Lecture 19 - Crop, Soil, and Environmental Sciences

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Dec 12, 2012 (4 years and 11 months ago)

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Molecular strategies for gene containment in transgenic crops


Nature Biotechnology


20
, 581
-

586 (2002)

The potential of genetically modified (GM) crops to transfer foreign genes
through pollen to related plant species has been cited as an environmental
concern. Until more is known concerning the environmental impact of novel
genes on indigenous crops and weeds, practical and regulatory considerations
will likely require the adoption of gene
-
containment approaches for future
generations of GM crops. Most molecular approaches with potential for
controlling gene flow among crops and weeds have thus far focused on maternal
inheritance, male sterility, and seed sterility. Several other containment strategies
may also prove useful in restricting gene flow, including apomixis (vegetative
propagation and asexual seed formation), cleistogamy (self
-
fertilization without
opening of the flower), genome incompatibility, chemical induction/deletion of
transgenes, fruit
-
specific excision of transgenes, and transgenic mitigation
(transgenes that compromise fitness in the hybrid). As yet, however, no strategy
has proved broadly applicable to all crop species, and a combination of
approaches may prove most effective for engineering the next generation of GM
crops.

Nature Biotechnology


20
, 581
-

586 (2002)

Maternal Inheritance

Cytoplasmic organelles (plastids and mitochondria) are inherited
maternally in plants and animals. In majority of angiosperms,
chloroplasts are inherited maternally. However, there are
exceptions: e.g. tobacco could have 0.1
-

0.5% pollen
-
mediated
transfer of chloroplast encoded traits.


Triazine resistance conferred by mutant chloroplast gene (
psbA
)
was found to be inherited paternally at the rate of <0.5%.

Paternal inheritance of chloroplast encoded traits has been noted in
a few higher plants

Efficacy of plastid transformation technology in gene containment

Nature Biotechnology


17
, 390
-

392 (1999)

Low probability of chloroplast movement from oilseed rape (
Brassica napus)
into
wild
Brassica rapa

Susan E. Scott &

Mike J. Wilkinson

Department of Agricultural Botany, School of Plant Sciences, The University of Reading

We assess the probability of chloroplast exchange between conventional oilseed rape and
wild
Brassica rapa

to model the future behavior of transplastomic cultivars. Primers
specific to cpDNA were used to demonstrate maternal inheritance of chloroplasts in 47
natural hybrids between cultivated
B. napus

and wild
B. rapa
. We conclude that there
will be no or negligible pollen
-
mediated chloroplast dispersal from oilseed rape.
Transgene introgression could still occur in mixed populations, however, if
B. napus

acted
as the recurrent female parent. Rate of transfer would then depend on the abundance of
mixed populations, their persistence as mixtures, and hybridization frequency within
stands. A low incidence of sympatry (0.6−0.7%) between wild
B. rapa

and cultivated
B.
napus

along the river Thames, UK, in 1997 and 1998, suggests mixed stands will form
only rarely. Eighteen feral populations of
B. napus

also showed a strong tendency toward
rapid decline in plant number, seed return, and ultimately, extinction within 3 years.
Conversely, hybrid production is significant in mixed stands, and the absence of control
practices means that oilseed rape will have slightly greater persistence. We infer that
some introgression from transplastomic
B. napus

into
B. rapa

is inevitable in mixed
populations even though such populations will occur infrequently and will tend to lose
B.
napus

plants relatively quickly. Chloroplast exchange will be extremely rare and
scattered


Male sterility

Disrupt pollen development by expressing a lethal gene in pollen.

Promoter of a tapetum specific gene (TA29) was used to drive the

expression of RNase T1 gene, which disrupted pollen formation.


Barstar Barnase system

Barnase (110 amino acids) is a secreted ribonuclease from
Bacillus
amyloliquefaciens
. Barstar (89 amino acids) is a cytoplasmic
barnase inhibitor with which the host protects itself. RNase is linked
with
bar

gene (glufosinate tolerant), so glufosinate tolerant plant
will be male sterile.


GM canola containing barstar/ barnase system composes about 10%
of commercially cultivated crops in Canada and is one of the few
GMO cleared for agricultural use in Europe.

Induction of male sterility in plants by a chimaeric ribonuclease gene

Nature

347
, 737
-
741 (25 October 1990)

Chimaeric ribonuclease genes that are expressed in the anthers of transformed tobacco and
oilseed rape plants were constructed. Chimaeric ribonuclease gene expression within the
anther selectively destroys the tapetal cell layer that surrounds the pollen sac, prevents
pollen formation, and leads to male sterility. These nuclear male sterility genes should
facilitate the production of hybrid seed in various crop plants.

Isoltaion of a tapetum
-
specific gene, TA29, facilitated tapetum
specific expression of RNase gene

Tetrad stage: maturation of microspores depends on tapetal cells

SMC= spore mother cells

V= vascular bundle

Fi= filament

w= anther inner wall

t= tapetum

A Nove1 Cell Ablation Strategy Blocks Tobacco Anther Dehiscence

TA56 gene is stomium specific

Lectin gene is active in many vegetative and floral tissues.

Ablation of the Stomium (St) Region Leads to Anthers That

Fail to Dehisce

Cell Ablation Strategy Using Chimeric
Barnase
and
Barstar
Genes with Overlapping Cell Specificities.

Blocks represent cross
-
sections through a hypothetical organ system that has four different cell types. The
circular cells in the lower right quadrants are the targets of the ablation experiment.

(A)
Blue represents transcriptional activity of the promoter (lectin gene) fused with the anti
-
cytotoxic
barstar
gene.

(B)
Red represents transcriptional activity of the promoter (TA56 gene) fused with the cytotoxic
barnase
gene.

(C)
Combined transcriptional activities of the chimeric
barnase
and
barstar
genes. Both chimeric genes are
active within the dark gray cells in the upper right quadrant. Only the chimeric
barnase
gene is active in the
target cells present in the lower right quadrant.

(D)
Selective ablation of the target cells. Barnase/barstar complexes are formed within the dark gray cells in
the upper right quadrant protecting them from the cytotoxic effects of barnase. The target cells in the lower
right quadrant have been ablated selectively due to the cell
-
specific activity

A Nove1 Cell Ablation Strategy Blocks Tobacco Anther Dehiscence

Seed sterility

Genes involved in seed development may be used to deliver toxicity to seeds

A. Terminator technology:

Not published in scientific literature. Patented by USDA and licensed by Monsanto.



Seeds are treated with tetracycline before sale.

Following components have been described:

1.
Ribosomal inhibitor protein (RIP) gene under the control


of
LEA

promoter. LEA (Late Embryonic Abundance) is a seed
-
specific gene.

2.

A
loxP

flanked spacer between
LEA

promoter and the
RIP

ORF.

3.

Cre expression controlled by a “Tet
-
on” system.


B. Hormone manipulation

LEA

promoter controls
iaaH

gene.

The “Tet
-
on” system

In
E. coli
, the Tet repressor protein
(TetR) negatively regulates the genes
of the tetracycline (Tc)
-
resistance
operon on the Tn
10
transposon. TetR
blocks transcription of these genes by
binding to the tet operator sequences
(
tetO
) in the absence of Tc. TetR and
tetO
provide the basis of regulation
and induction for use in mammalian
experimental systems.

Promoter

tetO

TetR

No transcription

Tc

Tc captures TetR (promoting transcription)

Cleistogamy and Apomixis

Cleistogamy
: occurrence of self
-
pollination and fertilization with the

flowers remaining unopened. (soybean, rice, wheat)


Apomixis
: Formation of seeds without sexual pollination. Seeds are

vegetative. (
Pennisetum

and
Panicum
produce apomictic embryos)
.


Better understanding of these processes and isolation of the genes

involved may lead to engineering cleistogamy and apomixis for gene

containment in crops plants.

Apomixis







Copyright ©2001 American Society of Plant Biologists

Grossniklaus, U., et al. Plant Cell 2001;13:1491
-
1498

Scheme of Sexual and Apomictic Reproduction

Chasmogamous

(wild
-
type)

cleistogamous

cleistogamous

Chasmogamous

(wild
-
type)

cleistogamous

japonica

indica background

Genome incompatibility

Many cultivated crops have multiple genomes. Only one of these
genomes is compatible for interspecific hybridization with weeds. For
eaxmple, the D genome of wheat is compatible with the D genome of
Aegilops cylindrica
(goatgrass). Therefore it is much harder to produce
inter
-
specific hybrid between durum wheat (AABB) and goatgrass.


Similarly there is a possibility for gene transfer from the B genome of
Brassica juncea

to many
Brassica

weeds, which carry B genome.
However, so far most of the genetic engineering is done on
B. napus

which contains AACC genome.

Theor Appl Genet (1997) 95 : 442
-
450

The impact on biosafety of the phosphinothricin
-
tolerance transgene

in inter
-
specific
B. rapa

X
B. napus

hybrids and their successive backcrosses

The present study investigated the transfer of a phosphinothricin
-
tolerance transgene by inter
-
specific hybridization
between
B
.
rapa
and two transgenic
B
.
napus
lines. The expression of the transgene was monitored in the F1 hybrids
and in subsequent backcross generations. The transgene was transmitted relatively easily into the F1 hybrids and
retained activity. Large differences in the transmission frequency of the transgene were noted between offspring of the
two transgenic lines during backcrossing. The most plausible explanation of these results is that the line showing least
transmission during backcrossing contains a transgene integrated into a C
-
genome chromosome. Approximately 10% of
offspring retained the tolerant trait in the BC3 and BC4 generations.

Temporal and tissue specific control

Temporal

Use chemically induced promoter to turn on the transgene. Many

inducible promoters are available but only one (alcohol dehydrogenase)

is commercialized so far. Spray the plants with the chemical to turn

on the promoter and then spray with herbicide/ pesticide. This is not

useful for those genes which need to be on through out the life cycle

of plants.


Tissue Specific

Remove the transgene from non
-
target organs of the transgenic plant.

This can be done by a two component system: 1). tissue specific promoter

driving
cre

gene; 2). Transgenes flanked by the
loxP

sites.

Transgenic mitigation

Compromise the fitness of weeds (TM trait) that by introgression

have acquired positive traits from crop genes. Strategy is based on the


following facts:

1.
Tandem constructs are inherited as tightly linked genes.

2.
TM traits are neutral or positive for crops but deleterious for


weeds.

3.

Mildly harmful TM traits will be eliminated from weed population


because such plants strongly compete among themselves and have


large seed output.

Potential Transgenic Mitigation (TM) Traits

Seed dormancy
:

weed seeds exhibit secondary dormancy. Abolishing

sec. seed dormancy is neutral to crops but deleterious to weeds. Arabidopsis

Sleepy 1

(Abscisic acid insensitive) mutant completely lacks sec. dormancy


Shattering
:
Anti
-
shattering genes are useful for crops and deleterious for

weeds. Shattering is thought to be a hormone regulated process, thus

changing hormone balance in the abscission zone influences shattering

propensity. Cytokinin overproduction is believed to delay shattering.

SHATTERPROOF
gene of Arabidopsis has been isolated that prevents

shattering by delaying valve opening in siliques.


Dwarfing
:
This is positive for crops. Dwarf varieties were credited for

green revolution. Height reduction is possible by preventing gibberellin

synthesis. Recently a mutant GA receptor gene was isolated that does not

respond to GA.

It is called “green revolution” gene.

Molecular Ecology

13: 697


-

March 2004


Tandem constructs to mitigate transgene persistence: tobacco as a model

Dwarfism, which typically increases crop yield while decreasing the ability to compete,
was used as a mitigator. A construct of a dominant
ahas

R

(acetohydroxy acid synthase)
gene conferring herbicide resistance in tandem with the semidominant mitigator dwarfing
gai

(gibberellic acid
-
insensitive) gene was transformed into tobacco (
Nicotiana tabacum
).

Lack of survival of transgenic mitigation (TM) plants in competition
with wild type (WT). The proportion of live plants was measured
after 100

days of growth in experiment I (closed symbols) and after
111

days in experiment II (open symbols). The lines show the
average of the two experiments.

In this method, the gene(s) of interest is tagged with a RNA interference cassette, which
specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and
thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control.
We generated transgenic rice plants by this method using a new glyphosate resistant

5
-
enolpyruvylshikimate
-
3
-
phosphate synthase (EPSPS) gene from
Pesudomonas putida

as the
gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to
bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field
trial of these transgenic rice plants further confirmed that they can be selectively killed at
100% by one spray of bentazon at a regular dose used for conventional rice weed control.
Furthermore, we found that the terminable transgenic rice created in this study shows no
difference in growth, development and yield compared to its non
-
transgenic control.
Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene
containment, which appears simple, reliable and inexpensive for implementation.