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SID 5Research Project Final Report
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Project identification
1.Defra Project code
HH3709SHN
2.Project title
Techniques and breeding lines for the genetic improvement of
hardy nursery stock
3.Contractororganisation(s)
East Malling Research
New Road
East Malling, Kent
ME19 6BJ
54.Total Defra project costs
££447,636
(agreed fixed price)
5.Project:start date
01 April 2003
end date
31 March 2008
6.It is Defra’s intention to publish this form.
Please confirm your agreement to do so.YES FORMCHECKBOX NO
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confidential data, used in or generated by the research project,
which should not be disclosed. In these cases, such information
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Executive Summary
7.The executive summary must not exceed 2 sides in total of A4
and should be understandable to the intelligent non-scientist. It
should cover the main objectives, methods and findings of the
research, together with any other significant events and options
for new work.
The project aim was to use conventional and biotechnological
methods to develop tools of genetic improvement and breeding lines
for the benefit of the UK industry, and material for
independently-funded assessment and trialling. Target characters
include improved habit, foliage characteristics, flower colour and
size and disease resistance. The UK Hardy Nursery Stock (HNS) trade
has a farm gate value of approximately £350 million, of which 10%
is attributable to rose, and new cultivars of ornamental shrubs and
trees are needed to maintain the competitiveness of the
industry.
The specific objectives were to:
1. Clarify genetics of horticulturally important characters in
Sambucus, Buddleia, Sorbus and Jasminum to inform breeding
programmes
2. Identify sources of resistance to blackspot, and perhaps
aphids, in Rosa, and transfer breeding lines to a commercial
breeding programme, with a view to subsequently developing a LINK
research programme
3. Demonstrate that tetraploidy has restored fertility to
interspecific hybrids of Syringa and Pyrus × Malus and check for
self compatibility and mode of inheritance, and complete
backcrossing programme of tetraploid Buddleia globosa to B. davidii
to introgress yellow flower colour
4. Determine if tetraploidy in the climbers Clematis and
honeysuckle (Lonicera), and shrubs such as Physocarpus induces more
compact habit, useful for small gardens
5. Develop transformation methods for hardy ornamentals,
assessing the xylose isomerase and MAT vector systems in Buddleia
davidii, and establishing regeneration methods for two additional
subjects, for use in subsequent transformation experiments and
technology transfer to the industry
Controlled crosses were performed with various subjects to
clarify the inheritance of horticulturally important traits
concerning leaf colour, plant habit, etc., and various segregating
progenies were already available for genetic analysis. In this way
two recessive genes for dwarf habit were inferred in breeding lines
related to Sambucus nigra ‘Black Beauty’; genes for purple and for
yellow leaf colour were identified in Physocarpus opulifolius
‘Diabolo’ and ‘Aurea’ respectively and the genes were located on an
AFLP map; and genes for white flower and for twisted branches were
detected in Chaenomeles ‘Nivalis’ and ‘Tortuosa’. In addition,
microsatellite markers were developed for Sambucus which enabled
fingerprinting of a panel of cultivars held in a breeding
collection and were useful in identifying some synonyms and
homonyms that would otherwise have caused confusion. For study of
the genetics of lime-tolerance in Rhododendron a small mapping
progeny was produced; twenty-five selections of Rhododendron raised
in the previous project were identified as apparently tolerant to
lime and five have been propagated for trialling.
Various roses reputed to have blackspot tolerance were added to
the field collection and the collection was scored for resistance.
Crosses between resistant, e.g. Rosa rugosa or R. wichuriana, and
susceptible, e.g. R. arvensis or R. ecae, accessions were made and
seedlings successfully raised and assessed for resistance. Eight
rose microsatellites were developed from a genomic library of R.
filipes and used to fingerprint a panel of diploid species and
tetraploid cultivars. A stategy for introducing resistance to downy
mildew from diploid wild species into tetraploid garden roses was
formulated as part of a possible larger proposal for LINK
funding.
Evaluation of various tetraploid mutants induced in the previous
project demonstrated the restoration of fertility in interspecific
hybrids of Sambucus nigra × racemosa and Syringa vulgaris ×
pinnatifolia. Interspecific breeding lines of Buddleia deriving
from a cross of the naturally tetraploid Buddleia davidii
‘Nanhoensis Alba’ (large panicles) with an induced tetraploid form
of B. globosa (yellow flowers) were used in further crossing to
combine yellow flower colour with larger panicles. Tetraploid
versions of pear × apple and pear × quince hybrids were weaned and
grafted on to rootstocks; it is hoped that, when they flower, they
will be fertile and useful for further breeding.
Populations of tetraploid Clematis, Lonicera and Physocarpus
were generated. Initial observations showed that tetraploidy has
induced more compact habit and larger flowers in some of the
Lonicera clones, but delayed flowering in the Clematis and
difficulties with propagation of the Physocarpus meant that
detailed comparisons for these two were not made by the end of this
project.
Micropropagation and regeneration methods were developed for
Lonicera x americana, L. × heckrotii and three cultivars of L.
periclymenum. For Clematis, micropropagation methods were devised
for one species, C. flammula, and seven hybrids. Adventitious
shoots and somatic embryos were regenerated from leaf explants of
C. flammula and three hybrids. A protocol for somatic embryo
maintenance and proliferation was developed for one of the hybrids,
‘Pagoda’, which provides an ideal system for genetic
transformation. For transformation, a vector for ‘clean gene’
transformation was constructed, which will enable excision of the
selectable marker gene from transformed plants. Removal of the
selectable marker should increase public acceptance; it will also
allow subsequent transformation with one or more additional genes.
The vector incorporates a GFP (green fluorescent protein) reporter
gene (which is linked to the selectable marker gene) and the GUS
(ß-D-glucoronidase) reporter gene (representing the gene of
interest), to facilitate monitoring of the transformation process.
Transformation experiments using Agrobacterium were conducted and
transient and stable expression of both marker genes was observed
in leaf cells and callus of C. flammula and cultivar ‘Pagoda’.
Using a biolistic gun for direct DNA transformation and also to
assist transformation with Agrobacterium, transient and stable
expression of both marker genes was detected in somatic embryos of
‘Pagoda’ and GFP expression was observed in regenerating secondary
somatic embryos.
The methods, markers and genetic information from the project
were communicated to the public and industry through publications,
presentations and exhibits at horticultural events and open days.
Know-how relating to chromosome doubling was transferred to several
commercial organisations. Papers to publish in the scientific press
the in vitro methods that have been developed are being drafted.
Plant material from the programme has been passed to
industry-funded breeding programmes and further material is
available for use as parents or for selection, trialling and
commercialisation. Discussions about a possible LINK project on
rose have been held with colleagues and potential commercial
partners. In addition, techniques developed during this project may
be useful for developing other crops, e.g. for sustainable energy
production.
Project Report to Defra
8.As a guide this report should be no longer than 20 sides of
A4. This report is to provide Defra with details of the outputs of
the research project for internal purposes; to meet the terms of
the contract; and to allow Defra to publish details of the outputs
to meet Environmental Information Regulation or Freedom of
Information obligations. This short report to Defra does not
preclude contractors from also seeking to publish a full, formal
scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such
publications as part of the contract terms. The report to Defra
should include:
the scientific objectives as set out in the contract;
the extent to which the objectives set out in the contract have
been met;
details of methods used and the results obtained, including
statistical analysis (if appropriate);
a discussion of the results and their reliability;
the main implications of the findings;
possible future work; and
any action resulting from the research (e.g. IP, Knowledge
Transfer).
BACKGROUND & OBJECTIVES
Our aim was to use conventional and biotechnological improvement
methods to produce novel tools and breeding lines of various woody
ornamentals for the benefit of the UK industry; a spin-off was the
generation of progenies and variants, a few of which may, after
independently-funded selection and trialling, be worth
commercialising as new cultivars. The Hardy Nursery Stock (HNS)
trade, which supplies trees, shrubs and herbaceous perennials to
the gardening public and for amenity planting, has a farm-gate
value of approximately £350 million, of which 10% is attributable
to rose. Novelty is the life-blood of the trade, attracting
customers and stimulating sales. Improvements in e.g. flower size,
foliage form and colour or plant habit, improved quality and
disease resistance are vital for the continued economic growth of
the industry.
Opportunities range from, e.g., rose (Rosa), in which the
industry itself has long been undertaking genetic improvement but
has expressed interest in biotechnological tools we have been
developing, to minor genera such as elder (Sambucus), where major
advances have recently been achieved at East Malling Research (EMR)
with Defra and HDC funding, by conventional breeding and
genetics.
The project sought to address various issues needed to underpin
successful improvement programmes. By genetic studies it could
clarify understanding of the inheritance of key horticultural
characters – affecting, e.g., flowers, foliage, plant habit or
disease resistance – to inform future crossing strategies. It was
able to develop markers for particular characters that can be used
for marker-assisted selection to increase the efficiency of
breeding programmes. It investigated novel sources of disease
resistance (in Rosa) to reduce the need for pesticides. It overcame
blocks in various inter-specific breeding lines by using oryzalin
to double chromosome number and by making crosses to check that
fertility has been restored. It also explored if chromosome
doubling can be used as a generalised method of introducing more
compact habit, especially in climbing plants. In addition it opened
up opportunities for genetic manipulation by developing
regeneration methods and ‘clean’ transformation systems. In doing
this it capitalised on progress in the previous HNS improvement
project HH1026SHN and benefited from interaction with the
Defra-funded project on rosaceous genomics HH3724SSF.
The work was fully consistent with Defra’s Roame A for
sustainable horticulture. This specifically indicates support for
conventional and genome-based techniques to improve ornamentals
with respect to disease resistance and plant quality.
The specific objectives are to:
1. Clarify genetics of horticulturally important characters in
Sambucus, Buddleia, Sorbus and Jasminum to inform breeding
programmes
2. Identify sources of resistance to blackspot, and perhaps
aphids, in Rosa, and transfer breeding lines to a commercial
breeding programme, with a view to subsequently developing a LINK
research programme
3. Demonstrate that tetraploidy has restored fertility to
interspecific hybrids of Syringa and Pyrus x Malus and check for
self compatibility and mode of inheritance, and complete
backcrossing programme of tetraploid Buddleia globosa to B. davidii
to introgress yellow flower colour
4. Determine if tetraploidy in the climbers Clematis and
honeysuckle (Lonicera), and shrubs such as Physocarpus induces more
compact habit, useful for small gardens
5. Develop transformation methods for hardy ornamentals,
assessing the xylose isomerase and MAT vector systems in Buddleia
davidii, and establishing regeneration methods for two additional
subjects, for use in subsequent transformation experiments and
technology transfer to the industry.
METHODS AND RESULTS
Objective 1 - Genetics and Markers
Sambucus:
Two fastigiate purple leaved selections of S. nigra from the
previous programme were identified for possible release. Various
controlled crosses were made to study inheritance of dwarfing in
some breeding lines related to S. nigra ‘Black Beauty’, which comes
from a sib cross between two seedlings of ‘Fastigiata’ × ‘Guincho
Purple’. In 2003 and 2004, two dwarf siblings of ‘Black Beauty’,
528-157 and -175, were backcrossed to the two grandparents and the
resulting seeds sown. The resulting progenies were scored for dwarf
types; approximately 25% of the seedlings were dwarf, suggesting
that the dwarf character is controlled by recessive genes at two
loci. In addition, the dwarf selections were also crossed with
‘Black Lace’ and seedlings raised and then in 2005 two of the
resulting seedlings were selfed. In 2006, the two progenies were
scored. Of 87 surviving seedlings, nine were dwarf, consistent with
the control of this character by recessive genes at two loci. This
information would be useful for breeding dwarf Sambucus for small
gardens. To develop markers for Sambucus, in 2004-05 a library of
genomic DNA of S. nigra ‘Black Beauty’ cloned into bacteria was
prepared and colonies were screened for the likely presence of
simple sequence repeat sequences. ‘Positive’ colonies were
sequenced and, from regions flanking the repeat sequences, primers
were designed and tested for amplification and polymorphism in a
range of cultivars. In this way eight microsatellite primer pairs
were developed for use in fingerprinting and checking parentages
for genetic studies. The primers were employed to fingerprint a
panel of cultivars held in the breeding collection and they
identified some synonyms and homonyms that would otherwise have
caused confusion. This work was published in Molecular Ecology
Notes in 2006.
Buddleia:
A progeny of Buddleia davidii ‘Nanhoensis Alba’ (white flowers,
cloying scent) x B. heliophila (mauve flowers, sweet scent) was
scored for flower characters. The seedlings had sweet rather than
cloying scent, suggesting sweet scent is dominant, and they
segregated ~1:1 for flower colour, confirming a heterozygous
dominant gene for white flower in B. davidii ‘Nanhoensis Alba’.
Chaenomeles:
A progeny from the cross of Chaenomeles superba ‘Tortuosa’
(twisted branches, red flowers) x C. speciosa ‘Nivalis’ (normal
branches, white flowers) was scored for plant habit and flower
colour. The segregations indicated a dominant gene for twisted
branches and a dominant gene for white flowers.
Jasminum:
In 2004, pollen of Jasminum nudiflorum (winter-flowering, yellow
flowers, non-scented) was collected for crossing the following
summer with various scented species. Fruit set resulted from the
cross with J. officinale (summer flowering, white flowers) (See
Table 1); however, the fruit aborted and no seed was obtained. A
local private breeder embarked on a series of Jasminum crosses
after discussion with EMR staff and, to avoid duplication, it was
decided not to pursue further crossing within the project.
Physocarpus:
In 2006, Physocarpus opulifolius progeny Phy 5, from the
intercross of two siblings from ‘Diabolo’ (purple leaves) × ‘Aurea’
(yellow leaves) made in association with EMO, was scored for
purple, yellow, green or orange (i.e. purple and yellow) leaf
colour. On the basis of the segregation, the dominant genes Pur and
Au were proposed for purple and yellow leaf colour respectively.
Samples were provided to the Defra-funded rosaceous genomics
project HH3724SSF so that a genetic map of this representative of
the rosaceous sub-family Spiraeoideae could be developed. DNA
extracts were amplified with AFLP primers and from the
co-segregation data a map was constructed on which Pur and Au were
located. This research has been accepted for publication in Plant
Breeding.
Rhododendron:
In 2004, approximately 50 seedlings were raised from crossing
Rhododendron hirsutum (resistant to lime-induced chlorosis) × R.
ferrugineum 3379 (susceptible) to increase the mapping progeny for
studying the genetics of ‘lime-tolerance’. Twenty seedlings
survive. A larger population would be required for a mapping
project. Twenty-five selections of various Rhododendron progenies
raised in previous projects were identified in the field as
apparently resistant to lime-induced chlorosis (see Table 2). The
plants were dug up in autumn 2006 and cuttings were taken in
May/June 2007 to provide replicated material for trialling. There
are at least three surviving cuttings of each of clones 4, 5, 18,
19 and 20. These are available for trialling for lime tolerance and
garden worthiness.
Ribes:
Progenies of Ribes sanguineum ‘Pulborough Scarlet’ (white bloom
on fruit) × ‘White Icicle’ (black fruit) and ‘Brocklebankii’ (white
bloom on fruit) × ‘White Icicle’ were scored for fruit colour. In
both cases the segregations were consistent with ‘White Icicle’
being heterozygous for a dominant gene for black fruit.
Objective 2 – Rose
On recommendations from the industry, five more roses reported
to have high levels of disease resistance were added to the
collection of cultivars and species accessions that had been
initiated in the previous project. Field resistance to aphids,
blackspot and mildew was assessed in the collection over three
years. Softwood cuttings from resistant and susceptible accessions
were rooted successfully under mist, to provide replicate material
for screening for blackspot resistance. In collaboration with East
Malling’s pathologists, plants were inoculated in a controlled
environment chamber. However this proved unsatisfactory, with
minimal symptoms developing on susceptible controls.
Crosses were made in the field between blackspot resistant and
susceptible diploid rose accessions, e.g. of Rosa rugosa or R.
wichuriana (resistant) and of R. arvensis or R. ecae (susceptible),
and also within and between accessions of R. rugosa to raise
progenies for study of incompatibility. Adverse weather conditions
resulted in poor fruit set of some crosses in 2005 but better set
was obtained in 2006, resulting in some 1100 seeds; selfing of R.
rugosa accessions failed. Seeds were sown and stratified for 16
weeks to germinate in the spring. In the progeny from the cross of
R. arvensis x R. wichuriana about 25% of the seedlings that
germinated were lethal and in that from crossing R. rugosa ‘Typica’
x R. ecae about 50% of the seedlings were miniature and sub-lethal.
None of the species progenies flowered in their first season,
whereas seedlings from crosses among recurrent-flowering cultivars
of R. hybrida, made as part of the Defra-funded project on
branching in nursery stock, flowered within two or three months of
germination. As controlled inoculation with blackspot in a
controlled environment chamber had proved unsatisfactory, the
seedlings were assessed for resistance under natural conditions.
However, assessment should be repeated to distinguish resistant
seedlings from ‘escapes’. The R. rugosa progeny from intercrossing
‘Alba’ (white flowers) and ‘Typica’ (pink flowers) was scored for
flower colour; the segregation of white to pink, approximately 1:1,
indicated that this white flower colour phenotype is attributable
to a dominant gene. Styles from seedlings of this progeny were
collected for ribonuclease analysis, previous work at EMR having
established that incompatibility in cherry and apple is mediated by
stylar ribonucleases.
An in vitro culture of one of the rose genotypes, R. wichuriana,
was initiated as a pre-requisite for e.g. chromosome doubling with
oryzalin.
In collaboration with the rosaceous genomics project HH3724SSF
it was possible to make good progress in the development of rose
microsatellites that would be useful for fingerprinting and/or for
mapping. Clones from a genomic library of Rosa filipes ‘Kiftsgate’
enriched for microsatellites that had been prepared previously were
screened for simple sequence repeats. Primers flanking the repeat
sequences were designed and ‘pigtailed’ with an M13 sequence and
tested for amplification. In all, 10 new microsatellite primer
pairs were developed and tested in 16 accessions, two of each of
the diploid species R. filipes, R. multiflora, R.rugosa and R.
xanthina and eight cultivars of tetraploid R. hybrida.
Interestingly the tetraploid accessions frequently showed only two
alleles and rarely showed four alleles. An account of this work has
been published in Plant Breeding.
Various discussions took place with members of the rose industry
and with colleagues working on disease control and plant habit
about opportunities for a LINK-funded project on rose.. It was
agreed that embryo rescue from difficult crosses would be of use to
commercial breeders, as would chromosome doubling of diploids and
the development of breeding lines and markers for disease
resistance. In response to an expressed need for resistance to
downy mildew, a scheme was proposed in which resistant diploids
identified from the literature and in consultation with colleagues
overseas would be crossed with recurrent-flowering diploids and the
resulting hybrids, which would be seasonal flowering as the
recurrent-flowering gene is recessive, backcrossed to
recurrent-flowering diploids; then resistant recurrent flowering
selections from the backcross generation could be treated with
oryzalin to produce tetraploids for crossing with tetraploid
cultivars so that the resistance can be introgressed into R.
hybrida.
Objective 3 – Assessing restoration of fertility and inheritance
in tetraploids
Buddleia
In 2004, with a view to combining yellow flower colour and large
inflorescence in Buddleia, three second generation tetraploid
hybrids between B. globosa (yellow) flowers and B. davidii (large
inflorescences) were variously intercrossed or selfed (rather than
back-crossed), and seed sown. In 2005 the seedlings were assessed.
None of those that flowered in 2005 had good yellow colour and so
no further crosses were undertaken that year. However, in 2006 when
the remaining seedlings flowered, several were relatively yellow
and were used in further crossing with the aim of improving
inflorescence shape. An infestation of light brown apple moth
inside the pollinating bags destroyed some capsules but some seed
was rescued and subsequently germinated. There is a population of
~200 seedlings that should flower in 2008 for assessment of
horticultural merit.
Pear × apple hybrid
Five tetraploid clones of the pear × apple hybrid PyMa2 were
successfully weaned from in vitro culture into the glasshouse and
four were subsequently propagated successfully on to M26 rootstock.
Likewise, one tetraploid clone of PyMa4, which had been weaned
previously, was grafted on to M26. By the end of the project the
plants had not flowered. When they do flower they should be crossed
with tetraploid Pyrus to show that red leaf and columnar habit can
be introgressed into that species. Diploid material of some PyMas
was supplied to the Apple & Pear Breeding Club for evaluation
as interstocks that may allow pears to be grown on dwarfing apple
rootstocks.
Pear × quince hybrid
Plants of a tetraploid clone of the pear × quince hybrid Pyronia
veitchii were raised by grafting in 2004 onto Quince C but did not
flower before the end of the project. When they do flower they
could, if fertile, be useful to the Rootstock Breeding Club for
breeding rootstocks for pear.
Sambucus
In 2004 several tetraploids of two sterile interspecific hybrids
from the cross Sambucus nigra ‘Guincho Purple’ (purple leaves) × S.
racemosa ‘Tenuifolia’ (dwarf habit, dissected leaves) (209/68 and
209/71), were raised, to check fertility and facilitate further
breeding. When the clones flowered in 2007 they were selfed and
intercossed. Two tetraploid clones of line 209/68 produced seed
pods and four clones of line 209/71 successfully set berries,
whereas the diploids did not set. Future work would entail raising
seedlings and performing further crosses if considered necessary,
to produce dwarf plants with red dissected foliage.
Syringa
In 2003, ten tetraploid clones of Syringa vulgaris × S.
pinnatifolia selection 104-18 and two tetraploid clones of
selection 104-25, along with diploid controls, were replicated by
grafting on to rootstocks for horticultural assessment and testing
fertility. In 2004, six tetraploid clones of a third selection,
104-1, were likewise replicated.
In spring 2003, flowers of one tetraploid clone of selection
104-18 set seeds following pollination with S. vulgaris ‘Primrose’.
One pod developed, and one of the four seed germinated. To allow a
second attempt within the reporting year, plants were forced in
late winter and in 2004 three tetraploid clones of 104-18 were
selfed, intercrossed and backcrossed to the parents, at least 50
flowers per cross (Table 3); however, limited seed set occurred
only when S. vulgaris was pollinated with three of the tetraploid
clones. It is possible that seed set was adversely affected by high
temperatures in the glasshouse, so further crosses were made in a
gauze tunnel. Viability of pollen was assessed by in vitro
germination: approximately 10%, 20% and 22% of pollen grains from
three tetraploid lines (2BC1, 1BB2 and 2BC6) germinated compared to
less than 1% of pollen grains from the diploid control (and 47% of
S. pinnatifolia pollen grains). Flower size in some of the
tetraploid clones was noticeably larger. Mean measurements (cm) of
corolla depth and diameter of the tetraploid line with the biggest
flowers (Clone 2BC1) were 1 and 2, compared with diploid
measurements of 0.7 and 1.5.
In 2005, in a gauze tunnel, seven tetraploid clones of Syringa
vulgaris × S. pinnatifolia selection 104-18 were selfed, up to 70
flowers each, to check fertility; of these, four produced seed pods
(numbering from 4 to 14). In addition six clones were intercrossed,
two crosses resulting in seed pods (Table 4). Seed was sown and
stratified but no seedlings were obtained on this occasion.
In 2006, five tetraploid clones of Syringa vulgaris × S.
pinnatifolia selection 104-1, were intercrossed in a gauze tunnel
with each other and with the original sterile diploid, and four of
these clones and the diploid were selfed, approximately 50 flowers
for each cross (Table 5). Some 400 seeds were harvested from 11 of
the intercrosses and one of the tetraploid selfs. The cross of
104-1 clones 3Fa4 × 3Dc1 (plant 1) was the most fertile. In
addition more than 6000 seeds were collected from pods of 104-1
after open pollination (in all likelihood with other Syringa
vulgaris × S. pinnatifolia tetraploids or by selfing). Furthermore,
four clones of selection 104-18 and two clones of 104-25 were
selfed, between 150 and 650 flowers of each (Table 6). Between 1
and 22 seeds were collected from each of these six selfings and
between 1 and 13 seeds following open-pollination of three 104-18
and one 104-25 clones. Clone 2Bb2 of selection 104-1 and O3-3 of
104-25 were the most fertile. Seed was stratified prior to
germination but only limited germination resulted, apparently
because of infestation with peat fly larvae. A summary of clones
exhibiting fertility, i.e. producing seed pods from controlled or
open pollination, is given in Table 7.
In 2003, tetraploid populations of two hybrids S. × chinensis
'Saugeana' and 'Bicolor', which are sterile as diploids, were
produced by in vitro application of oryzalin, and nine lines of
each were bulked, rooted and weaned. In 2005, eight clones of
‘Saugeana’ and nine of ‘Bicolor’, were replicated by grafting onto
rootstocks for subsequent assessment of fertility.
A chromosome doubling experiment was performed with S. vulgaris
‘Vestale’ × S. pinnatifolia (selection 48-36); however, no
tetraploid clones were produced.
We now have extensive knowledge of the compatibilities and
potential fertility of the tetraploid lines and this knowledge
should be applied to produce pinnate-leaved large-flowered
lilacs.
Objective 4 -– Tetraploid induction in climbing plants and
shrubs
In 2003, in vitro cultures of two climbing plants, Clematis
flammula and Lonicera periclymenum ‘Graham Thomas’, and the shrub
Physocarpus opulifolius 764/3 (= ‘Diabolo’ × ‘Dart’s Gold’) were
established and multiplied to provide sufficient material for
chromosome doubling experiments to investigate the effects of
tetraploidy on plant habit. In vitro explants were exposed to a
range of levels of the anti-mitotic herbicide oryzalin (0-100 µM)
for a period of 1 to 3 days using liquid medium or for 3 days to 2
weeks using semi-solid medium. The response and survival of the
material in these initial experiments enabled targeting of a
narrower range of treatments using a larger number of explants in
subsequent experiments. In C. flammula, tetraploid shoots were
successfully obtained from two experiments and weaned. Likewise
tetraploid shoots of L. periclymenum were produced and weaned. The
first three experiments with P. opulifolius produced only mixoploid
(2x-4x) shoots, but a fourth experiment yielded tetraploid shoots
for rooting and weaning. The tetraploid clones of Clematis,
Lonicera and Physocarpus were replicated by cuttings in 2005 for
comparison with the respective diploids from which they derived.
Six tetraploid clones C. flammula were successfully replicated;
however by the summer of 2007 none of these had flowered so
comparisons were not possible. In the case of Lonicera there are 15
replicated tetraploid clones, some of which flowered and underwent
preliminary evaluation in 2007 and, of these, four appeared to have
a more compact habit and larger flower size. Because flowering of
the tetraploid L. periclymenum was poor in 2007, sufficient data
for statistical analysis could be collected from only the diploid
clone and one tetraploid clone, clone 10 (Table 8): the tetraploid
clone produced significantly broader and longer flowers overall
than the diploid and the flowering shoots comprised fewer
internodes and were shorter in overall length, although differences
in mean internode length were not statistically significant. The P.
opulifolius proved difficult to keep alive once weaned and also to
propagate in the greenhouse; from eight tetraploid clones weaned
only three remain, clones 49, 111, 150, from which further cuttings
were taken in 2007. Because of the difficulties experienced in
propagation, replicated plants of sufficient maturity of the
Physocarpus were not available by 2007 for comparison.
The tetraploids may prove to be superior to their diploid
equivalents in some subjects. Several of the Lonicera are more
compact and have larger flowers but replicated trialling would be
required to assess their garden worthiness. There were no obvious
differences in plant habit between the diploid and tetraploid
Clematis; however these plants had not flowered by the final year
of the project. It is not possible to comment on the effects of
tetraploidy in Physocarpus, because of the difficulties encountered
with propagation.
Objective 5 – Transformation and regeneration methods
Micropropagation and Regeneration
In all regeneration experiments the abaxial leaf epidermis was
placed in contact with the medium, with 10 explants per 9 cm Petri
dish, and cultures were maintained at 240C in dark unless otherwise
stated. Media pH was 5.6 before autoclaving. All components were
autoclaved except ancymidol and STS. Oxoid No. 3 agar (0.75%) or
Sigma Agar (0.8%) were used for shoot proliferation and
regeneration experiments, respectively, unless otherwise
stated.
In 2003, eleven subjects were chosen for regeneration studies
and established in vitro: three Malus, Choisya ternata, Syringa
vulgaris, three Lonicera periclymenum and three Clematis. Following
initial observations and regeneration experiments (with Syringa,
Lonicera and Clematis), representatives of the two horticulturally
important genera Lonicera and Clematis were chosen for further
regeneration experiments (Table 9). Initial experiments with both
genera compared basal media, carbohydrate source and plant growth
regulators; the percentage of in vitro leaf explants regenerating
shoots and shoot primordia were: Clematis ‘The President’ 10%;
Clematis flammula 90%; Lonicera ‘Belgica’ 20%; Lonicera ‘Graham
Thomas’ 7%; and Lonicera ‘Honeybush’ 7%. Regeneration experiments
focused initially on optimising conditions for adventitious shoot
regeneration (Figure 2).
In 2004, improved protocols for shoot regeneration were
developed for Lonicera ‘Belgica’, ‘Graham Thomas’ and ‘Honeybush’.
Four other Lonicera species (× americana, × heckrotii, implexa and
sempervirens) were initiated in vitro and shoot regeneration
experiments were begun. In 2005, using the protocol developed for
Lonicera periclymenum, in vitro shoot regeneration was successfully
achieved with two more honeysuckles, L. × americana and L. ×
heckrotii but not with L. implexa and L. sempervirens (Figure 1).
Shoot proliferation medium for L. periclymenum is WPM (McCown Woody
Plant Medium) basal medium with cytokinin BAP (6-benzylaminopurine)
0.131 mg/l and 3% sucrose (gelling agent Sigma Agargel 0.6%). L. ×
heckrottii and L. × americana performed satisfactorily on this
medium, but L. implexa, L. sempervirens and L. tellmanniana did
not; so in 2006 shoot proliferation experiments were performed with
these three subjects. WPM medium was compared with MS (Murashige
and Skoog basal medium) with BAP 0.131 mg/l, and 3% sucrose was
compared with 3% fructose. On all media, shoot quality and
proliferation was poor but overall it appeared that L. implexa
performed best on WPM with sucrose, L. sempervirens on WPM with
fructose and L. tellmanniana on MS (less chlorosis) with fructose.
As the healthiest shoots were observed to have roots, in the next
experiment the media determined to be best for each cultivar were
supplemented with 0.1 mg/l auxin, IBA (indole-3-butyric acid) or
NAA ((-naphthaleneacetic acid), or with 0.1 mg/l IBA plus 162 mg/l
phloroglucinol (Ph), in an attempt to stimulate rooting. For L.
implexa there were more actively growing shoot tips on NAA, but
otherwise no treatment difference was observed for any of the
species. In a third experiment, using the same basic media for each
species as for experiment 2, the treatments were 0.1 mg/l NAA, 0.1
mg/l NAA plus 162 mg/l Ph, 162 mg/l Ph, 0.1 mg/l GA (gibberellic
acid), and 0.1 mg/l GA plus 0.1 mg/l NAA. L. implexa showed
increased axillary branching and overall quality (greener leaves,
shoot growth); L. sempervirens shoot quality was poor generally,
especially on NAA with Ph; and L. tellmanniana shoots were poor,
with no overall treatment differences observed. A final experiment
compared the cytokinins BAP, 2-iP (2,4-dichlorophenoxyacetic acid)
and kinetin (at 0.2 and 1 mg/l), with basal medium MS plus 3%
sucrose for all species. There was no obvious treatment for L.
implexa whereas shoot quality for L. sempervirens and L.
tellmanniana seemed best on BAP 1 mg/l. There were no further
attempts at optimisation for Lonicera as a decision was made to
concentrate on clematis, which is of more commercial
importance.
In 2004, two regeneration experiments were carried out with
Clematis flammula and Clematis ‘The President’ which demonstrated a
requirement for auxin for shoot regeneration. Twelve more
large-flowered hybrids representing five major recognised Clematis
groups were obtained and meristems initiated in vitro; meristems of
eight hybrids (from all five groups) survived. A range of media
were subsequently tested and efficient protocols established for
five cultivars which were then bulked up for regeneration
experiments. Shoots were successfully regenerated from explants of
three hybrids, ‘Pagoda’, ‘Princess Diana’ and ‘The President’
(Tables 12 to 16). All cultivars regenerated a mixed callus, a
proportion of which was of globular structure resembling
early-stage somatic embryos; a germinated embryo with secondary
embryos structures was observed in ‘Pagoda’. Papers were drafted
detailing regeneration procedures for clematis and honeysuckle and
experiments to provide further data were still in progress before
submission to a journal for publication. In 2007-08, shoot
proliferation and regeneration experiments with clematis were
completed. Micropropagation protocols were developed for C.
flammula and for the hybrids ‘Henryi’, ‘Madame Julia Correvon’,
‘Mrs Cholmondeley’, ‘Niobe’, ‘Pagoda’, ‘Princess Diana’ and ‘The
President’. Further optimisation of media would be required for
some of these. See Table 9 for a summary of all clematis initiated
in vitro and micropropagation media developed for each.
Experimental details for micropropagation experiments with C.
flammula, ‘Madame Julia Correvon’, ‘Pagoda’, ‘Princess Diana’ and
‘The President’ are presented in Tables 10-16.
In addition, sufficient clean cultures of the crab apple Malus
‘Red Sentinel’ were bulked up for regeneration experiments. ‘Red
Sentinel’ proved recalcitrant for adventitious shoot regeneration
on media previously found to be successful for other Malus. On 1
mg/l cytokinins TDZ (1-phenyl-3-(1,2,3,-thiadiazol-5-yl) urea) or
BAP with 0.1 mg/l auxins NAA or IBA, a white friable callus
developed, but very few shoots were observed.
The shoot proliferation and regeneration protocols developed
within this project provide useful tools for improvement by methods
such as chromosomal doubling, mutation induction and genetic
modification.
Somatic Embryogenesis
When somatic embryos were observed on some treatments,
experiments were also conducted to develop methods for somatic
embryo induction and maintenance. The main parameters investigated
were basal media, sugar type, growth regulators, ethylene
inhibition and incubation in light or dark. It was decided to
concentrate on the subjects, C. flammula, ‘Princess Diana’, ‘The
President’, and ‘Pagoda’ which were amenable to adventitious
regeneration. An experiment comparing basal media and sugar type
was performed for C. flammula: there was no overall difference in
adventitious shoot regeneration between basal media MS, DKW and
WPM; however overall regeneration was greater on sucrose than on
glucose (Table 17). To investigate the effect of sucrose
concentration, ethylene inhibition and illumination on development
of apparently pro-embryogenic callus in ‘Princess Diana’, calli
excised from leaf explants were transferred to media (MS) with the
cytokinin kinetin 1 mg/l, no auxin, earlier observations having
indicated that explants on cytokinins kinetin and BAP responded
similarly. The variables were sucrose 30 g/l or 60 g/l and 30 g/l +
20 µm STS (for inhibition of ethylene) and culture was in light or
dark for each medium variable. Calli were scored for the
development of pro-embryonic ‘globular stage’ (early and advanced
stages) callus. More explants regenerated advanced stage globular
callus in the light than in the dark (see Table 18) and on 30 g/l
sucrose than on 60 g/l sucrose but STS did not have an effect.
In a further experiment, leaf explants of C. flammula, ‘Princess
Diana’ and ‘The President’ were inoculated onto high and low auxin
first stage treatments (dark incubation) and transferred after four
weeks onto a second stage treatment (light incubation) with low
auxin or no auxin for four weeks before assessment. The cytokinin
and auxin used for each cultivar was dependent on preliminary
observations with small scale experiments. Explants were scored for
callus type, somatic embryos and shoots, the type of callus being
categorised as early globular (EG, very early stage pro-embryonic),
translucent globular (TG, later stage pro-embryonic) or opaque
globular (OG, later stage pro-embryonic). An attempt was made to
correlate callus type with somatic embryos and shoot regeneration,
on an explant basis.
For C. flammula (Table 19), somatic embryo regeneration was
greatest on treatments with BAP and a high auxin first stage
(followed by a second stage with low or no auxin). Very few shoots
regenerated on kinetin and very few somatic embryos regenerated on
the cytokinin TDZ. In a subsequent experiment (Table 20) a high
auxin first stage of four weeks (as for the preceding experiment)
was compared with one week and the effects of light and dark
incubation and STS were assessed. It was difficult to distinguish
between shoots and germinated embryos so scoring was for
‘regenerants’. Significantly more explants regenerated shoots or
embryos when illuminated compared to incubation in the dark. There
was an apparent trend for increased regeneration with STS and after
a four week high auxin pulse, but the differences were not
statistically significant.
For ‘Princess Diana’ (Table 21), overall, explants on BAP from
high or low auxin first stage (with no auxin second stage)
regenerated more advanced stage pro-embryonic callus than those on
media with auxin in second stage media. Only one somatic embryo
regenerated, on BAP low auxin first stage, no auxin second stage
and shoots regenerated only on BAP. Treatments with BAP and auxin
second stage regenerated no OG callus. There is a highly
significant positive correlation between explants with advanced
stage (OG and TG) pro-embryonic callus, and those with embryos and
shoots.
For ‘The President’ (Table 22), overall, explants on a BAP +
high auxin first stage (and low auxin second stage) regenerated
more OG callus compared to kinetin with high auxin or low auxin
first stage treatments. Only very few somatic embryos and shoots
regenerated; shoots developed only on BAP media. There was no
observable effect of adding STS to the kinetin media. On an explant
basis, there is no statistically positive correlation between
advanced (OG) callus and embryo regeneration, but there was a
strong negative correlation between less advanced pro-embryogenic
(TG) callus and embryo regeneration.
For ‘Pagoda’ initial small scale experiments indicated that, on
kinetin (with low auxin), explants exhibited less browning than on
BAP (with low auxin) or media with high auxin, and developed more
globular stage callus. A few somatic embryos were observed on this
medium, without a need for a high auxin first stage. Various
parameters were investigated to develop an improved protocol for
somatic embryo regeneration. Culture was in darkness throughout due
to severe browning of explants when cultured in a lit incubator.
Leaf and stem (internodal) explants responded similarly, and
addition of STS was detrimental to somatic embryo regeneration
(Table 23). Response of leaf explants to the auxins NAA and 2,4-D,
kinetin no auxin, and the gibberellic acid inhibitor ancymidol was
determined (Table 24). There is a strong negative correlation
between the presence of embryos and early stage (EG) and mid-stage
(TG) callus. Explants on media with ancymidol 0.1 mg/l and 1 mg/l,
on 2,4-D and without auxin all regenerated more TG callus compared
to NAA and significantly more explants on 2,4-D than on NAA and
medium without auxin regenerated OG callus. There was a requirement
for auxin for embryo regeneration – on medium without auxin very
few embryos developed. There was an indication that 2,4-D was more
effective than NAA in promoting somatic embryo regeneration, so in
a subsequent experiment these were again compared, as were
increased levels of ancymidol, ancymidol in combination with 2,4-D
and the sugars sucrose, glucose and fructose (Table 25). Explants
on medium with both 2,4-D and ancymidol 1 mg/l regenerated
significantly more embryos than those on NAA, NAA with 1 or 5 mg/l
ancymidol, but not compared to 2,4-D with no ancymidol. Increasing
the level of ancymidol to 5 mg/l appeared to be detrimental.
Significantly more explants regenerated embryos on sucrose than on
glucose and fructose.
Proliferating embryogenic cultures of ‘Pagoda’ can be maintained
indefinitely on kinetin 1 mg/l, NAA 0.1 mg/l by subculture of
embryos, embryo clusters, and detached maturing and mature
cotyledons, from which secondary embryos regenerate. Embryos of C.
flammula, ‘Princess Diana’ and ‘The President’ did not proliferate
on the second-stage medium that enabled somatic embryo
regeneration; however they may proliferate by manipulating growth
regulator regimes. Liquid culture (shaken) of embryos and leaf
material of ‘Pagoda’ showed increased organogenesis compared to
culture on semi-solid medium, but regeneration was very slow from
previously submerged embryonic structures after transfer to
semi-solid medium, and abnormal structures predominated; however
use of a liquid culture system with improved aeration may result in
increased proliferation with normal development. Detached embryos
of ‘Pagoda’ developed into apparently normal rooted shoots upon
transfer to MS medium (3% sucrose) without growth regulators.
Transformation – Agrobacterium
In all transformation experiments using Agrobacterium, bacterial
cultures were grown to OD600, pelleted and resuspended in liquid MS
medium with 2% sucrose for inoculation. After blotting to remove
excess bacterial suspension, explants were transferred to the
appropriate regeneration medium and co-cultivated in the dark at
240C for three days, then transferred to regeneration medium
containing ticarcillin/clavulanic acid (TCA) 400 mg/l (unless
otherwise stated) for inhibition of Agrobacterium re-growth and the
appropriate selection agent.
To develop non-antibiotic transformation protocols with Buddleia
davidii ‘Nanhoensis Alba’, a binary vector pNOV2819 with T-DNA
construct for constitutive expression of phosphomannose isomerase
(Figure 5) was obtained from Syngenta and transformed into
Agrobacterium EHA101. Regeneration of in vitro leaf explants was
assessed on media with various levels of mannose and sucrose:
approximately 10% of explants regenerated shoots on medium with 2%
mannose (plus 3% sucrose), compared with 80% regeneration on medium
with 3% sucrose only and no regeneration on 3% mannose plus 3%
sucrose. An initial transformation experiment was performed,
comparing two selection strategies based on these mannose tolerance
results but transformed shoots were not obtained.
In 2004, four further transformation experiments were performed
with Buddleia davidii ‘Nanhoensis Alba’ employing various mannose
selection strategies (see Table 26). All plates were cultured in a
dark incubator at 240C. Ticarcillin/clavulanic acid, which is more
effective at suppressing Agrobacterium growth than Cefotaxime
(carbenicillin), was used from experiment 2 onwards. A comparison
with Cefotaxime indicated a small increase in regeneration with
TCA. Because of this and the slight beneficial effect of
filter-sterilised mannose compared to autoclaved mannose, the level
of mannose selection was increased in experiments 3 and 4. The
selection was based on a successful strategy published for sugar
beet. Explants were cultured initially on the mannose concentration
that allowed shoot regeneration from 10% of explants, and mannose
selection was increased in subsequent passages. Sucrose was kept at
30 g/l throughout. Organogenic calli developed from 0.3-1.5% of
explants (but not from control explants) on selection. Calli
developing shoot primordia (including calli with shoots
regenerating on plates without selection) were transferred to shoot
development medium with 30 g/l sucrose, mannose 50 g/l and ‘TCA
200’ under lights. Control (non-transgenic) plates on selection did
not develop organogenic calli. None of the putative transformed
shoots survived due to Agrobacterium overgrowth; however control
(non-transgenic) calli with shoots from non-selection medium
inoculated onto this medium did survive and become green and
because of this it is highly likely that any putative transgenic
shoots would not be transformed. A possible explanation for the
survival of non-transgenic shoots is that photosynthesis in vitro
occurs at a level sufficient to overcome the inhibitory effects of
the mannose, and/or that callus and regenerated shoots have a
different mannose/sucrose tolerance.
The mannose selection strategy was based on the response of leaf
material to a range of mannose and sucrose concentrations
determined initially, and the regeneration of organogenic calli on
selection only from leaf inoculated with Agrobacterium indicates
that the strategy is basically sound; however the problems of
selection and survival beyond this stage needed to be addressed. As
the callus was comprised of a mixture of transformed and
untransformed tissue an experiment was devised to determine the
response of calli (on regeneration medium in the dark) and
regenerated shoots (on shoot proliferation medium under lights) to
a range of mannose and sucrose concentrations (Table 27). It was
decided therefore to determine the mannose dose response of
regenerated calli and regenerated shoots prior to further
transformation experiments (Figure 4).
Mannose dose response was determined with Lonicera periclymenum
‘Honeybush’ using the optimised regeneration protocol (Figure
5).
The MAT (multi-auto-transformation) vector components were
requested in August 2004 and eventually obtained in mid-March 2005
from Nippon Paper Industries, Japan. Following selection of
transformed tissue this should enable excision of the selectable
marker and production of marker-free transformed plants.
In 2005, the MAT vector was successfully constructed for
production of marker-free transformed plants using phosphomannose
isomerase (PMI) for initial selection. This was a more substantial
piece of work than originally expected: as limited information was
received for the constructs pNPI128 and pNPI300 (Figure 6), it was
necessary to clone them into a well-characterised cloning vector to
determine restriction enzyme sites and fragment sizes before
proceeding with cloning. Subsequently, the PMI construct was
excised from the binary vector pNOV2819 and cloned into the
recombination construct, and the inducible promoter construct was
inserted upstream of this. This recombination/selection construct
was then inserted between the left and right borders of the T-DNA
in the binary vector (from which the PMI construct had previously
been excised). An initial transformation experiment using this MAT
vector was carried out on Lonicera periclymenum ‘Honeybush’ and
experiments with Buddleia were planned for spring 2006. A marker
gene encoding ß-D-glucoronidase (GUS) under a constitutive promoter
was cloned into pNOV2819, to use as a control vector for
transformation experiments. In addition, the GUS gene was being
inserted between the left T-DNA border and the recombination system
in the MAT vector, where it would represent the ‘gene of interest’
to prove the efficacy of the system. In both cases the presence of
the GUS gene will facilitate the detection of transformed
tissue.
In 2006, initial experiments were performed with Lonicera and
Buddleia using the PMI gene encoding for selection of transformed
tissue tolerant to mannose. However, mannose proved inefficient as
a selection agent in both subjects. Information was received from
other workers experiencing difficulty in developing mannose
selection systems in other subjects. Therefore the MAT vector was
re-constructed, replacing the PMI gene with the neomycin
phosphotransferase (npt II) gene for selection using kanamycin. A
marker gene encoding a modified green fluorescent protein (GFP),
sGFP(S65T)-NOS construct (Sheen et al., 1995), was obtained and
inserted within the recombination/selection (R/RS) construct to
facilitate monitoring of transformation events and subsequent
marker gene excision. MAT vector construction was completed by
cloning the npt II-GFP R/RS construct between the left and right
borders of the T-DNA in the binary vector and a visible (GUS)
marker gene, representing the ‘gene of interest’, was cloned within
the right T-DNA border. The binary vector was called pEM-RS1
(Figure 7).
Transformation experiments using Agrobacterium EHA101/pEM-RS1
were performed with ornamental crab apple (Malus x robusta ‘Red
Sentinel’), honeysuckle (Lonicera periclymenum ‘Honeybush’) and
clematis (C. flammula) and four days after inoculation transient
expression of the GFP gene was observed only in clematis by
fluorescence microscopy. However, GUS gene expression was observed
in regenerating calli of Malus ‘Red Sentinel’ and Lonicera
‘Honeybush’. As clematis is of most commercial importance, it was
decided to focus on transformation of C. flammula and hybrids for
which there were shoot regeneration protocols. Three transformation
experiments were performed with C. flammula using different
kanamycin selection strategies (kanamycin concentrations were
increased to 75 and 100 mg/l, which inhibited regeneration from
control explants). GUS gene expression was observed in most calli
(although most of these also comprised a varying proportion of
non-transgenic tissue) and some calli were seen to fluoresce.
However, no transformed shoots were regenerated.
In a transformation experiment with Clematis ‘Pagoda’, leaves
and stem pieces were inoculated with EHA101 pEM-RS1. Selection was
on medium containing kanamycin 50 mg/l. Fluorescent calli
regenerated and also some embryos but these were not
transformed.
Transformation – Biolistics
A different approach was adopted in subsequent experiments with
‘Pagoda’: a large stock of somatic embryos had been generated and
it was decided to inoculate embryogenic cultures. If stable
transformation could be achieved in a single cell of a developing
cotyledon, this could develop into a transgenic sector from which
transformed secondary embryos may regenerate. For targeting
individual cells a Biorad Biolistic PDS-1000/He gun was used,
essentially following the manufacturer’s instructions. Embryogenic
cultures were inoculated onto the centre of a Petri dish (covering
an area 2.5 cm in diameter) and 3 mg of prepared 1µm gold
microcarrier particles were coated with 5 µg of pEM-RS1, sufficient
for six shots. Vacuum pressure used was 28 inches Hg, rupture disc
pressure was 1100 psi. The Petri dishes were placed variously at 6,
9 and 12 cm distance from the stopping screen. At 6 cm sample
disruption occurred. Following bombardment the samples were
incubated at 240C in the dark. After four days, scattered
fluorescing cells were observed. The highest number were seen on
explants from the 6 cm treatment, with obviously fewer resulting
from the 9 cm stopping distance and fewer still from the 12 cm
stopping distance. After a further three days transient expression
was still seen; however subsequently no stable fluorescence was
observed.
Transformation –Agrobacterium versus Biolistics
It was decided to compare Agrobacterium-mediated transformation
with direct DNA bombardment. To facilitate wounding and access of
Agrobacterium the biolistic gun was used, i.e. the microcarriers
were prepared as for DNA except that the DNA precipitation steps
were omitted. Following the 100% ethanol wash step they were mixed
with 60 µl bacteria suspended in MS 2% sucrose to OD600 1.0
(instead of 60 µl ethanol as for the DNA-coated microcarriers). It
was not possible to spread this aqueous suspension over the
macrocarrier surface in an even, continuous layer, so 10 µl was
spotted onto the surface of each macrocarrier in evenly spaced
aliquots of approximately 0.5 µl volume. A total of twelve plates
were bombarded, six with DNA-coated microcarriers (as before) and
six using microcarriers mixed with EHA101/pEM-RS1. The stopping
distance was 6 cm throughout. After four days the Agrobacterium
treated material was briefly washed in a solution of TCA 400 mg/l,
blotted and transferred to medium with TCA 400 mg/l. Kanamycin
selection was not applied to any of the treated embryos. It was
observed that the number of cells transiently expressing GFP were
much greater in the embryos bombarded with DNA but were very few in
embryos treated with Agrobacterium. One month after bombardment a
few fluorescing sectors were seen on both DNA and Agrobacterium
treated material. Two months after bombardment green fluorescence
was observed in an embryo from Agrobacterium treated material. This
is taken to be GFP expression and indicates stable transformation;
however confirmation of this would require molecular analysis. In
addition, three months after bombardment embryogenic cultures were
destructively assayed for GUS expression, using X-gluc
(5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid), sodium salt, as a
substrate. The product of cleavage of the substrate was a water
insoluble blue precipitate at the site of enzymatic cleavage. In
both DNA and Agrobacterium bombarded cultures, blue sectors were
visualised on cotyledons. This further indicates that stable
transformation of embryo tissue has occurred and that transgenic
cotyledonary sectors have subsequently developed from which
transgenic secondary embryos may develop. It shows that it would be
possible to use this method to generate a population of
transgenics.
For this work to be publishable it would be necessary to perform
further experiments to generate a population of proven transgenics.
Excision of the cassette that includes the selectable marker gene,
transgenic lines would be accomplished by culture on medium
containing the herbicide Safener®. Optimisation of this procedure
would be facilitated observation of GFP expression, i.e.
marker-free tissue would not fluoresce. Analysis of marker-free
lines would be by detection of the GUS gene, PCR and Southern
blotting.
ACTIONS ARISING
Intellectual Property
Sambucus nigra ‘Black Lace’, which was developed in the previous
project, proved to be a great commercial success, earning
substantial royalties in the UK and overseas. European Plant
Breeders Rights and a US Plant Patent were granted. Two other
selections of S. nigra from the previous programme, with bronze
leaves and fastigiated habit, were identified for possible release
and multiplied. Twenty-five selections of Rhododendron identified
as apparently tolerant to lime were lifted from the field in autumn
2006 and propagated for commercial evaluation. Various selections
were made available to East Malling Ornamentals (EMO) for use in
breeding. Pear × apple (PyMa) hybrids were supplied to the Apple
& Pear Breeding Club for evaluation as interstocks and a
tetraploid pear × quince hybrid is available for breeding
rootstocks for pear.
Publications
· Tobutt, K.R. and Wilson F (2003) Defra’s HNS improvement
project at HRI-East Malling. Plant It! Newsletter 3:6
· Clarke, J.B. and Tobutt, K.R. (2006) Development of
microsatellite primers and two multiplex polymerase chain reactions
for the common elder (Sambucus nigra). Molecular Ecology Notes, (in
press)
· Wilson, F.M. ‘Selectable Marker-Free Transformation of
Vegetatively Propagated Woody Ornamental Plants using a GST-MAT
Vector System’. Abstract published in Programme and Abstracts of
‘Plant Transformation Technologies’ conference, University of
Vienna, 4-7 February 2007
· Sutherland, B.G., Tobutt, K.R., Marchese, A., Paternoster, G.,
Simpson, D.W., Sargent, D.J. (2008). A genetic linkage map of
Physocarpus, a member of the Spiraeoideae (Rosaceae), based on
RAPD, AFLP, RGA, SSR and gene specific markers. Plant Breeding (in
press)
· Sutherland, B., Clarke, J.B., Wilson, F, Sargent D.J., Tobutt
K.R. (2008) Development and characterisation of ten polymorphic
microsatellite markers for fingerprinting Rosa accessions. Plant
Breeding (in press)
Presentations
· Tobutt, K.R. ‘Breeding fruit, broad-leaved timber trees and
woody ornamentals’, talk to visitors attending the launch of EMR,
East Malling, 17 March 2004
· Hardie, I.W. & Twomey, U. Launch of Sambucus nigra ‘Black
Lace’, on Notcutts stand at Chelsea Flower Show, London, May
2004
· Wilson, F.M. ‘In vitro methods for improving ornamentals’.
Presentation to Institute of Biology, June 2005
· Tobutt, K.R. & Wilson, F.M. ‘Rose-related research at EM’.
Presentation to British Rose Growers Association committee at East
Malling Research, July 2005
· Tobutt, K.R. & Wilson, F.M. ‘Improving woody ornamentals
at East Malling’. Talk to Kent Nursery Stock Group, at East
Malling, August 2005
· Wilson, F.M. ‘Defra-funded Hardy Nursery Stock Improvement’.
Poster presentation at ‘Trade’ Open Day, East Malling Research,
September 2005
· Wilson, F.M. ‘Introducing Blackspot Resistance from Diploid
Rose Species’. Poster presentation at ‘Trade’ Open Day, East
Malling Research, September 2005
· Wilson F.M. ‘Techniques and breeding lines for the genetic
improvement of hardy nursery stock’. Presentation to Defra Project
Reviews, London, September 2005
· Tobutt, K.R. & Wilson, F.M. ‘Defra-funded HNS improvement
at East Malling Research’. Presentation to the Horticulture
Development Council HNS panel, at East Malling Research, September
2005
· Tobutt, K.R. ‘Aspects of perennial plant breeding relevant to
A-level syllabus’. Presentation to pupils of Sutton Valence School,
at East Malling Research, February 2006
· Wilson, F.M. ‘Applications of in vitro techniques in the
improvement of ornamental plants’. Presentation to pupils of Sutton
Valence School, at East Malling Research, February 2006
· Wilson, F.M. ‘Selectable Marker-Free Transformation of
Vegetatively Propagated Woody Ornamental Plants using a GST-MAT
Vector System’. Poster presentation at ‘Plant Transformation
Technologies’ conference, University of Vienna, 4-7 February
2007
· Tobutt, K.R. ‘Opportunities for Hardy Nursery Stock
Improvement at East Malling – conventional breeding’. Presentation
to the Horticultural Trades Association (HNS Committee), at East
Malling, February 2007
· Wilson, F.M. ‘Opportunities for Hardy Nursery Stock
Improvement at East Malling – biotechnology’. Presentation to the
Horticultural Trades Association (HNS Committee), at East Malling,
February 2007
· Tobutt, K.R. ‘Genetic improvement of rose’. Talk to Kent
Horticultural Discussion Group, at East Malling, 31 July 2007
· Wilson, F.M. ‘Hardy Nursery Stock Improvement at East Malling
Research’. Presentation to Kent Horticultural Discussion Group, at
East Malling, 31 July 2007
· Sosa, P.A., Gonzales-Perez, M.A. & Clarke, J.B. ‘Genetic
characterisation of Sambucus palmensis in the Canary Islands’.
Poster presentation at International Biogeography Meeting,
Tenerife, December 2007
· Wilson, F.M. ‘Ploidy Manipulation’. Presentation at EMRA
Members’ Day, ‘A Day of DNA’, 8 November 2007
· Tobutt, K.R. ‘Rosaceous genetics and genomics research at
EMR’. Presentation at Rosaceous Genomics Workshop, Aylesford
Priory, 5 December 2007
Other technology transfer
· The launch of ‘Black Lace’ at the Chelsea Flower Show (2003)
was reported widely in the trade and national press and on
television
· David White (Paul Chessum Roses) presented an overview of
current and proposed work on roses at EMR to leading UK rose
producers, spring 2005
· Opportunities for Jasminum improvement were discussed with a
private breeder
· Benefits arising from chromosome from doubling, e.g. increased
flower size and restoration of fertility in lilac, have been
communicated to the industry at several of the presentations listed
above
· The in vitro techniques have been applied and further
developed by East Malling Ornamentals Ltd with the aim of producing
a range of novel garden plants
· An adaptation of the chromosome doubling techniques to
germinating seed was communicated to a commercial seed breeding
company
· Microsatellite markers developed for Sambucus nigra were used
by a Spanish group to study the population genetics of S.
palmensis, an endangered species in the Canary Islands; an EMR
staff member was included as a co-author
· In February 2007, a proposal outlining opportunities for hardy
nursery stock improvement resulting from this project was submitted
to the Horticulture Development Council HNS panel for
consideration
· Meetings were held with representatives of the rose
propagation and wholesale industry involved with the Royal National
Rose Society and the British Rose Growers Association, to discuss
how our work on rose could be applied to the needs of the industry:
there was particular interest in opportunities for molecular and
biotechnological methods for developing pest and disease resistant
breeding lines
· Using techniques developed within the project as a basis for
work on other subjects, EMO produced tetraploid versions of a range
of subjects including Syringa vulgaris, Sambucus nigra ‘Black Lace’
and Kerria japonica ‘Golden Guinea’
· Opportunities for chromosome doubling were discussed with a
commercial breeder
· The techniques were also being applied in an EMO Defra-funded
project on production of polyploid clones of lavender for improved
oil quality and yield
OPPORTUNITIES ARISING FROM WORK
The project has been successful in developing breeding lines,
information and techniques that should be taken up and carried
forward by the industry. As specified above, there are now a number
of breeding lines and a wealth of genetic information that are
likely to be useful in future commercial breeding programmes. In
vitro protocols developed within the project during this project
may also be useful for other crops. For example, the clones of
Miscanthus currently favoured for biofuels are mostly sterile
interspecific triploids and must be propagated vegetatively; using
chromosome doubling techniques it could be possible to create
fertile hybrids that could be propagated by seed. In addition,
polyploid characteristics such as fewer (but larger) stomata,
altered root systems, shorter internodes and thicker leaves might
enhance crop performance under conditions of environmental stress
by affecting traits such as water use efficiency, photosynthesis
and carbon fixation. And the transformation systems developed using
the MAT vector could be applied to various crops to produce
selectable marker free plants expressing traits such as, e.g.,
compact habit, disease resistance, drought tolerance, improved
nitrogen use efficiency or increased yield.
As explained, there is considerable interest from the rose
industry in opportunities for markers and novel resistance lines
and there have been various discussions to explore LINK
funding.
References to published material
9.This section should be used to record links (hypertext links
where possible) or references to other published material generated
by, or relating to this project.
Refereed publications
· Clarke, J.B. & Tobutt, K.R. (2006) Development of
microsatellite primers and two multiplex polymerase chain reactions
for the common elder (Sambucus nigra). Molecular Ecology Notes (in
press)
· Sutherland, B.G., Tobutt, K.R., Marchese, A., Paternoster, G.,
Simpson, D.W. & Sargent, D.J. (2008). A genetic linkage map of
Physocarpus, a member of the Spiraeoideae (Rosaceae), based on
RAPD, AFLP, RGA, SSR and gene specific markers. Plant Breeding (in
press)
· Sutherland, B., Clarke, J.B., Wilson, F, Sargent D.J. &
Tobutt K.R. (2008) Development and characterisation of ten
polymorphic microsatellite markers for fingerprinting Rosa
accessions. Plant Breeding (in press)
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