Genetic Transformation of Petunia for Delayed Leaf and Flower Senescence Progress Reports - June 2000
Genetic Transformation of Petunia for
Delayed Leaf and Flower Senescence
Michelle L. Jones and David Clark
Colorado State University,
Department of Horticulture and Landscape Architecture,
111 Shepardson, Fort Collins, CO 80523;
University of Florida, Environmental Horticulture Department,
P.O. Box 110670,
Gainesville, FL, 32611
Progress Report- Year 1 (9-99 to 9-00)
The post-production quality of many ornamentals is influenced
by both flower and leaf senescence. The gaseous hormone ethylene causes
premature senescence of flowers and leaves while the cytokinins have been shown
to delay leaf senescence. Cytokinins have also been shown to increase the vase
life of flowers although this effect is more variable and highly dependent on
concentration. While the generation of transgenic plants that are insensitive to
ethylene has been very successful at extending the life of flowers, we are
taking a different approach to increasing the postproduction quality of
ornamentals. The goal of this three-year research project is to increase the
postproduction quality of ornamentals by delaying leaf and flower senescence. We
have taken two different approaches to reaching this goal. The first involves
over-expressing isopentenyl transferase (IPT), the rate limiting enzyme in
cytokinin biosynthesis, in senescing tissue using a senescence-specific promoter
(sag). The second approach is to delay senescence by targeting
senescence-related genes- i.e. genes that are responsible for carrying out the
degradative processes that predominate during senescence. We will decrease the
endogenous levels of the senescence-related gene cysteine protease in order to
delay the senescence process.
Objective 1: to delay flower and leaf senescence by
decreasing the endogenous levels of cysteine protease in Petunia hybrida
cv. Mitchell.
We have cloned a cysteine protease cDNA (phcp1- Petunia
hybrida cysteine protease 1) and have made a genetic construct that includes
this gene in the antisense orientation driven by the CaMV-35S (35S-phcp1)
constitutive promoter. The constitutive promoter will result in expression of
the gene all the time in all tissues. The antisense nature of the gene construct
will result in the production of mRNA molecules that are in the opposite
orientation or complement of the endogenous RNA. These two complementary mRNA
will bind to each other and prevent translation of the endogenous mRNA into an
active enzyme, thereby decreasing the cysteine protease activity within the
entire plant. Using Agrobacterium tumefaciens we have successfully
transformed Petunia leaf disc explants with this construct. However,
transformed leaf pieces have formed callus and regenerated shoots, but have not
regenerated roots. Control plants were transformed with a construct containing
the CaMV-35S promoter and a reporter gene gus at the same time. These
transformants have successfully formed shoots and roots in tissue culture,
confirming that our transformation and regeneration protocol is successful. We
have successfully rescued these 35S-phcp1 transgenic shoots by grafting them
onto wild type rootstocks. Additional leaf discs have been infected with the
same construct in order to generate more shoots for grafting and increase the
number of lines that we will have to evaluate.
In addition to using the 35S constitutive promoter to drive
the antisense expression of the cysteine protease we also proposed to isolate
the native promoter from the cysteine protease gene and use this to drive
expression of the gene. Using the cysteine protease cDNA as a probe, the phcp1
gene has been cloned from a Petunia hybrida cv Mitchell genomic library
after 3 rounds of screening. In light of the above results and the finding that
the cp gene is expressed at a low level in roots (Jones, unpublished), we have
decided that we need a more specific promoter that does not drive expression in
the roots.
One of the problems with generating transgenic plants in
which a specific developmental event, like senescence, is manipulated by genetic
engineering is the ability to specifically target the transgene to a specific
plant organ or drive expression only at a certain developmental stage. One
example of this can be seen with ethylene insensitive petunias. These plants
have flowers with delayed flower senescence, but also have defects in seed
germination and adventitious rooting due to ethylene insensitivity in the entire
plant. We see this as a continuing obstacle to creating plants that have delayed
flower senescence.
In order to create transgenic plants in which we can delay
flower senescence and not effect other processes we need to have gene promoters
that will drive the expression of the transgenes in a flower specific manner.
Currently we have gene promoters from 2 SR (senescence-related) genes from
carnation flowers (obtained from W.R. Woodson, Purdue University). In order to
confirm that they will drive senescence specific expression in Petunia
flowers we have made a construct that includes the SR promoters and the reporter
gene GUS. The gus gene encodes an enzyme $ -glucuronidase. When plant tissue is
supplied with the substrate for this enzyme a blue color is formed that
indicates which tissue your promoter is driving gene expression in. One of these
SR-GUS constructs has been transformed into Petunia and the plants are
currently flowering. Preliminary research indicates that this promoter is flower
senescence specific in Petunia (Figure 1).
Objective 2: to over-express isopentenyl transferase (IPT-the
rate limiting enzyme in cytokinin biosynthesis) in senescing tissue by using a
senescence-specific promoter (sag).
Part 1: Genetic transformation of Petunia hybrida
Cytokinins have been shown to delay the onset of leaf
senescence. The focus of this part of the project was to generate petunia (Petunia
x hybrida) plants that over-produce cytokinins in leaves in a senescence
specific manner. This was achieved by transforming plants with the IPT (isopentenyl
transferase) gene driven by the senescence-associated promoters, sag12 and
sag13. This was done in an attempt to delay leaf senescence without
negatively altering the horticultural performance of the plants. Preliminary
studies with the sag12-IPT construct in Petunia demonstrated that
the autoregulatory over-expression of cytokinins in Petunia effectively
delays leaf senescence (Dervinis et al., 1999). sag12-IPT petunia plants
have been shown to have delayed leaf senescence, increased lateral branching and
increased flower number compared to nontransformed plants. In the past year, we
conducted studies to determine the effects of this transgene on the
horticultural performance and reproductive characteristics of Petunia to
help determine the potential impact of this technology to the floriculture
industry. A second sag promoter, sag13 (sag13-IPT) was used in
addition to sag12-IPT to transform Petunia because it was thought
to have a tighter regulation than sag12 (Amasino, personal
communication).
Part 2: Identify transformed lines and breed to homozygosity
using traditional breeding methods
The highly inbred ‘Mitchell Diploid’ Petunia was
transformed with the sag13-IPT T-DNA on a binary vector.
Approximately 75 transformed sag13-IPT T0 plants were
self-pollinated to produce the T1 generation. The T1
plants were screened by drought-stress for a delayed leaf senescence phenotype.
Non-senescent T1 plants were self-pollinated to produce the T2
generation. Four stable lines of sag13-IPT petunias have demonstrated that they
stably transmit the transgene and a delayed senescence phenotype to progeny.
These plants are currently being bred to homozygosity, and it is hoped that we
will have isogenic inbred lines of all of these lines by the end of 2000.
Experiments are being conducted to evaluate the horticultural performance of
these transformed plants as we breed them.
Part 3: Evaluate horticultural performance of transgenic
lines
Previously two sag12-IPT lines produced seedlings that
demonstrated the desired non-senescent phenotype in the T1 and T2
generations. These lines are being grown in Colorado and Florida to evaluate the
horticultural performance of sag12-IPT petunia plants in terms of rooting
of vegetative cuttings, lateral branch growth, flower number, floral timing, and
biomass distribution. In experiments conducted to date, we have determined that
the two transgenic lines differ from each other in terms of their horticultural
performance.
Line I97-1-7 demonstrated a delayed leaf senescence
phenotype. This line also demonstrated a decrease in adventitious rooting and an
increased number of branches during vegetative propagation and during plant
production. The altered horticultural performance of these plants is thought to
be due to ‘leaky’ expression of the transgene.
Line I97-3-18 also demonstrated a delayed leaf
senescence phenotype. However, members of this line were not significantly
altered in any horticultural performance characters when compared to wild-type
‘V26’. These results demonstrate that it is possible to use biotechnology to
delay leaf senescence without altering other important horticultural traits. It
is likely that all of the data produced on sag12-IPT petunias will be
written into a refereed manuscript and submitted for publication in late summer
or early fall of 2000.
Application for continued funding:
Objective 1: To delay senescence by decreasing endogenous
levels of the senescence-related gene cysteine protease, in Petunia hybrida
cv. Mitchell.
Part 1: constitutive down-regulation of cysteine protease
We will continue to generate shoots for the 35S-phcp1
(constitutive down-regulation of the senescence-related gene cysteine protease)
lines and graft them to wild type rootstock until we have 50-100 lines (T0 plants)
to evaluate. Transformants will be selected on the basis of their ability to
grow on tissue culture media containing kanamycin (a characteristic encoded for
by the nptII gene built into the genetic construct) and the presence of the
transgene construct will be confirmed using ELISA assays for the nptII gene. T0
plants will be screened for a delayed flower senescence phenotype and the best
lines will be selected. After selfing the T0 lines, the T1
will be screened for the desired phenotype of increased flower longevity to show
that the trait is heritable. These selected lines will be bred to homozygosity.
Evaluation of the horticultural performance of these plants will occur on the
heterozygous T1 and homozygous T2 lines and this will be
conducted in both Florida and Colorado to allow for proper evaluation in
different greenhouse production environments. Plants will be evaluated to
determine the effects of the transgene on flowering, sexual reproduction,
vegetative reproduction and leaf yellowing.
Part 2: Flower specific down regulation of cysteine protease.
We have generated 30 lines of SR-GUS plants and have
additional lines coming out of tissue culture. We will continue to analyze the
expression patterns of GUS to confirm that the SR promoter drives flower
senescence specific gene expression in Petunia. This will include
assaying for gus activity (presence of blue color when supplied with substrate)
in senescing and non-senescing flowers, leaves, roots, and stems. We will also
analyze temporal patterns of expression to determine when during flower
senescence the gene is turned on, its regulation by ethylene, and what parts of
the flower it is expressed in. A construct has been created that includes the
antisense cysteine protease gene and the SR promoter. This will be transformed
into Petunia following confirmation of the senescence specific nature of
the SR promoter. This will begin prior to September. These plants will be
analyzed and bred as described above for the 35S-phcp1 plants. Horticultural
performance will be conducted in both Florida and Colorado.
Objective 2: to over-express isopentenyl transferase (IPT-the
rate limiting enzyme in cytokinin biosynthesis) in senescing tissue by using a
senescence-specific promoter (sag).
Part 1: Genetic transformation of Petunia hybrida
This part was completed in 2000 for the objectives listed
previously. However, it is likely that we will continue looking for new
genetic constructs that may confer delayed leaf senescence to petunia plants.
For example, Ori et al (1999) showed that controlled expression of the maize
transcription factor gene knotted (kn1) with the sag12 promoter conferred
delayed leaf senescence in tobacco. We are currently making and evaluating
transgenic petunia plants containing this construct, and hope to be able to
report success in next year’s report.
Part 2: Identify transformed lines and breed to homozygosity
using traditional breeding methods
Currently we are selecting homozygous lines of sag12-IPT
petunias, and preparing to plant the T2 generation of sag13-IPT plants.
It is hoped that we will have homozygous lines of plants containing both
constructs within the next 6 months. Once we have these inbred lines, we will be
setting up experiments in Colorado and Florida to test both types of plants side
by side in a traditional cultivar trial format. A graduate student is currently
in place in Dr. Clark’s laboratory to take over this part of the project.
Part 3: Evaluate horticultural performance of transgenic
lines
We are currently conducting experiments on horticultural
performance of all plants produced for this project as we breed them to
homozygosity. Once all transgenic lines are homozygous for their given genetic
construct, seeds will be sent to Colorado for further evaluation. We expect to
start producing a large amount of data in the next year with applied greenhouse
production experiments.
Anticipated Benefit to the Floriculture Industry
Research like that proposed above has the potential to lead
to improved floriculture crops that will increase our international
competitiveness as an industry. The development of bedding plants and potted
plants that retain blooms longer and have decreased leaf yellowing will allow
growers to increase the range of shipping and open up new markets. It will also
increase the amount of time that these plants are saleable in the retail market
and decrease post production losses. The increased quality of these plants will
also create a product with more aesthetic value in the landscape. This research
has many important applications to the Floriculture industry. While this
research is initially being conducted using petunias, the technology will be
transferable to other crops of interest.
Professional or published information:
1. Dervinis, C., D.G. Clark, H.J. Klee, J.E. Barrett and T.A.
Nell 1999 Prevention of Leaf Senescence via Genetic Transformation with
sag-IPT. Proceedings of the Florida State Horticultural Society Annual
Meetings 111:12-15.
2. Johnson, B., C. Dervinis and D.G. Clark. 2000
Genetic Transformation of Petunia x hybrida for Delayed Leaf
Senescence. 13th Annual Penn State Symposium in Plant Physiology
“Plant Signaling 2000″, State College, PA (poster).
