg L-1 Casein enzymatic hydrolysate (Sigma), 3% sucrose, 30 μM dicamba, 2.2

μM BA, and Gel-Gro™ (MP biomedical). After 4-5 wk at 25 ºC in the dark, embryogenic callus lines were selected and subcultured onto fresh medium every 4-6 wk.

Transformation and regeneration

Embryogenic callus was placed on 9-cm filter disks (0.3g tissue per disk) in plates of MSI medium containing 0.3 M mannitol and 0.3 M sorbitol at least 4 hours prior to bombardment(Kern et al., 1993). Gold particles plasmid DNA containing

PHYB1 construct coated were delivered into embryogenic creeping bentgrass callus

cultures using a Bio-Rad PDS-1000/He biolistic delivery system at 900 psi (Vain et

al., 1993). Bombarded callus was selected on MSI medium containing 2mM

glyphosate for about 6-8 wk in dark. Afterwards, embryogenic callus was regenerated

on MSS medium, MS medium including 3mg L-1 BA and 0.2 mM glyphosate. Shoots were transferred to MSR medium, MS medium containing 0.2 mM glyphosate for rooting. Finely rooted plants were grown in metromix under fluorescent light for 1wk before they were moved into greenhouse.

Plant propagation and maintenance

Plant material for the study was established from stolon nodes of seven primary

transgenic events, one no-short-control line, and 2 conventional lines (Table 3.2 ) in

7-cm square plastic pots filled with metro-mix 350 (Scotts Co., USA). Throughout the

trial, plants were kept at 21°C /16°C (Day/Night) and 13-14 hours day length. Pots

were weekly fertilized at a rate of 100ppm N with a 20N-10P2O5-20K2O soluble

fertilizer (Scotts/Sierra, USA). Turf was irrigated twice daily (0900 and 1300 hr) with

automatic irrigation system. Fungicide and insecticide were applied monthly to

prevent disease and insect problems. All plants were trimmed weekly to the height of

2.5 cm and clippings were taken away.

A randomized split-plot greenhouse study with 3 replicates was performed at

The Ohio State University, Columbus, OH between May 30 and July 11, 2005. Each

replicate was split into 4 treatments covered with non-photoselective polyethylene

film (controls), mature soybean plants that are 60-70 cm in height (canopy shade),

black shade cloth (neutral shade), and photoselective AFR (far-red light absorbing)

polyethylene film (Mitsui Chemicals, Inc., Tokyo, Japan). PPF (Table 3.1) was averaged over weekly readings taken during the study using a quantum light meter

(Spectrum Technologies, Plainfield, IL). Red:far-red (R:FR) values (Table 3.1) were calculated from the weekly measurements at 660±5nm and 730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience, Lincoln, NE).

Data collection

Overall quality ratings, erectness ratings, leaf extension rates, and vertical growth rates were obtained weekly, starting from wk 3. Visual quality ratings were based on

RNA isolation and RT-PCR analysis

Two-wk-old leaf tissue of transgenic and non-transgenic plants was collected fresh and snap frozen in liquid nitrogen. Total RNA was isolated from approximately

1.5g frozen tissue using TRIzol® reagent (Invitrogen, Carlsbad, CA) and further treated with DNaseI (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. First strand cDNA was obtained through reverse

transcription of an aliquot of 1µg RNA from each sample using Oligo-dT primers

with the 2-step Reverse Transcription System (Promega, Madison, WI). An aliquot of

1µl cDNA out of each 20µl sample was analyzed by PCR using Taq DNA polymerase

(New England Biolabs, Ipswich, MA) and gene-specific primers. Primers for PHYB

were phyBPrimerF (5’- TGTTGGCCAGGATGTTACT-3’) and phyBPrimerR990 (5’-

TTGCCTTTGAACTCTTAGAGC-3’) which generated a 680bp DNA fragment. PCR

reaction was conducted under the following conditions: 94℃ for 5 min for one cycle,

followed by 94℃ for 30 sec, 55℃ for 40 sec, and 72℃ for 1 min for 30 cycles, and

a final incubation at 72 ℃ for 5 min. Furthermore, primers actin-5

(5'-GAGAAGATGACCCAGATCATGTTTG-3') and actin-3

(5'-TCCTAATATCCACGTCGCACTTCAT-3') were used to amplify the actin gene

(Wang and Luthe, 2003).

Data analysis

Data were analyzed with a PROC MIXED procedure of SAS (PC version 9.1, the

SAS Institute, Cary, NC). LSMEANS and CONTRAST procedures were performed to

test hypothesis of gene transformation, light effect, and their interaction.

RESULTS

Selection and regeneration of transgenic clones

Bombarded and non-bombarded calli were recovered on MSI medium for 1 wk in dark before they were subject to glyphosate selection. All calli were maintained on

MSI medium supplemented with 2mM glyphosate for 2-3 wk, and then transferred to fresh MSI medium with 2mM glyphosate for another 2-3 wk selection. During the

first round of selection, nontransgenic tissue gradually stopped growing and finally

turned brown after the second selection (Figure 3.2D). Next, green, glyphosate-resistant creeping bentgrass tissue appeared 1 wk after survived

embryogenic callus were plated on MSS medium with 0.2 mM glyphosate (Figure

3.2E). Transgenic plantlets were smaller and had lower shoot and root growth rates than non-transgenic plantlets on rooting medium (Figure 3.2F&G). When grown in the greenhouse, some transgenic plant was much shorter and had more horizontal leaf orientation compared with the non-transgenic control plant (Figure 3.2H). All together

4 transgenic lines, named PB0501, PB1101, PB1102, PB0701, were obtained and multiplied for further analysis. Simultaneously, control plants were recovered from non-bombarded embryogenic calli after culturing on the similar induction and differentiation media, but with the omission of glyphosate selection. Herbicide resistance of transformants was confirmed by spraying plants with 0.9g/m2 Roundup® herbicide (Scotts Miracle-Gro Co., Marysville, OH).

Growth analysis under various light conditions

The overall effect of gene transformation on visual quality was non-significant

(Table 3.2). However, visual quality varied significantly among transgenic creeping bentgrass plants. Particularly, trangenic line PB0701 had the highest quality rating among all lines under all light conditions (Figure 3.3A-C &3.4A-D). The various light treatments had significant impact on visual quality of all creeping bentgrass plants.

Plants grown in full sun had significantly higher visual quality ratings than those grown under either shade conditions. Light transmitted through the AFR film significantly increased plants visual quality as compared to the control film and shade treatment. Nonetheless, light quality change under low light conditions did not have

significant impact on plants visual quality (Table 3.2, Figure 3.4A-D).

Despite the significant variation among transgenic plants, they generally

exhibited more horizontal growth habit than wild type and tissue culture control plants

(Table 3.2). Besides, erectness of creeping bentgrass plants was significantly

influenced by light treatments. Leaf orientation of plants grown in full sun was

significantly more horizontal than those grown under low light conditions. Increase in

R:FR did not have significant influence on leaf orientation of the plants in full sun.

Similarly, decrease in R:FR did not change the leaf orientation of the plants under low

light conditions (Table 3.2, Figure 3.5A-D). Leaf orientation of PB0701 remained

constantly horizontal across all light treatments, while LS44 and tissue culture control

plants became more upright when they were moved from full sun to low light conditions. Change in light quality seemed to have greater impact on LS44 and NSC

plants than PB0701 plants (Figure 3.3A-C).

Leaf growth rate was not significantly different between transgenic and control

plants, neither did it vary among transgenic lines. (Table 3.2). However, trangenic line

PB0701 had the lowest leaf growth rate among all lines under all light conditions

(Figure 3.3A-C &3.6A-D). Plants grown in full sun had significantly lower leaf

growth rate than those grown under shade conditions. However, leaf growth rate was

not slowed down by far-red light absorbing film, as compared to the control film. It

was not affected by the canopy shade either, as compared to neutral shade.

Generally, PHYB1 gene transformed creeping bentgrass plants had significantly

lower vertical growth rate compared with wild type and tissue culture control plants

(Table 3.2). Vertical growth rate differed significantly among PHYB1 transgenic

plants. PB0701 had the lowest vertical growth rate among all lines under all light

conditions (Figure 3.3A-C &3.7A-D). Moreover, vertical growth rate of creeping

bentgrass plants differed significantly under various light treatments. Plants grown in

full sun had significantly lower vertical growth rates than those grown under low light

conditions. Far-red light absorbing film significantly lowered plant vertical growth

rates, as compared to shade treatments. However, no significance was found between

the vertical growth rates of plants grown under control and AFR films. Furthermore, decrease in both PPF and R:FR significantly promoted plant vertical growth rates, as

compared with reduction in PPF only (Table 3.2; Figure 3.7A-D). It was also noticed

that light quality and quantity changes had greater effects on control plants than

transgenic plants, especially PB0701 (Figure 3.3A-C& 3.7A-D).

RT-PCR

Transcription of the PHYB1 gene in creeping bentgrass was studied by RT-PCR

analysis of RNA samples isolated from glyphosate-resistant and control plants.

Results indicated that the expected PHYB1 band of the right size (680bp) appeared in

all experimental lines, including glyphosate-resistant lines, tissue culture control line,

and the conventional lines (Figure 3.8). Glyphosate-resistant line PB0701 exhibited higher transcription level of PHYB1 compared with other transgenic and non-transgenic lines. Primers for actin were used as a positive control and its transcript was present in all samples (Figure 3.8).

DISCUSSION

Genetic engineering has been successfully utilized to develop traits of interest in

bentgrass such as disease resistance (Chai et al., 2002; Dai et al., 2003; Fu et al., 2005;

Guo et al., 2003) and herbicide resistance (Gardner et al., 2004; Gardner et al., 2003).

Our studies expands the usage of biotechnology in creeping bentgrass by introducing the system of developing transgenic creeping bentgrass plants transformed with

Arabisopsis PHYB1 gene and evaluating phenotypic traits of plants under different

PPF and R:FR.

During regeneration process, false plantlets appeared on shoot regeneration medium. They were characterized by light green or bleached leaf color. Besides, they stopped growing when adapted to extended glyphosate selection. True trangenic lines survived glyphosate selection and were regenerated eventually. The whole process of callus induction and plant regeneration from the induced callus are lengthy and difficult. It took about 8 months to finish one round of bombardment experiment. A recently developed callus-free transformation system using stolon nodes only takes one-third of the time required for other reported transformation systems (Ge et al.,

2006; Wang and Ge, 2005a; Wang and Ge, 2005b). We may consider using it as an alternative method in future studies.

Reduction of R:FR stimulated shade avoidance response which led to elongation of stem and petiole, increase in leaf angle, and reduction of tillering in grasses (Casal et al., 1986). Turfgrass species perform better in shade if they can decrease vertical growth and be adapted to horizontal growth habit (Tegg and Lane, 2004). Our results suggests that overexpression of tobacco PHYB gene induces dwarfism in transgenic creeping bentgrass by delaying root and shoot growth, as well as altering shoot architecture. It confirmed the report about role of phytochromes in controlling the increase of shoot zenith angles (the angles between the tillers the vertical line) in

annual ryegrass (Lolium multiflorum Lam) and dallies grass (Paspalum dilatatum

Poir.) with the rise of R:FR (Casal et al., 1990; Gibson et al., 1992). Leaves maximize absorption of sunlight when the leaf surface is perpendicular to the incident light (Taiz and Zeiger, 2002).

Besides, there is ample red light under full sun conditions and phytochromes might have been saturated by red light, which explains why increase of R:FR under full sun conditions did not lead to significant changes of vertical growth rate or growth habit of creeping bentgrass plants.

Creeping bentgrass plants with PHYB1 overexpression, especially PB0701, displayed improvement of turf quality and alleviation of shade response under low light conditions. The changes may be due to the over-production of phyB apoprotein which leads to increased sensitivity of transgenic plants to red light. The drawback of the studies is that number of regenerated plants was small and thus limited the statistical evidence of tobacco phyB effect in trangenic creeping bentgrass.

High PHYB1 transcription level of transgenic line PB0701 may account for its highest ranking among all lines under altered PPF and R:FR. However, PHYB1 band appeared in all experimental lines. There are two possible reasons for that: the RNA sample of the control plants might have been contaminated by trace amount of RNA from transgenic plants during the isolation process. Replacing the common pipette tips with filter tips might be a solution to reduce cross contamination in the future.

Moreover, there might be endogenous gene in creeping bentgrass genome that has high level of homology to the tobacco PHYB1 gene. Further molecular analysis needs to be done to dissect the details of the PHYB1 band, which may lead to the discovery of homological PHYB sequence in creeping bentgrass. However, little information is

available for the creeping bentgrass genome. The first linkage map of creeping bentgrass was recently published, which may ultimate facilitate marker-assisted breeding of some economically important quantitative traits(Chakraborty et al., 2005).

Another limitation of the studies is lack of large quantities of regenerated transgenic lines. Only 18 plates of tissue were bombarded, and most of them were affected by fungus during the selection process. Sterile condition is critical during the transformation and tissue culture process. More bombardment experiments need to be carried out to generate candidate lines for phenotypic and molecular analysis for the best possible shade-tolerant creeping bentgrass plants.

Percentage of FS Treatment Percentage of FS (R:FR) ‡ (instant PPF) †

Non-covered full sun (FS) 100 100 Non-selective cover (control) 43 103 Canopy shade (CS) 7 55 Neutral shade (NS) 11 102 A filter (A ) 48 FR FR 108 †Instant PPF(mol m-2s-1) was averaged over weekly readings taken using a quantum light meter (Spectrum Technologies, Plainfield, IL) around 2pm during the study from May 30 through July 11, 2005. ‡Red:far-red (R:FR) values were calculated from the weekly measurements at 660±5nm and 730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience, Lincoln, NE) around 2pm during the study from May 30 through July 11, 2005.

Table 3.1: Instant photosynthetic photon flux and red:far-red ratio of the four light treatments.

Leaf growth rate Vertical growth Visual quality† Erectness‡ (mm/day)§ rate(mm/day)¶ Source of variance Pr > F Pr > F Pr > F Pr > F Cultivar *** *** NS *** Among PHYB1# *** *** NS *** PHYB1 vs. NSC†† NS *** NS *** PHYB1 vs. WT‡‡ NS *** NS *** Treatment *** *** * *** Neutral vs. Canopy NS NS NS ** Shade vs. Full sun * *** ** ***

FRED vs. Full sun ** NS NS NS

FRED vs. Shade *** *** NS *** Cultivar×Treatment NS NS NS *** † Visual quality ratings are based on 1-9 scale; 1 is poor density and uniformity, dormant/dead turf or bare soil, and coarse texture, 9 is ideal turf, dark green color, excellent density and uniformity, and fine texture; acceptable turf quality≥6.Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05). ‡ Erectness is visual rating of angles between turf stand and the ground. It is based on 1-9 scale: 9, 90°; 1=0°. Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05). §Values are means of 15 numbers; 3 replicates averaged across 5 dates (6 June 2005, 21 June 2005, 30 June 2005, 8 July 2005, and 14 July 2005). ‡Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, and 13 July 2005). #Transgenic plants were regenerated from embryogenic callus following bombardment with PHYB1 DNA coding sequence. ††Tissue culture control (NSC) plants were regenerated from un-bombarded embryogenic callus. ‡‡Wild type controls are non-transgenic creeping bentgrass. *, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively. NS, not significant at P<0.05.

Table 3.2: Effects of full sun, neutral shade, canopy shade, and far-red light absorbing filter treatments on overall quality, erectness, leaf growth rate, and vertical growth rate of 4 PHYB1 transgenic, 1 NSC control, and 2 wild type control cultivars in 2005.

P35S Ract-Intron PHYB1 nos P35S Ract-Intron CP4 EPSPS nos

5 4.0 kb 3’

NotI BamHI SalI NotI

Figure 3.1: Schematic map of PHYB1 final construct

Figure 3.2A-H: Production of transgenic creeping bentgrass (Agrostis palustris) plants using biolistic bombardment of embryogenic callus cells. A Callus induction from mature caryopses of creeping bentgrass on callus induction medium. B Embryogenic callus maintained on fresh callus induction medium in dark. C Suspension cells of creeping bentgrass 1 d after particle bombardment. D Bombarded calli (left) and tissue culture control calli (right) of creeping bentgrass both subjected to selection with 2mM glyphosate in selection medium for 6-8 wk. E Shoot differentiation of glyphosate-resistant callus of creeping bentgrass 2 wk after transfer onto regeneration medium supplemented with 0.2 mM glyphosate. F Transgenic creeping bentgrass plantlets rooting on rooting medium supplemented with 0.2 mM glyphosate. G Tissue culture control creeping bentgrass plantlets rooting on rooting medium without glyphosate. H Transgenic (right) and tissue culture control (left) creeping bentgrass plants grown in metro-mix 3 mo after bombardment.

100

A B

C D

E F

A G H

101

Figure 3.3A-C LS44 (A), tissue culture control (B), and PB0701 (C) under canopy shade, neutral shade, full sun, and far red light (AFR) filter (left to right).

102

10 10 A B

8 8

6 6

4 4 Visual quality Visual quality Visual

2 2

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 10 10 C D 8 8

6 6

4 4 Visual quality Visual Visual quality

2 2

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 s

Visual quality ratings are based on 1-9 scale; 1 is poor density and uniformity, dormant/dead turf or bare soil, and coarse texture, 9 is ideal turf, dark green color, excellent density and uniformity, and fine texture; acceptable turf quality≥6.Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05).

Figure 3.4A-D: Visual quality ratings of wild type cultivars (Crenshaw and LS44), tissue culture control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) under A Neutral shade, B Canopy shade, C

Full sun, and D far-red light absorbing (AFR) filter.

103

10 10 A B 8 8

6 6

4 4 Erectness Erectness

2 2

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 10 10 C D 8 8

6 6 Erectness 4 4 Erectness

2 2

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701

Erectness is visual rating of angles between turf stand and the ground. It is based on 1-9 scale: 9, 90°; 1=0°. Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05).

Figure 3.5A-D: Erectness ratings of wild type cultivars (Crenshaw and LS44), tissue culture control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) under A Neutral shade, B Canopy shade, C

Full sun, and D far-red light absorbing (AFR) filter.

104

8 8 A B 6 6

4 4

2 2 Leaf rate (mm/day) growth Leaf Leaf growth growth rate (mm/day) Leaf

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 8 8 C D 6 6

4 4

2 2 Leaf growth rate (mm/day) Leaf Leaf growth rate (mm/day) Leaf

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Values are means of 15 numbers; 3 replicates averaged across 5 dates (6 June 2005, 21 June 2005, 30 June 2005, 8 July 2005, and 14 July 2005).

Figure 3.6A-D: Leaf growth rate(mm/day) of wild type cultivars (Crenshaw and LS44), tissue culture control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) under A Neutral shade, B Canopy shade, C Full sun, and D far-red light absorbing (AFR) filter.

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5 5 A B 4 4

3 3

2 2

1 1 Vertical growth rate (mm/day) rate Vertical growth Vertical growth rate (mm/day) rate Vertical growth

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701

5 5 C D 4 4

3 3

2 2

1 1 Vertical growth rate (mm/day) rate Vertical growth Vertical growth rate (mm/day) rate Vertical growth

0 0 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701 Crenshaw LS44 NSC PB0501 PB1101 PB1102 PB0701

Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, and 13 July 2005).

Figure 3.7A-D: Vertical growth rate(mm/day) of wild type cultivars (Crenshaw and LS44), tissue culture control (NSC), and transgenic lines (PB0501, PB1101, PB1102, PB0701) of creeping bentgrass (Agrostis palustris) under A Neutral shade, B Canopy shade, C Full sun, and D far-red light absorbing (AFR) filter.

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aw 01 02 01 01 sh 11 11 07 05 en 44 C kb PB PB PB PB Cr LS NS

0.68 PHYB1

0.5 Actin

Figure 3.8: Reverse-transcription (RT-PCR) of RNA samples from putative PHYB1 transformants (PB1101, PB1102, PB0701, PB0501), conventional control plants (Crenshaw and LS44), and tissue culture control plant (NSC). Arrows specify the expected GA2ox and Actin bands.

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REFERENCES

Bell, G., and K. Danneberger. 1999a. Managing creeping bentgrass in shade. Golf Course Management:56-60.

Bell, G.E., and T.K. Danneberger. 1999b. Temporal shade on creeping bentgrass turf. Crop Sci. 39:1142-1146.

Casal, J.J., and H. Smith. 1989. The function, action and adaptive significance of phytochrome in light-grown plants. Plant Cell Environ 12:855–862.

Casal, J.J., R.A. Sanchez, and D. Gibson. 1990. The significance of changes in the red/far-red ratio, associated with either neighbour plants or twilight, for tillering in Lolium multiflorum Lam. New Phytologist 116:565-572.

Chakraborty, N., J. Bae, S. Warnke, T. Chang, and G. Jung. 2005. Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L). Theor Appl Genet 111:795-803.

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Dai, W.D., S. Bonos, Z. Guo, W.A. Meyer, P.R. Day, and F.C. Belanger. 2003. Expression of pokeweed antiviral proteins in creeping bentgrass. Plant Cell Rep. 21:497–502.

Emmons, R. 2000. Turfgrass science and management. 3 ed. Delmar Publishers, Albany, NY.

Franklin, K.A., and G.C. Whitelam. 2005. Phytochromes and shade-avoidance responses in plants. Annals of Botany 96:169-175.

Fu, D., N.A. Tisserat, Y. Xiao, D. Settleb, S. Muthukrishnanc, and G.H. Liang. 2005. Overexpression of rice TLPD34 enhances dollar-spot resistance in transgenic bentgrass. Plant Sci. 168:671–680.

Gardner, D.S., T.K. Danneberger, E.K. Nelson, W. Meyer, and K. Plumley. 2003. Relative fitness of glyphosate-resistant creeping bentgrass lines in Kentucky bluegrass. HortScience 38:455 – 459.

Ge, Y., T. Narton, and Z.-Y. Wang. 2006. Transgenic zoysiagrass ( Zoysia japonica ) plants obtained by Agrobacterium -mediated transformation. Plant Cell Rep.

Gendreau, E., H. Höfte, O. Grandjean, S. Brown, and J. Traas. 1998. Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl. Plant J. 13:221-230.

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Gibson, D., J.J. Casal, and V.A. Deregibus. 1992. The effects of plant density on shoot and leaf lamina angles in Lolium multiflorum and Paspalum dilatum. Ann. Bot. 70:69-73.

Guo, Z., S. Bonos, W.A. Meyer, P.R. Day, and F.C. Belanger. 2003. Transgenic creeping bentgrass with delayed dollar spot symptoms. Molecular Breeding 11:95-101.

Halliday, K.J., B. Thomas, and G.C. Whitelam. 1997. Expression of heterologous phytochromes A, B or C in trangenic tobacco plants alters vegetative development and flowering time. Plant J. 12:1079-1090.

Kern, R., A. Gasch, M. Deak, S.A. Kay, and N.-H. Chua. 1993. phyB of tobacco, a new member of the phytochrome family. Plant Physiol. 102:1363-1364.

Lee, L., C.L. Laramore, P.R. Day, and N.E. Tumer. 1996. Transformation and regeneration of creeping bentgrass (Agrostis palustris Huds) protoplasts. Crop Sci. 36:401-406.

McCormac, A.C., M. Kobayashi, D. Wagner, M.T. Boylan, P.H. Quail, H. Smith, and G.C. Whitelam. 1993. Photoresponses of transgenic Arabidopsis seedlings expressing introduced phytochrome B-encoding dDNAs: Evidence that phytochrome A and phytochrome B have distinct photoregulatory functions. Plant J. 4:19-27.

Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15:473-497.

Neff, M.M., C. Fankhauser, and J. Chory. 2000. Light: an indicator of time and place. Genes and Dev. 14:257-271.

Quail, P.H. 1994. Phytochrome genes and their expression, p. 71–104, In R. E. Kendrick and G. H. M. Kronenberg, eds. Photomorphogenesis in plants, 2 ed. Kluwer, Dordrecht.

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Reicher, Z., and C. Throssell. 1998. Improving lawns in the shade. Purdue University Cooperative Extension Service.

Shirley, L.H. 1945. Light as an ecological factor and its measurement. Bot. Rev. 11:463-524.

Smith, H. 1982. Light quality, photoperception, and plant strategy. Ann. Rev. Plant Physiol. 33:481–518.

Somers DE, S.R., Tepperman JM, Quail PH. 1991. The hy3 long hypocotyl mutant of Arabidopsis is deficient in phytochrome B. Plant Cell 3:1263–1274.

Taiz, L., and E. Zeiger. 2002. p. 171-192 Plant physiology. Sinauer Associates, Inc., Sunderland, MA.

Tegg, R.S., and P.A. Lane. 2004. A comparison of the performance and growth of a range of turfgrass species under shade. Aust. J. Exp. Agric. 44:353-358.

Thiele, A., M. Herold, I. Lenk, P.H. Quail, and C. Gatz. 1999. Heterologous expression of Arabidopsis phytochrome B in transgenic potato influences photosynthetic performance and tuber development. Plant Physiol 120:73-81.

Vain, P., M. D., McMullen, and J.J. Finer. 1993. Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12:84-88.

Wagner, D., J.M. Tepperman, and P.H. Quail. 1991. Overexpression of Phytochrome B i a short hypocotyl phenotype in transgenic Arabidopsis. The Plant Cell 3:1275-1288.

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Wang, D., and D.S. Luthe. 2003. Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance. Plant Physiol 133:319-327.

Wang, Z.-Y., and Y. Ge. 2005a. Agrobacterium -mediated high efficiency transformation of tall fescue ( Festuca arundinacea Schreb.) J. Plant Physiol. 162:103 – 113.

Wang, Z.-Y., and Y. Ge. 2005b. Rapid and efficient production of transgenic bermudagrass and creeping bentgrass bypassing the callus formation phase. Funct. Plant Biol. 32:769–776.

Zheng, Z.-l., Z. Yang, J.-C. Jang, and J.D. Metzger. 2001. Modification of plant architecture in chrysanthemum by ectopic expression of the tobacco phytochrome B1 gene. J. Amer. Soc. Hort. Sci. 126:19-26.

Zhong, H., C. Srinivasan., and M.B. Sticklen. 1991. Plant regeneration via somatic enbryogenesis in creeping bentgrass (Agrostis palustris Hunds.). Plant Cell Rep. 10:453-456.

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CHAPTER 4

DEVELOPMENT AND CHARACTERIZATION OF TRANSGENIC CREEPING

BENTGRASS TRANSFORMED WITH ARABIDOPSIS BAS1 GENE

ABSTRACT

The purpose of the study was to develop transgenic creeping bentgrass

(Agrostis palustris) plants with dwarfism and good quality under low light conditions.

Arabidopsis BAS1 gene was inserted into creeping bentgrass by biolistic bombardment, with CP4EPSPS gene as the selectable marker. Fifteen transgenic plants were recovered following transformation and glyphosate selection. Tissue culture control (NSC) were regenerated from non-bombarded callus at the same time.

Together with wild type and NSC plants, six transgenic lines were multiplied and subjected to 4 light treatments: full sun (FS), canopy shade (CS), and neutral shade

(NS). Our results showed reduction in R:FR increased vertical growth and erectness of both transgenic and control plants, however, overexpression of BAS1 gene induced dwarfism in transgenic creeping bentgrass by delaying vertical shoot growth and altering shoot architecture. True BAS1 transformants (BS1305, BS1307, BS1701) had the best performance in most traits even under reduced PPF and R:FR.

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INTRODUCTION

Sunlight is not only the primary energy source for plants, but also as a stimulus to trigger photomorphogensis, series of developmental events including germination, deetiolation, leaf expansion, stem and petiole elongation, circadian clock, and transition from vegetative to reproductive growth (Nagy and Schäfer, 2002; Quail,

2002). Turfgrass performs best in full sun with a minimum of four to six hours of full sun per day(Reicher and Throssell, 1998). However, both photosynthetic photon flux(PPF)and red:far red ratio (R:FR) drop dramatically when the sunlight penetrates the tree canopies and reaches the surface of the turf. Shade stress causes increase of vertical growth and succession of harmful physiological and morphological changes in plants, such as thinner, more delicate leaves, reduced tillering, poor shoot density, altered pigment concentration and affected carbohydrate reserve (Bell and

Danneberger, 1999b). Creeping bentgrass usually has good shade tolerance compared to other turfgrass. However, when grown on golf courses, intensive mowing removes most of leaf surface area and reduces turfgrass photosynthesis and recuperative ability under low light conditions (Bell and Danneberger, 1999b). Besides, reduction of air flow under trees usually increases humidity and occurrence of turfgrass disease (Bell and Danneberger, 1999a).

Plant growth is regulated both by light and plant hormones (Azpiroz et al., 1998).

Brassinosteroids (BRs) are plant hormones that influence plant growth and development at extremely low concentration(Clouse and Sasse, 1998). Brassinolide

(BL), the most bioactive form of BR, was isolated and characterized as a plant steroid

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compound promoting cell division and plant growth (Grove et al., 1979). BR

biosynthesis mutants exhibit dwarf and de-etiolated phenotype of typical BR

deficiency (Bishop and Yokota, 2001). Additionally, activation-tagged Arabidopsis

bas1-D (phyB activation-tagged suppressor1-D) mutant exhibited dwarf phenotype,

contained no detectable BL, and accumulated biologically inactive

26-hydroxybrassinolide in feeding experiment. Genetic analysis showed that bas1-D

acted downstream of phyA and cry1, as a bypasssuppressor of phyB signalling. BAS1 gene was found to encode cytochrome P450 (CYP72B1) that inactivated BL by

C26-hydroxylation (Neff et al., 1999).

The purposes of this study were to develop transgenic Agrostis palustris

‘Crenshaw’ transformed with Arabidopsis BAS1 gene using biolistic bombardment, and to explore the phenotypic changes of the transgenic plants under altered PPF and

R:FR conditions.

MATERIALS AND METHODS

Callus induction and maintenance

Induction and maintenance of embryogenic callus cells were described previously

(Busov et al., 2003). Mature seeds (caryopses) of creeping bentgrass cultivar,

‘Crenshaw’, were surface sterilized in 70% ethanol for 1 min, followed by 30-min treatment of 50% commercial bleach and 0.1% Tween-20 under vacuum. Surface sterilized seeds were rinsed three times with sterile distilled water and then cultured on MSI medium (Lee et al., 1996; Zhong et al., 1991), MS basal medium supplemented with 500 mg L-1 Casein enzymatic hydrolysate (Sigma), 3% sucrose, 30

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μM 3,6-dichloro-o-anisic acid (dicamba), and 2.2 μM 6-benzylaminopurine (BA), solidified with Gel-GroTM (MP biomedical). After 4-5 wk at 25 ºC in the dark, calli were selected and transferred to new MSI media. Embryogenic calli were subcultured onto fresh media every 4-6 wk.

Transformation, selection, and regeneration

The transformation plasmid was generously provided by Dr. Lisa Lee (Scotts

Miracle-Gro Co., Marysville, OH). It contained the BAS1 gene coding sequence downstream of the cauliflower mosaic virus (CaMV) 35S promoter and rice ract1 intron, with the nos termination sequence. The final construct also included

CP4-EPSPS gene as the selective marker (Fig4.1). Embryogenic calli were placed on

9-cm filter disks (0.3g tissue per disk) in plates of MSI medium containing 0.3 M mannitol and 0.3 M sorbitol at least 4 hr prior to bombardment (Kern et al., 1993).

Plasmid DNA containing BAS1 gene construct was delivered into embryogenic creeping bentgrass callus cultures using the biolistic transformation system (Harriman et al., 2002; Lee et al., 2002; Vain et al., 1993). After 6-8 wk on selective MSI media containing 2mM glyphosate, embryogenic callus was regenerated on MSS medium,

MS medium including 3mg L-1 BA and 0.2 mM glyphosate. Appearing shoots were transferred to MSR medium, MS medium containing 0.2 mM glyphosate for rooting.

Finely rooted plants were grown in metro-mix 350 (Scotts Miracle-Gro Co.,

Marysville, OH) in the greenhouse for trait analysis. Simultaneously, control plants were recovered from non-bombarded embryogenic calli after culturing on the similar induction and differentiation media, but with the omission of glyphosate selection.

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Herbicide resistance of transformants was confirmed by spraying plants with 0.9g/m2

Roundup® herbicide (Scotts Miracle-Gro Co., Marysville, OH).

Greenhouse evaluation of transgenic, tissue culture control, and conventional control plants under various light conditions

A randomized split-plot study with 3 blocks was performed in the greenhouse at

The Ohio State University, Columbus, OH between May 30 and July 11, 2005. Three light treatments were assigned at random to the main plots within each block, including non-photoselective polyethylene film (full sun control), mature soybean plants that were 60-70 cm in height (canopy shade), and black shade cloth (neutral shade). Creeping bentgrass lines were assigned random to the subplots within each main plot. PPF was averaged over weekly readings taken during the study using a quantum light meter (Spectrum Technologies, Plainfield, IL). Red:far-red (R:FR) values were calculated from the weekly measurements at 660±5nm and 730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience, Lincoln, NE).

Plant material for the greenhouse study was established from stolon nodes of 6 primary transgenic events, one tissue culture control line, and 2 conventional lines

(Table 4.2) in 7-cm square plastic pots filled with metro-mix 350 (Scotts Miracle-Gro

Co., Marysville, OH). Throughout the trial, plants were kept at 21°C /16°C (day/night)

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under a 14/10-hr (day/night) photoperiod. Pots were weekly fertilized at a rate of

100ppm N with a 20N-10P2O5-20K2O soluble fertilizer (Scotts Miracle-Gro Co.,

Marysville, OH). Turf was irrigated twice daily (0900 and 1300 hr) with automatic

irrigation system. Fungicide and insecticide were applied monthly to prevent disease

and insect problems. All plants were trimmed weekly to the height of 2.5 cm with

clippings autoclaved and discarded.

Data collection

Overall quality ratings, erectness ratings, leaf extension rates, and vertical growth

rates were obtained weekly, starting from wk 3. Visual quality ratings were based on

1-9 scale; 1 was bare soil or dormant/dead turf with poor density and uniformity, as

well as coarse texture, 9 was ideal turf with dark green color, excellent density, good

uniformity, and fine texture; acceptable turf quality≥6. Erectness was visual rating of

angles between turf stand and the ground. It was based on 1-9 scale, 9= 90° branching

angle; 1=0°. All evaluations were conducted following NTEP turfgrass evaluation

guidelines. Leaf extension rate was defined as the difference of leaf length measured

24 and 36 hr after trimming. Vertical growth rate was determined by the increase of

representative plant height of each pot (height from soil level to plant apex) between

two clipping events, divided by number of days.

RNA isolation and RT-PCR analysis

Two-wk-old leaf tissue of transgenic and non-transgenic plants was collected fresh and snap frozen in liquid nitrogen. Total RNA was isolated from approximately

1.5g frozen tissue using TRIzol® reagent (Invitrogen, Carlsbad, CA) and further

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treated with DNaseI (Invitrogen, Carlsbad, CA) in accordance with the

manufacturer’s instructions. First strand cDNA was obtained through reverse

transcription of an aliquot of 1µg RNA from each sample using Oligo-dT primers

with the 2-step Reverse Transcription System (Promega, Madison, WI). An aliquot of

1µl cDNA out of each 20µl sample was analyzed by PCR using Taq DNA polymerase

(New England Biolabs, Ipswich, MA) and gene-specific primers. Primers for BAS1

were BAS1-F (5'-AGGTTCTTGTTCTGTCTGTAATC-3') and BAS1-R

(5'-TCGACCATCTTCATAGCTACTTC-3'), which generated a 641 bp DNA fragment.

PCR reaction was conducted under the following conditions: 94℃ for 5 min for one

cycle, followed by 94℃ for 30 sec, 55℃ for 40 sec, and 72℃ for 1 min for 30 cycles, and a final incubation at 72℃for 5 min. Furthermore, primers actin-5

(5'-GAGAAGATGACCCAGATCATGTTTG-3') and actin-3

(5'-TCCTAATATCCACGTCGCACTTCAT-3') were used to amplify the actin gene

(Wang and Luthe, 2003).

Data analysis

Data were analyzed with a PROC MIXED procedure of SAS (PC version 9.1, the

SAS Institute, Cary, NC). A base index weights each trait based on its economic importance, and is calculated by the following equation: I= a1P1+a2P2+…+anPn, with a representing the economic weight and P representing the standized phenotypic value. Economic weights were 3 for visual quality rating, -1 for vertical growth rate,

-1 for erectness rating, and -1 for leaf growth rate. LSMEANS and CONTRAST procedures were performed to test hypothesis of gene transformation, light effect, and

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their interaction.

RESULTS

Selection and regeneration of transgenic clones

Bombarded and non-bombarded calli were recovered on MSI medium for 1 wk in dark before they were subject to glyphosate selection. All calli were maintained on

MSI medium supplemented with 2mM glyphosate for 2 rounds of 3-wk selections.

Survived embryogenic callus were then plated on MSS medium with 0.2 mM glyphosate and 10 d later, green, glyphosate-resistant creeping bentgrass tissue appeared. Transgenic plantlets were smaller and had lower shoot and root growth than non-transgenic plantlets on rooting medium (Fig 4.2A). After being transferred to metro-mix, some transgenic plants were much more compacted and had more horizontal leaf orientation compared with the non-transgenic control plant (Fig 4.2B).

Fifteen glyphosate-resistant transgenic clones were recovered, named BS1101,

BS1102, BS0701, BS0703, BS1301, BS1302, BS1303, BS1304, BS1305, BS1306,

BS1307, BS1311, BS1701, BS0606, and BS0101. They were maintained under fluorescent light for 1 wk (Fig 4.2C) before moving to the greenhouse bench (Fig

4.2D). Due to unexpected drought accident in greenhouse, only 6 lines survived which were BS1302, BS1305, BS1307, BS1701, BS0606, and BS0101.

RT-PCR analysis

Transcription of the BAS1 gene in creeping bentgrass was studied by RT-PCR analysis of RNA samples isolated from glyphosate-resistant and control plants.

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Results indicated that the expected BAS1 band of the right size (641bp) appeared in glyphosate-resistant lines BS1305, BS1307, and BS1701, but not in BS0101, tissue culture control line (NSC), or the 2 conventional lines Crenshaw and LS44 (Fig 4.3).

Primers for actin were used as a positive control and its transcript was present in all samples (Fig 4.3).

Growth analysis under various light conditions

Visual quality

Visual quality ratings of plants grown in full sun were significantly higher than

those grown under shade conditions. R:FR change under low PPF conditions did not

significantly affect visual quality. Besides, visual quality did not differ significantly

between glyphosate-resistant and conventional plants, or between glyphosate-resistant

and NSC plants. However, visual quality varied significantly among

glyphosate-resistant plants with positive BAS1 transformants BS1307 and BS1701

rated the highest among all transgenic and nontrangenic plants, while negative

transformant BS0101 the lowest (Table 4.2).

Both changes in PPF and R:FR significantly affected erectness ratings of creeping

bentgrass plants. On average, plants had 32% and 21% higher erectness ratings in

canopy shade and neutral shade than in full sun, respectively. Furthermore, as group,

glyphosate-resistant plants tended to grow more horizontally than NSC or traditional

plants (Table 4.2). The ranking of erectness was

BS1305

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Plants grown in shade had significantly higher leaf growth rate than those grown

in full sun. However, change in R:FR did not have significant effect on leaf growth

rates of in low PPF (Table 4.2). Leaf growth rate was significantly different among all

lines, with BS0606 having the lowest rate and BS1305 the highest. As group, NSC

and conventional controls had lower leaf growth rates than most glyphosate resistant

lines (Table 4.2).

Vertical growth rates of creeping bentgrass plants were significantly lower in full

sun than in the shade. Additionally, decrease in R:FR significantly increased the

vertical growth under low PPF. Moreover, on average, glyphosate-resistant plants

had significantly lower vertical growth rates than either NSC or conventional plants.

For example, vertical growth rates of Crenshaw, LS44, and NSC were 10-30% and

30-80% higher than BS1305, BS1307, and BS1701 under neutral shade and canopy

shade, respectively (Fig 4.4). While in full sun, vertical growth rates of control plants

were either equal to or slightly different from glyphosate-resistant plants (Fig 4.4).

In a word, true BAS1 transformants (BS1305, BS1307, BS1701) had the best

performance in most traits even under reduced PPF and R:FR.

DISCUSSION

Gene transformation has been successfully performed in several Agrostis species, including Agrostis alba (Asano and Ugaki, 1994), Agrostis stolonifera (Asano et al.,

1998; Chai et al., 2002; Dai et al., 2003; Dalton et al., 1998; Fu et al., 2005; Guo et al.,

2003; Han et al., 2005; Hartman et al., 1994; Lee et al., 1996; Luo et al., 2004; Wang and Ge, 2005a; Xiao and Ha, 1997; Yu et al., 2000; Zhong et al., 1994), and Agrostis

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tenuis (Chai et al., 2004). Genetic engineering has been utilized to develop traits of

interest in bentgrass such as disease resistance (Chai et al., 2002; Dai et al., 2003; Fu

et al., 2005; Guo et al., 2003) and herbicide resistance (Gardner et al., 2004; Gardner

et al., 2003). Our studies expanded the usage of biotechnology in creeping bentgrass

by inserting Arabisopsis BAS1 gene which was reported to alter endogenous bioactive

BR level and affect photomorphogenesis (Neff et al., 1999).

During regeneration process, false plantlets appeared on shoot regeneration medium. They were characterized by light green or bleached leaf color. Besides, they stopped growing when subject to extended glyphosate selection. True transgenic lines survived glyphosate selection and were regenerated eventually. The whole process of callus induction and plant regeneration from the induced callus are long and tough. It took about 8 months to finish one round of bombardment experiment. A recently developed callus-free transformation system using stolon nodes only takes one-third of the time required for other reported transformation systems (Ge et al., 2006; Wang and Ge, 2005a; Wang and Ge, 2005b). We may consider using it as an alternative method in future studies.

Results from this study shows that shading adversely promotes vertical shoot growth and affects turfgrass visual quality and color, which is similar to the previous findings (Tegg and Lane, 2004). Choosing shade tolerant creeping bentgrass is critical

in turfgrass management under shade. Our results show that overexpression of BAS1

gene induces dwarfism in positive transformants by delaying vertical shoot growth

and altering shoot architecture. Early studies displayed that BR counteracted the effect

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of light on stem elongation and morphogenesis (Krizek and Mandava, 1983). When grown in the dark, many brassinosteroid mutant seedlings showed light grown phenotype of short hypocotyls with developing cotyledons, while adults exhibited dwarfism with dark green epinastic leaves, short stems and petioles, and delayed senescence (Li and Chory, 1999).

Follow-up work for this studies would include four major parts: first, due to somaclonal variation, more transgenic plants need to be regenerated and screened to select the economically important cultivars (Sharp et al., 2002); second, further detailed molecular analysis should be done to characterize the BAS1 true transformants; third, homozygous descendents of BAS1 transformants need to be made through breeding programs; fourth, growth response is a complicated process and interaction between multiple photoreceptors and BR effect needs to be further explored (Neff et al., 1999).

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Percentage of FS Percentage of FS Treatment (instant PPF) † (R:FR) ‡

Non-selective cover (full sun control) 100 100 Canopy shade (CS) 16 53 Neutral shade (NS) 26 99 †Instant PPF(mol m-2s-1) was averaged over weekly readings taken using a quantum light meter (Spectrum Technologies, Plainfield, IL) around 2pm during the study from May 30 through July 11, 2005. ‡Red:far-red (R:FR) values were calculated from the weekly measurements at 660±5nm and 730±5nm, respectively using LI-1800 spectroradiometer (LI-COR Bioscience, Lincoln, NE) around 2pm during the study from May 30 through July 11, 2005.

Table 4.1: Instant photosynthetic photon flux and red:far-red ratio of the three light treatments.

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Leaf growth rate Vertical growth Visual rating† Erectness‡ (mm/day)§ rate(mm/day)¶ Cultivar Glyphosate-resistant lines(BAS1)# BS1302 5.9 5.3 4.4 2.9 BS1305 5.9 4.6 5.2 2.5 BS1307 6.2 5.8 4.6 2.5 BS1701 6.1 6.0 3.9 2.7 BS0606 6.0 6.3 3.2 2.6 BS0101 5.4 7.6 3.6 3.6 Conventional wild type control(WT)†† Crenshaw 5.7 7.0 3.5 2.9 LS44 5.8 7.1 3.8 3.2 Tissue culture control(NSC)‡‡ NSC 5.8 7.9 3.4 3.4 Light treatment Neutral 5.8 6.6 4.0 3.0 Canopy 5.7 7.2 4.4 3.4 Full sun 6.1 5.4 3.4 2.4 Analysis of variance Source Pr > F Pr > F Pr>F Pr>F Cultivar NS *** ** *** Among BAS1 * *** *** *** BAS1 vs. NSC NS *** * *** BAS1 vs. WT NS *** * ** Treatment *** *** ** *** Neutral vs. Canopy NS * NS *** Shade vs. Full sun *** *** ** *** Cultivar×Treatment NS NS NS ** †Visual quality ratings are based on 1-9 scale; 1 is poor density and uniformity, dormant/dead turf or bare soil, and coarse texture, 9 is ideal turf, dark green color, excellent density and uniformity, and fine texture; acceptable turf quality≥6. Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05). ‡Erectness is visual rating of angles between turf stand and the ground. It is based on 1-9 scale: 9, 90°; 1=0°. Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, 13 July 05). §Values are means of 15 numbers; 3 replicates averaged across 5 dates (6 June 2005, 21 June 2005, 30 June 2005, 8 July 2005, and 14 July 2005). ¶Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, and 13 July 2005). #Transgenic plants were regenerated from embryogenic callus following bombardment with plasmid DNA containing BAS1 gene coding sequence. ††Conventional controls are non-transgenic creeping bentgrass plants obtained from seeds. ‡‡Tissue culture control (NSC) plants were regenerated from un-bombarded callus through tissue culture.

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*, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively. NS, not significant at P<0.05.

Table 4.2: Visual rating and erectness of 9 creeping bentgrass cultivars in the greenhouse.

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3’ 5’ P35S Ract-Intron BAS11.5 kb nos A

Figure 4.1: Schematic map of BAS1 gene expression cassette. Final construct also contained CP4-EPSPS gene as the selective marker. 128

A B

C D

Figure 4.2A-D: Production of transgenic creeping bentgrass (Agrostis palustris) plants using biolistic bombardment of embryogenic callus cells. A Shoot differentiation of glyphosate-resistant callus of creeping bentgrass 2 wk after transfer onto regeneration medium supplemented with 0.2 mM glyphosate (right) and tissue culture control(NSC) callus on regeneration medium without glyphosate. B Transgenic (left and right) and NSC (middle) creeping bentgrass plants grown in metro-mix 3 mo after bombardment. C. Transgenic and NSC creeping bentgrass plants moved to metro-mix from rooting medium. D. Transgenic and NSC plants grown in the greenhouse.

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Transgenic glyphosate resistant plants BS1305 BS1307 BS1701 BS0101 Crenshaw LS44 NSC

0.6 BAS1

0.5 Actin

Figure 4.3: Reverse-transcription (RT-PCR) of RNA samples from transgenic glyphosate resistant plants, conventional control plants (Crenshaw and LS44), and tissue culture control plants (NSC). Arrows specify the expected BAS1 and Actin bands.

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Neutral 5 Canopy Full sun

2 Vertical growth rate(mm/day) Vertical 1

0 BS1305 BS1307 BS1701 BS0101 Crenshaw LS44 NSC

Values are means of 15 numbers; 3 replicates averaged across 5 dates (13 June 2005, 20 June 2005, 29 June 2005, 7 July 2005, and 13 July 2005).

Figure 4.4: Vertical growth rate(mm/day) of transgenic lines (BS1305, BS1307, BS1701, BS0101), conventional cultivars (Crenshaw and LS44), tissue culture control (NSC) plants of creeping bentgrass (Agrostis palustris) under neutral shade, canopy shade, and full sun.

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