**The *Populus* Genome Science Plan**

**Tissue Culture & Transformation Panel**

Panel members: Rick Meilan, Malcolm Campbell, Shujun Chang, Takashi
Hibino,

Ove Nilsson, and Gilles Pilate

# 

# **Background**

Although scientists often generically refer to the procedure by which
plants are genetically engineered as "transformation", it really entails
two separate processes. The first, transformation, involves stable
integration of DNA into the chromosome of an individual plant cell. The
second step, regeneration, is the process by which individual
transformed cells are coaxed, through hormonal and cultural
manipulations, to develop into a whole plant. Generally, regeneration is
the most limiting of the two processes.

Transformation is essential for introducing novel traits, particularly
those that are not available in the gene pool of sexually compatible
species. In addition, transformation is an important tool for the
analysis of gene functionality. By constitutive or conditional up- or
down-regulation (knock-in/knock-out, KI/KO) of the target gene,
important information about its function and downstream targets can be
obtained. Constitutive expression from the cauliflower mosaic virus
(CaMV) *35S* promoter is still the dominant approach, but there is a
growing demand for more refined methods using tissue- and
temporal-specific promoters. Few other promoters have been characterized
and used in the poplar system; however, promoters active during various
stages of wood formation or floral development are in high demand, both
for basic and applied research. Inducible systems are also very
important tools for the study of gene function. We lack a good
understanding of how the systems that have been developed in
*Arabidopsis* perform in poplars. It is especially important to have
reliable inducible systems for use with transcript profiling or
proteomics, in order to identify early downstream targets of
transcription factors.

While reliable transformation systems have been developed for pure
species and hybrids in the section *Populus*, the genotypes in other
sections have been found to be recalcitrant. To date, only a limited
number of genotypes in sections *Tacamahaca* and *Aigeros* have been
successfully transformed. Most notably the genotype being sequenced,
Nisqually-1, which is in the section of *Tacamahaca*, has been very
resistant to regeneration. Regardless of section, the most commercially
and experimentally important genotypes tend to be the least
transformable.

Transformation barriers can be divided into two broad categories:
biology and maturity. Biological barriers have not received much
attention, but they are likely the reason that many clones with good
organogenic potential are not transformable. Maturation-related
obstacles, on the other hand, have been well documented as a major
limitation in the transformation of many important genotypes. However,
rootability is often confused with regenerability. In the case of *P.
deltoides*, commercial clones are screened for their rootability before
clonal testing. As long as cuttings root well, axillary buds flush and
the propagation cycle is complete. However, *in vitro* regenerability is
dependent on the ability to induce adventitious shoots from cells.
Several studies have shown that seedlings from recalcitrant clones can
be easily regenerated. The apparent lack of hereditary control implies
that the decline in regeneration potential is related to maturity. In
another case, leaf explants from a recalcitrant clone were easily
regenerated after a year of *in vitro* culture on medium containing
cytokinin. The regenerated plants turned out to be highly organogenic.
This suggests that the recalcitrance was related to maturation.
Comparing expression profiles in tissues that differ in regeneration
potential, after growth on regeneration medium, could help identify
genes involved in regulating the process. In addition, gene expression
profiling done on tissues gathered during the juvenility-to-maturity
transition could help identify genes affecting regeneration.

During transformation, a selectable marker gene is usually introduced
simultaneously with the gene of interest. This marker gene is often one
that imparts resistance to an antibiotic or herbicide. As a result, a
transformed cell can be isolated on a medium containing the appropriate
selection agent. While this method is convenient, it can be problematic.
First, performing subsequent rounds of transformation may not be
possible because there are a limited number of selectable marker genes
available. Second, various selection agents can dramatically affect
transformation efficiency. Finally, the presence of a selectable marker
gene is an impediment to gaining public acceptance of genetically
engineered plants.

Recently, alternative selection systems have been developed. One such
system is based on the growth medium lacking a substance needed for
proper metabolic activity or physiological development. A particularly
attractive option is based on the inability of a cell to regenerate a
whole plant without the addition of a phytohormone or phytohormone
derivative to the culture medium at a precise step in the regeneration
process. This is true for most plant transformation protocols, which
rely on the addition of cytokinin to the medium for inducing the
differentiation of adventitious buds from transgenic callus. This
supplement has to be transitory because cytokinin can have an inhibitory
effect on subsequent root development.

New vectors have been developed that carry a gene critical for the
biosynthesis of cytokinin from adenine. Cells transformed with these
vectors will continuously produce cytokinin; only they will be able to
regenerate into shoots, whereas untransformed cells will remain in an
undifferentiated state. Further, these new vectors carry an excision
system for removal of the cytokinin biosynthetic gene once transgenic
shoots have been regenerated, allowing for rooting of these shoots.
Excision systems consist of a recombinase gene, often under the control
of a reliable inducible promoter**,** and recognition sites flanking the
DNA fragment to be removed. These alternative selection systems are
attractive because they: 1) have the potential to increase both the
yield and speed with which transgenic poplars can be produced,
facilitating the use of various high-throughput strategies, such as
KI/KO, and 2) may eliminate the need for both specific selection and
regeneration conditions, making it possible to transform a wider array
of genotypes.

Finally, a versatile system for long-term storage of poplar genotypes is
critically needed. Plant preservation has traditionally relied on seed
banks or collections in gardens, orchards, or clone banks. Seed storage
is the most common mode of preservation for many angiosperms and
gymnosperms, but for species with long generation times, actively
growing samples are the preferred form of preservation. However,
maintenance of actively growing collections is labor-, time- and
space-consuming, not to mention the potential for pests infestations and
microbial contamination. In addition, plants or tissues maintained *ex
vitro* or *in vitro* are subject to growth constraints and environmental
fluctuations that can reduce viability and inhibit propagation
potential. Field planting transgenic trees for long-term germplasm
preservation suffers from the additional burden of regulatory
constraints, ecological and security concerns, and difficulties in
maintaining juvenility. Cryopreservation is viewed by many as the only
viable method for long-term storage and distribution of important
germplasm stocks.

This portion of the Science Plan has two overall objectives: 1)
improving *Populus* transformation/regeneration efficiency and 2)
developing tools to increase the utility of *Populus* as a model plant
system. Thus, under each of two Scientific Objective headings below,
there are several numbered sub-tasks.

# 

# **Scientific Objective #1**

# Develop a more robust transformation protocol, applicable to a wide array of genotypes, by improving our understanding of the fundamentals underlying transformation and regeneration. This system should allow for significant reduction in the time needed to regenerate transgenic poplars. The ultimate goal is to develop a high-throughput transformation protocol with over 50% transformation efficiency and rapid turnover (*e.g.*, co-cultivation to soil in 3-4 months). 

**Highly efficient *Populus* transformation**

It is essential to develop a highly efficient transformation protocol in
order to over- or under-express single or multiple genes. The key is to
be able to rapidly test a large number of constructs in the shortest
time possible. Several aspen clones such as 717-1B4 (*P. tremula* x *P.
alba*) and a few *P. tremula* x *P. tremuloides* clones, which have
transformation efficiencies equivalent to that of tobacco, can serve the
gene-testing purpose. However, their genetic distance from Nisqually-1
and their restricted planting ranges could possibly limit the utility of
these clones for high-throughput analysis of gene functionality or
field-testing.

### *Short-term goals*:

-   Agree on the genotypes to be used

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-   Select a clone that closely related to Nisqually-1 that has a
    transformation efficiency similar to that of tobacco (another clone
    entirely or progeny of Nisqually-1)

-   Select a relatively early-flowering genotype (until better clones or
    systems are identified) not only for testing flowering-related
    genes, but also as a method for testing other genes and producing
    viable transgenic seeds

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-   Improve the transformation of the genotype being sequenced,
    Nisqually-1, which could also be used as the recalcitrant clone for
    testing regeneration- and transformation-related genes

-   Identify poplar genes that interfere with or promote regeneration

-   Identify poplar genes related to transformation

*Mid-term goals*:

-   Test (over- or under-express) various genes thought to affect
    regeneration and transformation

-   Develop a better gene-testing model system, which has consistently
    high transformation efficiency and a shorter infection-to-soil cycle
    (similar to that of tobacco)

-   Develop an efficient gene-testing model with an early-flowering
    genotype

# *Future applications*: 

### Produce a complete collection of lines (saturate the genome) through: activation tagging, enhancer or gene trapping, KI/KO, and insertional mutagenesis

### Develop the publicly accessible database for the collection of transgenic lines and link the information with functional analysis and sequence data

### Achieve a global understanding of the genes related to morphogenesis, maturation, rooting, and *Agrobacterium* susceptibility

**Universal vectors**

It would be useful to have some universal shuttle and binary vector
backbones for assembling both over-expression and KO constructs.
However, these progenitor constructs need to be freely available, which
is now frequently not the case (*e.g.*, recent reports of difficulties
with agreements governing the use of vectors that are commonly utilized
in the assembly of RNAi vectors).

# *Short-term goals*:

-   Evaluate usefulness of resources developed by the *Arabidopsis*
    community

*Mid-term goals*:

-   Assemble shuttle and binary vector backbones for over-expression and
    suppression

# *Future applications*:

-   The ability to easily produce constructs (high throughput) needed to
    verify the function of individual genes through the use of KI/KO
    constructs

**Transformation without tissue culture**

The goal is to produce an *in planta* transformation method similar to
the one that has been established for *Arabidopsis*.

# *Short-term goals*:

-   Establishment of a basic transformation protocol of using reporter
    genes

*Mid-term goals*:

-   Optimize the protocol by testing various transformation parameters

*Future applications*:

-   A faster, more efficient transformation system that has lower risk
    of somaclonal variation than traditional methods

## Scientific Objective #2

Develop molecular tools that will allow *Populus* to become an even
better model species and a more attractive system for doing comparative,
developmental biology research.

# 

**Inducible promoters**

Plant genetic engineering has largely been performed using strong
constitutive promoters, the aim being to obtain maximum levels of
expression and, consequently, maximal effect. Such a strategy may be
useful for imparting certain commercial traits (*e.g.*, insect
resistance or herbicide tolerance), but is not practical when the aim is
to alter the expression of an endogene. Indeed, numerous cases of gene
silencing have been reported. In addition, altering the expression of
key genes, such as transcription factors or other regulatory genes, may
have lethal or at least strong negative effects on plant development.
For the IPGC, the major application of genetic engineering is
large-scale evaluation of gene function via KI/KO strategies. The
production of transgenic plants altered in the expression of numerous
genes vital to plant growth and development will be impossible unless a
reliable system exists for conditional transgene expression. It is
imperative for the inducer to be highly specific to the target promoter
and lack phytotoxicity. It is also important for the target promoter to
be tightly regulated (*i.e.*, no "leaky" expression) and that high-level
expression is conferred upon induction.

# *Short-term goals*:

-   Obtain existing constructs for various systems (ethanol,
    dexamethasone, estrogen, etc.)

-   Transform constructs into *Populus*

# *Mid-term goals*: 

-   Test for toxicity, inducibility, specificity of inductive agent

-   Determine the level and how tightly regulated expression is

*Future applications*:

-   The ability to over- or under-expression selected genes may be
    lethal or have otherwise undesirable consequences

**Gene excision**

In any genomics project, the availability of a reliable excision system
is valuable both for applied and fundamental studies. An excision system
will not only permit removal of selectable marker genes, to alleviate
public concern, but it will also allow for easy retransformation using
vectors derived from a common backbone. Moreover, the alternative
transformation systems described above depend on a reliable excision
system.

Because transposon systems have proven difficult to control, alternative
excision systems were developed. These systems exploit naturally
occurring recombinases, such as cre/loxP or R/RS. Gene excision vectors
include: 1) a recombinase gene under the control of an inducible
promoter, and 2) recognition sites that flank the targeted DNA. The goal
of this project is to evaluate various excision systems that are
available in order to determine which is the most appropriate for use
with poplar. For each system, it is important to assess the efficacy of
induction, the level of inducibility, and leakiness of the promoter (as
described above). In addition, it is necessary to ascertain the efficacy
of the recombinase, and how clean excision is, in various poplar
genotypes.

# *Short-term goals*: 

# Obtain existing constructs for various systems

-   Transform constructs into *Populus*

# *Mid-term goals*: 

-   Evaluate transgenic plants for clean excision of target sequence

*Future applications*:

-   The ability to selectively remove unwanted genetic material (*e.g.*,
    selectable markers)

**RNA interference**

Because poplars have a long juvenile period and are dioecious, classical
mutagenesis or insertional mutagenesis are not practical tools to study
loss of gene function in poplar. The poplar community is, therefore,
dependent on indirect methods, such as anti-sense RNA or RNA
interference (RNAi), for down regulation of gene expression. Due to the
dominant nature of this approach, and the relative ease with which large
numbers of RNAi constructs can be generated, it should be possible to
design large-scale programs with the ultimate goal of down-regulating
all poplar genes. Several approaches to RNAi have been described, but
these need to be thoroughly tested in poplar.

*Short-term goals*:

-   Test various approaches to RNAi for efficiency

-   Determine the extent to which similar gene family members can be
    targeted individually or collectively (specificity)

-   Test the use of tissue- and temporal-specific promoters to drive
    RNAi constructs

-   Test the use of inducible RNAi constructs (especially important for
    the analysis of genes whose loss-of-function proves to be lethal or
    in other ways prevents *in vitro* regeneration, and in combination
    with transcript profiling and proteomics approaches)

*Mid-term goals*:

-   Evaluate stability over time (*i.e.*, will the silencing be
    maintained over several cycles of growth and dormancy?), which is
    especially relevant for perennial plants

-   Development of stock centers where RNAi constructs for all poplar
    genes can be deposited and shared with the rest of the poplar
    community

*Future applications*:

-   The ability to effectively silence any gene in a given tissue or at
    a specific point in time

-   Development of clonal archives of transgenic plants where all
    individual poplar genes have been targeted

**Floral induction**

# Most genotypes of poplar have a five- to seven-year juvenile period. This is a serious impediment for the many researchers working on flowering control. In order to make more rapid progress in this area, an early-flowering genotype or a simple, reliable method of floral induction is needed.

*Short-term goals*:

-   Conduct an exhaustive search for genotypes that are known to flower
    early

-   Experiment with various inductive treatments (chemical, physical,
    cultural) that have been shown to be successful with other woody
    angiosperms

-   Identify genes involved in controlling the timing of floral
    development

*Mid-term goals*:

-   Transform putative flowering-time genes into normal-flowering
    genotype(s)

-   Verify that induced flowers are functional

*Future applications*:

-   An important tool for breeding and marker-aided selection

-   With a highly efficient early-flowering system, it would even be
    realistic to generate homozygous insertional mutants

-   It might also be possible to generate populations for rapid mapping
    of a specific trait

# **Cell culture** 

Synchronized cell culture systems, such as tobacco BY2 cells, in which
it is possible to obtain an absolutely homogenous response to added
compounds or treatments, is an important tool for studying the
regulation of growth and development.

*Short-term goals*:

-   Further development of stable *Populus* cell cultures that are
    highly homogenous

-   Develop methods to efficiently induce differentiation of tracheary
    elements with a high degree of synchronization

*Mid-term goals*:

-   Development of a highly synchronized system for the study of
    cell-cycle regulation

*Future applications*:

-   The possibility of inducing relatively undifferentiated cell
    cultures to differentiate into specific cell types, as with the
    *Zinnia* tracheid development system, is a powerful tool for
    analyzing developmental transitions and cell-cycle regulation

**Virus-induced gene silencing**

Virus-induced gene silencing (VIGS) has emerged as a powerful approach
to modify gene expression in herbaceous annual plants. Several viral
vectors have been described, which function to silence endogenous genes
within a range of plant genomes. VIGS is a systemic and epigenetic
phenomenon, which means that gene silencing occurs throughout the plant,
but is not transmitted from generation to generation. Due to the
dominant nature of this loss-of-function approach, and the relative ease
with which large numbers of viral constructs can be generated, it should
be possible to design large-scale programs with the ultimate goal of
down-regulating all poplar genes via VIGS. Several approaches to VIGS
have been described, but these need to be thoroughly tested in poplar.

*Short-term goals*:

-   Test various VIGS vectors for efficacy with poplar

-   Development of new VIGS vectors for poplar, based on tree viruses,
    such as poplar mosaic virus (PopMV)

-   Determine the extent to which similar gene family members can be
    targeted individually or collectively (specificity)

*Mid-term goals*:

-   Evaluate stability over time (*i.e.*, will the silencing be
    maintained over several cycles of growth and dormancy?), which is
    especially relevant for perennial plants

-   Development of stock centers where VIGS constructs for all poplar
    genes can be deposited and shared with the rest of the poplar
    community

*Future applications*:

-   The ability to effectively silence any gene in a given tissue or at
    a specific point in time

-   Development of clonal archives of transgenic plants where all
    individual poplar genes have been targeted

**Cryopreservation**

It is vital to develop and test protocols to enable transgenic stocks to
be stored at low cost and with low rates of somaclonal variation.
Cryogenics is the most logical means for long-term storage of transgenic
germplasm (especially KO/KI lines). Vitrification is a simplified
cryostorage procedure that eliminates the need for expensive cooling
devices. It relies on the controlled application of cryoprotective
solutions that desiccate and penetrate cells at low temperatures,
leading to increased viscosity of the cytosol and formation of an
immobilized solution state. When vitrification is optimally achieved,
even the most delicate tissues can survive direct immersion and storage
in liquid nitrogen without ice crystal damage. Because of its simplicity
and low cost, vitrification has become a preferred method in recent
years.

# *Short-term goals*:

-   Optimize the key steps: 1) conditioning, 2) pre-culture regimes, 3)
    cryoprotection, 4) warming, and 5) recovery regimes

-   This will include testing factors known to affect successful storage
    and recovery such as sorbitol concentration in the pre-culture
    media, composition of vitrification solution, and PGRs in the
    recovery media, etc.

*Mid-term goals*:

-   Evaluate genetic fidelity of re-established plants (*e.g.*, by using
    morphology, NIR spectra, and simple sequence repeat (SSR) markers
    previously developed for *Populus* spp.)

-   Develop inventory-tracking software (*e.g.*, to record species or
    genotype origin, genetic modifications, number of propagules stored,
    cryostorage parameters, viability, recovery regimes,
    micropropagation procedures, genetic fidelity, and other
    characteristics)

-   Establish a mechanism to fund a long-term storage and distribution
    system

*Future applications*:

-   The infrastructure to maintain and distribute important stocks to
    the poplar research community