Transgenics for

improved quality
traits in turf grass

Pavani.U
RHD/09-
02
 Introduction
• Breeding for pest and disease resistance, greater adaptability
to environmental conditions – objectives of turfgrass
breeders.

• Genetic control of these traits not clear.

• Absence of genetic recombination in apomictic types.

• Genetic transformation has become important strategy.

• Generation of transgenic plants, coupled with breeding .

 Introduction contd..
• Transgenic approaches - accelerate conventional breeding,
-offer the opportunity to generate unique genetic variation.

• Resistance to the herbicide glufosinate in creeping bentgrass

– first trait used for genetic engineering.

• First - by direct gene transfer to protoplasts.

• Protoplast-independent transformation techniques,

microprojectile bombardment and Agrobacterium-mediated
transformation.
Potential applications of transformation technology
I. Genetic engineering for low mow grass

II. Genetic engineering for biotic stress

A. Genetic engineering for weed control
B. Genetic engineering for insect resistance
C. Genetic engineering for disease resistance

III. Genetic Engineering for abiotic Stress Tolerance

A. Freezing
B. Heat tolerance
C. Salt and drought tolerance
D. Shade tolerance
E. Herbicide tolerance
 Potential applications of transformation technology contd..

• IV. Genetic Engineering for other quality traits

A. Self-elimination by pro-herbicide
B. Genetic manipulation of flowering time
C. Improvement of phosphorus uptake and utilization
D. Development of Hypo-Allergic Grasses
E. Grasses as bioreactors
 I. Genetic engineering for low mow grass

• Howard Hughes Medica Institute Scientists deciphered the

brasinosteroid signaling path way - regulates growth and
development in plants.

• By manipulating the steroid path way, regulation of grass

growth may be possible.

• It can be achieved in one of three ways viz., i) modifying

the levels of hormone ii) manipulating the chemical
structures of hormones and iii) recording the signals sent
along the pathway.
 II. Genetic engineering for biotic stress
A. Weed control:
• Genomics has been used in studying weed invasions and
hybridization between different species.

• Used to identify the genes that would weaken the weeds

competitive ability.

• Isolated genes - introduced into an out crossing weeds - result

in a less fit weed type.

• Resource utilization of crops - allow the development of crops

that produce allelochemical compounds for natural weed
control.
 II. Genetic engineering for biotic stress
contd..
B. Insect resistance:

• Traditionally - using Integrated Pest Management

• Indiscriminate use of pesticides - development of insect

resistance and the elimination of other beneficial insects.

• Transgenic crops expressing Bacillus thuringiensis (Bt) δ-

endotoxins - natural choice.
 Insect resistance contd…

 Introduction of cry1Fa gene in bahiagrass:

Gabriela Luciani et al. (2007)
• Bahia grass - low maintenance requirements and tolerance to
drought, heat, many diseases, and overgrazing.

• susceptible to : mole crickets (Scapteriscus spp.) and fall

armyworm (Spodoptera frugiperda).

• Stable expression of minimal synthetic cry1Fa expression

constructs in bahiagrass conferred resistance to the difficult to
control, and important insect pest, fall armyworm.
 Insect resistance contd…

 Resistance against certain coleopteran pests:

• A transgenic grass was engineered with the cry8Da gene from
B. thuringiensis strain called SDS-502.

• The cry8Da gene was cloned in a vector, pBI221.

• GFP was used as an indicator of transformed grass cells.

• The transgenic grass containing the cry8Da gene showed

strong resistance against the feeding attack by the Japanese
beetle.
II. Genetic engineering for biotic stress contd..
C. Disease resistance:

• The major infectious diseases in turf grasses are caused by

fungi.

• Transgenic creeping bentgrass plants expressing a receptor

protein kinase gene, PR5K, from Arabidopsis thaliana
delayed disease expression by 29 to 45 days - dollar spot
(Sclerotinia homoeocarpa).

• PR5K transcripts accumulate in the extracellular spaces of

plants challenged with pathogenic microorganisms and
acidifies the tissue.
 Disease resistance contd…

• The rice thaumatin-like protein (TLPD34) gene was

introduced into creeping bentgrass cv. Crenshaw.

• Thaumatin-like proteins (TLPs) - pathogenesis-related (PR)

proteins of group 5 - involved in plant defense mechanisms.

• Field tests demonstrated that transgenic lines displayed

improved resistance to dollar spot.

• No improvement against Rhizoctonia solani, the causative

agent of brown patch disease
 Disease resistance contd…

• Transgenic creeping bentgrass expressing a chitinase-like

gene from American elm showed significantly increased
resistance to brown patch disease in greenhouse trials.

• The introduction of a rice chitinase gene into Italian

ryegrass resulted in increased resistance to crown rust
disease in studies using in vitro inoculation of detached
leaves.
 Disease resistance contd…

 Pokeweed antiviral protein:

• PAP - ribosome-inactivating protein from the plant Phytolacca

americana. - inactivates ribosomes by inhibiting translation.

• Scientists have generated transgenic creeping bentgrass plants

expressing three forms of ribosome- inactivating proteins viz.,
PAP-Y and PAP-C and PAPII.

• PAPII transformants could accumulate stable levels of the

protein- protected from infection by - Sclerotinia homoeocarpa
II. Genetic engineering for biotic stress contd..
D. Virus resistance:

• The potential of RNA-mediated virus resistance was explored by

introducing a ryegrass mosaic virus (RgMV) coat protein
(RgMV-CP) gene I.

• Resistance against high-dose virion inocula was shown in primary

transformants nto perennial ryegrass.

• The underlying resistance mechanism is associated with targeted

degradation of homologous virus RNA, commonly described as
post-transcriptional gene silencing (PTGS).
 
 Virus resistance contd…
 Bacterio-opsin:
• Bacterio-opsin is a proton pump protein from the bacterium
Halobacterium halobium.

• Transgenic plants expressing bacterio-opsin - block the

replication of tobacco mosaic virus and the growth of the
bacterial pathogen Pseudomonas syringae pv tabaci.

• Expression of bacterio-opsin activates the plant defence

mechanisms resulting in constitutive expression of
pathogenesis-related proteins.
 III. Genetic engineering for abiotic stress
tolerance
• Abiotic stresses- temperatures and supplies of water and
inorganic solutes, frequently limit growth and productivity
of forage and turf.

A. Freezing tolerance:
• Transgenic ryegrass plants with altered fructan metabolism
brought about by the expression of chimeric levansucrase
genes were generated.

• The expression of sacB genes in transgenic ryegrass and the

concomitant accumulation of low levels of bacterial levan -
distorted the native grass fructan synthesis pattern.
 Freezing tolerance contd..

• Transgenic perennial ryegrass plants - with wheat

fructosyltransferase genes, wft1 and wft2, encoding 6-SFT
and 1-SST, respectively, under the control of CaMV 35S
promoter.

• Significant increases in both fructan content and freezing

tolerance at the cellular level were detected in the transgenic
perennial ryegrass plants.

• Thus transgenic over expression of fructan genes may serve

as a strategy to produce freezing-tolerant grasses.
 III. Genetic engineering for abiotic stress
tolerance contd..
B. Heat tolerance with ipt gene: Xing et al. (2007)
• Creeping bent grass (Agrostis palustris) is widely used on greens but
it is not very heat tolerant.
• Heat stress results in plant decline (Summer Bent grass Decline).
• Cytokinins may be the key in controlling SBD.

• Two types of transgenic bent grasses

i) Bacterial cytokinin synthesis gene ipt controlled by promoter -
activated at the start of leaf senescence (pSAG12-ipt)
ii) Heat shock promoter (pHSP 18-ipt) that is activated by exposure to
temperatures of > 35°C.
• pSAG12-ipt plants produced more cytokinins in response to heat
stress
 III. Genetic engineering for abiotic stress
tolerance contd..
C. Salt and drought tolerance:

• Traditional breeding - a hybrid between Lolium multiflorum

and F. glaucescens.

• Salt and drought tolerance in Kentucky blue grass and

Bermuda grass - enhanced by introducing the gene encoding
Betaine Aldehyde Dehydrogenase.

• Under osmotic stress, betaine aldehyde dehydrogenase

oxidises glycine betaine aldehyde into the osmoprotectant
glycine betaine
 Salt and drought tolerance contd…
 Expression of antiporter genes to improve salinity tolerance:

• Salt tolerant plants - compartmentation of Na+ into vacuoles,

through Na+/H+ antiport, provides an efficient mechanism to
avert the deleterious effects of Na+ in the cytosol.
• A vacuolar membrane Na+/H+ antiporter gene was cloned
from rice and introduced into perennial ryegrass.
• The leaves of transgenic ryegrass plants accumulated higher
concentrations of Na+, K+ and proline than those of the
control plants.
• The transgenic plants displayed significantly enhanced salt
tolerance
 Salt and drought tolerance contd…

 Mannitol-1 -phosphate dehydrogenase: Zhang and Mian 2006

• Accumulation of mannitol has been associated with protection

of plant cells from osmotic stress.

• Scientists at Rutgers University are currently constructing an

expression vector for creeping bent.

• However, transgenic creeping bentgrass plants carrying mt1D

gene showed no difference in their drought and salt tolerance
with the controls.
 III. Genetic engineering for abiotic stress
tolerance contd..
D. Shade tolerance: Pill-Soon Song 2007
• Lawn grass grows in compact situation-this dense growth
creates shadow on its neighbors and triggers the grass to grow
vertically as fast as it can, to avoid shade.

• Leads to frequent mowing and less chlorophyll development.

• If grasses are made tolerant to shade instead of avoiding the

shade, they remain green and can absorb and use shade light.

• This can be done by chemically and genetically modifying the

plant’s phytohormones.
 Shade tolerance contd..

 Transgenic Zoysia grass with reduced shade avoidance:

• The phytochrome absorbs 660 nm but not 730 nm of shade
light.

• If length absorption of phytochrome is changed towards the

shade light - effectively utilize the shade light and grow
slower.

• Transgenic zoysiagrass transformed with a modified oat

phytochrome A gene (S598A) in which the serine-598 codon
was replaced with an alanine codon -significantly suppressed
shade avoidance reactions.
 III. Genetic engineering for abiotic stress
tolerance contd..
• E. Genetic engineering for herbicide resistance:

• Herbicide resistance may be used as a tool for controlling

weeds, thus allowing easier maintenance of golf courses and
lawns.

• Genetically transformed cultivars of creeping bentgrass

(Agrostis palustris) - resistant to glyphosate have been
developed by a collaboration of Scotts and Monsanto
companies.

• Glyphosate - inhibits the enzyme 5- enolpyruvylshikimate-3-

phosphate synthase (EPSPS).
• Genetic engineering for herbicide resistance contd..

• ESPS provides substrate for biosynthetic path ways for

aromatic amino acids, vitamins and many secondary
metabolites.

• The transgenic bent grass was developed by the insertion of a

bacterial EPSPS gene - derived from the soil bacterium
Agrobacterium sp. strain CP4.

• It is tolerant to glyphosate and tightly binds to PEP.

 Genetic engineering for herbicide resistance contd..

 Glufosinate-Resistant Creeping Bentgrass

Lisa Lee et al. (1995)

• Glupfosinate inhibit the enzyme glutamine synthetase, causing

rapid accumulation of ammonia and cell death.

• The bar gene encodes an enzyme phosphinothricin N-

acetyltransferase (PAT) that inactivates the active ingredient of
the herbicide PPT

• Thus, transformed plants that carry the bar gene are resistant.
 Genetic engineering for herbicide resistance contd..
 Apomictic bahia grass expressing bar gene:
Sandhu et al. (2007)
• The bar gene was originally cloned from Streptomyces
hygroscopicus - a self protecting mechanisms to avoid the
toxicity of their own products.
• Bialaphos is commercially produced from S. hygroscopicus,
which is being used in agriculture as a non-selective
herbicide.
• If bar is introduced and used as a resistance gene in crop
plants, bialaphos could be used more selectively in the field.
• Transgenic bahia grass cv. Argentine with bar gene resistant
to herbicide bialophos was generated.
 IV. Genetic engineering for other quality traits
A. Self-elimination by pro-herbicide:

• The selective elimination of a grass species in sports fields .

• A major problem of overseeding bermudagrass with perennial
ryegrass is the ability of the ryegrass to persist after the
bermudagrass turns green.
• The E. coli arg E gene which encodes N-acetylornithinase was
introduced into perennial ryegrass.
• The enzyme is able to de-acetylate N-acetyl-Lphosphinothricin,
a non-toxic compound, to produce PPT.
• Perennial ryegrass plants expressing the arg E transgene were
selectively controlled upon application of a proherbicide, N-
acetyl-PPT.
 IV. Genetic engineering for other quality traits
contd…
B. Genetic manipulation of flowering time: Wang and Ge 2006

• In turf applications, flowering increases mowing

requirements and decreases turf quality.

• Flowering perennial grasses also produce pollen that in

certain instances can cause allergic reactions in humans.

• Inhibition of floral development was achieved in red fescue by

transgenically expressing a strong floral repressor TERMINAL
FLOWR1 (LpTFL1) isolated from perennial ryegrass.
 IV. Genetic engineering for other quality traits
contd…
• C. Improvement of phosphorus uptake and utilization:
Wang and Ge 2006
• Phosphorus fertilization can result in improved productivity
of forages and decrease the risk of grass tetany.

• Ectopic expression of a fungal phytase gene or a fungal-based

synthetic phytase gene resulted in increased P acquisition
and biomass production in transgenic Arabidopsis and
potato.

• Transgenic expression of phytase genes of plant origin has

great potential for improving plant phosphorus acquisition.
 IV. Genetic engineering for other quality traits
contd…
D. Development of Hypo-Allergic Grasses:

• Ryegrass (Lolium species) pollen is a major source of

airborne allergens and causes hayfever and seasonal allergic
asthma.
• The cDNAs for the major ryegrass pollen allergens Lol p 1,
Lol p 2 and Lol p 5 have been isolated.
• The antisense Lol p 1 and Lol p 2 transgene under the control
of a pollen-specific promoter were introduced into Italian
and perennial ryegrasses.
• The transgenic ryegrass plants showed a reduction in
accumulation levels of Lol p 1 and Lol p 2 allergens in pollen.
 Risk assessment.
• Due to the outcrossing nature of major forage and turf
grasses, field release of transgenics has become very
complicated for these species.

• To date, no transgenic forage or turf grasses have been

approved for commercialization.

• The threat of spreading herbicide resistance into weedy grass

species is a real possibility and one that could have significant
effects on agriculture and the environment.

• In a study of EPA, genetically engineered bent grass

pollinated test plants – 13 miles down wing from a test farm.
 Total transgene containment

• The strategy is simply to prevent flower formation in plants

that are released into the field.

• The mechanism makes use of a site-specific recombinase, in

this case the FLP/FRT system from yeast, to activate a gene
designed to down-regulate a critical gene in the initiation of
floral development.

• The targeted gene for down regulation is FLORICAULA/

LEAFY, which regulates the vegetative to reproductive
developmental transition of meristems.
 Total transgene containment contd…

• The mechanism operates by establishing a transgenic line

homozygous for both the transgene of interest and a genetic
construct containing the following linked elements:

• A constitutive plant promoter – an FRT site (recognition site

for FLP) – a blocking sequence – RNAi or antisense
construction for FLORICAULA/LEAFY.

• In the final seed production cycle, homozygous plants are

crossed to plants homozygous for a constitutively expressed
FLP gene to produce hybrid seed.
 Total transgene containment contd…

• When grown the hybrid seeds will generate plants that

constitutively express FLP resulting in the excision of the
blocking sequence contained in the initial construct.

• This will activate the constitutive expression of the RNAi or

antisense construction for FLORICAULA/LEAFY.

• This in turn will down regulate the expression of the

endogenous FLORICAULA/LEAFY genes rendering the plant
incapable of producing flowers.
 Conclusions
• The benefits that can come from this science is seemingly
endless:

• That means landscapers, sports field managers and golf course

superintendents can have grasses that are herbicide-resistant,
tolerant of drought, stress, salt and insects, grasses that have
better growth habits and color.

• The possibilities truly are endless.

• Thus, biotechnology may soon have a positive environmental
and economic impact on the turf and ornamental industry as
well, assuming successful completion of the regulatory process.