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Title Page
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Table of Contents
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Page vii
Chapter 1. Introduction
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Page 2
Page 3
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Chapter 2. Review of the literature
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Chapter 3. Materials and methods
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Chapter 4. Results and discussion
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Chapter 5. Summary and conclusions
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Appendix A. Tissue culture and protoplast media
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Appendix B. Molecular biology buffers
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Literature cited
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Biographical sketch
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|Full Text
There are many people I wish to thank for their contributions to
this work. First and foremost, I wish to thank my major professors- Dr.
Jude Grosser and Dr. Ken Derrick for their guidance, support and
friendship. I would also like to thank Dr. Chuck Niblett for his
contribution of the plasmids used in this study, Dr. Richard Lee for the
use of his lab and equipment, and both for their guidance and support
also. I also would like to thank Gary Barthe, Toni Ceccardi, Jiang
Jingrui, Adriana Quiros, J. L. Chandler, Mary Price, Manjunath Keremane
and Eliezer Louzada for their help in the lab.
There are several people whom I would like to offer a special thank
you. First, Francisco Mourao, my good friend and fellow student, who was
always there when I needed a shoulder to lean on. Also, Remei Albiach,
who helped me tremendously with all the molecular techniques, as well as
being a dear friend. Also, Beatriz Nielsen and all the Nielsen family,
Tim Widmer, Ana Mourao, Tom and Linda Fontana, my parents, Joel and Joan
Schell, my sisters- Joyce and Jane, my brother-in-laws-Cris and Dale, and
all my many friends who have supported me over the years. To all of you-
many Thanks.
ACKNOWLEDGEMENTS .............................................. i
ABSTRACT ....................................................... vi
Methods of Transformation .................... ..... 5
Agrobacterium tumefaciens-mediated
Transformation ............................ 5
Direct Gene Transfer Using Particle
Bombardment .......................... 8
Gene Transfer by Direct DNA-Uptake ........... 12
Other Methods of Transformation ............... 16
Microinjection ........................... 16
Liposome mediated transformation ..... 17
Transformation using silicon
carbide fibers .................... 18
Cross Protection ................................. 18
Cross Protection of CTV in Citrus ................... 20
Genetic Engineering for Virus Resistance ........... 22
Replicase Gene ............................. 23
Movement Protein ........................... 24
Protease ................................... 24
Antisense and sense-defective RNAs ............ 25
Coat Protein ............................... 26
Transformation of Citrus ......................... 31
Plasmid Preparation .............................. 34
Transformation of E. coli .................... 37
Quick miniprep plasmid preparation ........... 38
Large scale isolation and preparation
of plasmid .............................. .40
Establishment and Maintainance of Suspension
Cultures ................................... 42
Isolation and Transformation of Citrus
Protoplasts ................................... 43
Culturing and Regeneration of Treated
Protoplasts ................................... 45
Testing for Transformed Plants ..................... 46
Polymerase Chain Reaction for Detection
of Transformants ....................... 46
Testing for Transformed 'Rohde Red
Valencia' Sweet Orange Using
Dot Blot Analysis .................... 46
Isolation of DNA from Citrus leaves
for dot blot analysis .......... 46
Preliminary screening using dot
blots .... ....................... 49
Confirmation of Transformation ............. 49
Isolation of DNA for Southern Blot
Hybridization .................... 49
Southern Transfer of DNA Isolated
from Transformed Plants ...... 51
Transfer under neutral
conditions ............... 51
Transfer under alkaline
conditions ............... 53
Preparation of Radioactive Probes
by Random Primer for Southern
Hybridization .................. 54
Hybridization of DNA with a Labeled
Probe ............................ 56
Southern Blot Hybridization Using
the GENIUS non-radioactive
labeling system ................ 57
Western Immunoblot Analysis for
detection of the Coat Protein.. 59
Plasmid Preparation ................................ 62
Transformation of Citrus Protoplasts and
Plant Regeneration ......................... 62
Screening for Transformants ........................ 65
Selection During the Early Stages of
Protoplast Growth and Microcalli
Development .......................... 65
Polymerase Chain Reaction for Selection
of Transformants ..................... 68
Testing of 'Nova' tangelo and
'Valencia' sweet orange
regenerates ...................... 68
Testing of the 'Rohde Red Valencia'
sweet orange regenerates ....... 70
Screening for Transformants Using
Southern Dot Blot Analysis... 75
Confirmation of Transformation ...................... 76
Southern Hybridization Analysis Using
the Non-radioactive GENIUS
system ................................. 76
Southern Hybridization Analysis Using
Radioactive Probes .................... 80
Western Analysis ........................... 84
APPENDIX A Tissue Culture and Protoplast Media ................. 94
APPENDIX B Molecular Biology Buffers ........................ 102
Abstract of Dissertation Presented to the Graduate
School of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy
Jay L. Schell
December 1996
Chair: Jude W. Grosser
CoChair: Kenneth S. Derrick
Major Department: Horticultural Science
There has been considerable interest in using plant molecular
biology for crop improvement. The technology to isolate and clone a
specific gene is commonly utilized today. The methodology to insert these
genes has been well established for many crop species. One of these
methods is the use of PEG-mediated direct DNA uptake. This method relies
on the availability of a reliable protoplast isolation and plant
regeneration system, which is available for Citrus.
Protoplasts isolated from ovule-derived callus of 'Nova' tangelo
('Clementine' mandarin (Citrus reticulata Blanco) X 'Orlando' tangelo (C.
reticulata X C. paradisi Macf)) and 'Rohde Red Valencia' sweet orange (C.
sinensis L(Osbeck)) were
transformed by PEG-mediated direct DNA uptake using plasmids containing
the coat protein gene of citrus tristeza virus (CTV). Three transgenic
'Nova' tangelo plants were obtained using plasmid pMONI0098 containing the
coat protein gene of the Florida mild isolate T30. Twenty-two transgenic
'Rohde Red Valencia' sweet orange plants were obtained using plasmid
pBPFQ7 containing the coat protein gene of the Florida severe isolate T36.
Transformants were selected by PCR analysis or dot blot hybridization.
Confirmation of transformation was by Southern hybridization and western
immunoblot analysis. These transgenic plants will now be budded onto sour
orange rootstock and subjected to challenge inoculation with severe
isolates of citrus tristeza virus in the greenhouse, to determine if they
are resistant to CTV.
There is considerable interest in using plant molecular biology
techniques for crop improvement. The technology to isolate and clone a
specific gene is commonly utilized today. The number of genes and
proteins isolated and sequenced is in the thousands, and they are
cataloged in several databases. Latterich and Croy (1993) have published
a partial listing of these databases and many of the genes and proteins
Along with the advances in gene and protein isolation and
characterization has been an increased interest in using this knowledge to
clone and insert these genes into plants. Transgenic plants have been
engineered with several purposes in mind, including development of new and
more efficient transformation methods, for the basic study of a specific
gene, and for crop improvement for a specific purpose (Kung, 1993). The
first transgenic plants were produced in the early 1980s. Murai et al.
(1983) transferred the -Phaseolin gene from bean to sunflower and tobacco
plants using Agrobacterium tumefaciens. At around the same time, several
transgenic tobacco plants transformed using Agrobacterium tumefaciens
vectors and expressing foreign genes were reported (Horsch et al.,1984; De
Block et al., 1984).
These first transformation reports, as well as many that followed,
utilized the Agrobacterium tumefaciens vector system. However a number of
alternative methods have been developed. These include direct DNA uptake,
electroporation, particle bombardment, and microinjection. These methods
are discussed in Chapter 2.
Citrus tristeza virus (CTV) is the most economically important viral
pathogen of citrus (Lee and Rocha-Pena, 1992) This member of the
closterovirus group has a flexous rod shaped particle and a positive-
sense, single stranded RNA genome with a molecular weight of approximately
6.5 X 106. The genome of CTV contains 19,296 nucleotides constituting a
single messenger- sense RNA, making it the largest single-stranded, plus-
sense RNA plant virus known (Karasev et al., 1995). The coat protein gene
was first identified and sequenced by Sekiya et al. (1991). Of the nearly
20 kb in the genome, the 7292 nucleotides at the 3' end were sequenced and
eight open reading frames (ORFs) were identified, including the coat
protein gene, by Pappu et al. (1994). The remaining genome was sequenced
by Karasev et al. (1995), and it was thus determined that the complete
genome encodes 12 ORFs, and potentially codes for at least 17 gene
products. CTV is transmitted in a semipersistent manner by several
species of aphids, most notably Aphis gossypii and Toxoptera citricida.
T. citricida is a much more effective vector than is A. gossypii (Lee and
Rocha-Pena, 1992).
Citrus tristeza virus exists as a number of strains with different
biological activities. Mild isolates which show little to no symptoms are
often found in most citrus plantings. Mild strains will cause a slight
stem-pitting, little to no vein clearing, and flecking in the indicator
plant, Mexican Lime (Citrus aurantifolia) The more serious seedling
yellows symptoms can be found in seedlings of sour orange (C. aurantium),
lemon (C. limon), and grapefruit (C. paradisi). Seedling yellows is
characterized by a severe chlorosis and dwarfing. Stem pitting can occur
in both sweet orange and grapefruit. These trees will be stunted and
often chlorotic, with pitting under the bark of the stem and branches.
The stunted trees will often show pronounced longitudinal ridges or
depressions running up and down the trunk. Fruit size and productivity is
greatly reduced. One of the more severe form of the tristeza disease is
quick decline. This is a disease of sweet orange grafted on sour orange
rootstock. With quick decline the leaves turn yellow or golden in color,
wilt and then fall from the tree, leaving only the fruit hanging from the
tree. The tree quickly dies. Often the bud union will have an overgrowth
immediately above it (Lee and Rocha-Pena, 1992). Plants exhibiting quick
decline symptoms often succumb to the disease in as little as three to six
Since Beachy and his colleagues were able to transform tobacco with
the coat protein gene of tobacco mosaic virus (TMV) and obtain resistance
to TMV (Powell-Abel et al., 1986; Beachy et al., 1987) there has been
considerable interest in using this technology to develop plants resistant
to other viruses (See chapter 2 for a review of this work). The purpose
of the present study was to apply this technology to insert the coat
protein gene from CTV into citrus scion cultivars in an attempt to
engineer virus resistant plants. If successful, this could be a
significant step in eliminating one of the major disease problems in
Citrus, and could lead to the possibility of once again using the CTV
susceptible rootstock sour orange in areas where CTV is found.
Methods of Transformation
Agrobacterium tumefaciens-mediated Transformation
Agrobacterium tumefaciens is the pathogen that causes crown gall
disease in many plant species. The infection cycle of Agrobacterium is
very complex, involving a number of chemical signals emitted by both the
host and the pathogen. Infection occurs when the bacteria are attracted
to a number of chemicals secreted by wounded plant cells (Gelvin, 1993).
The virulent strains of A. tumefaciens contain large plasmids which are
responsible for the DNA transfer that subsequently causes the gall
formation. These plasmids have been termed tumor-inducing or Ti plasmids
(Schell et al., 1979; Chilton et al., 1980). Among the genes residing in
the Ti plasmids are the genes for opine synthesis in infected plants, and
opine catabolism in the Agrobacterium (Bomhoff et al., 1976; Genotello et
al., 1977). Early studies had shown a genetic linkage between
oncogenicity and opine metabolism (Lippincott et al., 1973; Genetello et
al., 1977). This led to the hypothesis that somehow the Ti plasmid was
involved in some sort of DNA transfer mechanism from the bacterium to the
plant. This DNA transfer mechanism was first shown to occur by Chilton et
al (1977) using hybridization experiments. This same group later showed
transcription of a part of this T-DNA (transferred DNA) in the crown gall
cells (Drummond et al., 1977).
The T-DNA covalently integrates into plant nuclear DNA, where genes
encoded by the T-DNA direct the synthesis of auxins and cytokinins, as
well as the low molecular weight opines. Opines are secreted from the
tumorous cells and can be used as a carbon, and sometimes, nitrogen source
by the Agrobacterium. Some of these opines also induce the conjugal
transfer of the Ti plasmid between Agrobacterium cells (Gelvin, 1993).
Thomashow et al. (1980 a,b) demonstrated that a majority to all of the T-
DNA is integrated into the plant DNA and that the T-DNA can be inserted in
more than one site in the plant genome. The excision of the Ti plasmid
and its subsequent transfer from the Agrobacterium to plant cells is
controlled by a group of genes on the Ti plasmid called the vir genes.
These vir genes are induced by a group of phenolic compounds produced by
the plant (Garfinkel and Nester, 1980; Klee et al., 1982; Hooykaas et al.,
1984; Lundquist et al., 1984; Gelvin, 1993).
The T-DNA contains specific DNA sequences called T-DNA borders which
have been shown by mutational analysis to be essential for tumorigenesis.
These T-DNA borders are made up of 25 bp highly conserved DNA sequences in
a directly repeated orientation (Schell, et al., 1979; Lemmers et al,
1980; Thomashow et al., 1980a,b; Zambryski et al., 1983; Bevan, 1984).
The mechanisms by which the DNA crosses the plant cell wall, crosses
through the cytoplasm, enters into the nucleus, and becomes integrated
into the plant DNA, are still unknown.
It was these T-DNA properties that led researchers to investigate
the use of Agrobacterium and its Ti plasmid as a tool for genetic
engineering. In order for this to be possible, two major modifications
were found to be necessary. First, the non-essential sequences (those not
involved in T-DNA transfer) of the T-DNA must be replaced with the foreign
genes of interest. Secondly, the tumor inducing capabilities of the T-DNA
need to be inactivated without inactivating its DNA transfer capabilities
(Van Montagu and Schell, 1982). In addition, the large size of the Ti
plasmid (>200 kb) makes it necessary to produce a series of autonomously
replicating or integrative vectors to use in gene transfer. These
integrative vectors are constructed in such a way that they recombine
specifically with a resistant Ti plasmid in A. tumefaciens which has been
disarmed. An example of a disarmed A. tumefaciens Ti plasmid is pGV3850,
a Ti plasmid mutant in which all the oncogenic functions have been deleted
and replaced by pBR322. (The plasmid pBR322 was developed for use in a
multipurpose cloning system prior to the identification of the Ti plasmids
of Agrobacterium (Bolivar et al., 1977). This plasmid (pBR322) is a
relaxed replicating plasmid that is sensitive to colicin El and also
carries resistance to the antibiotics ampicillin and tetracycline.
Plasmid pBR322 also contains five unique restriction sites- BamHI, EcoRI,
HindIII, PstI, and SalI. The resulting pGV3850 plasmid is still capable
of mediating efficient transfer and stabilization of its truncated T-DNA
into infected plant cells, but its integration and expression does not
interfere with normal plant function (Zambryski et al., 1983; Rogers et
al., 1987). At a minimum, the chimeric genes that are integrated
generally include a promoter such as the cauliflower mosaic virus gene VI
promoter (Comai et al., 1983; Potrykus et al., 1985a,b) or the nopaline
synthase promoter (de Block et al., 1984; Hain et al., 1985), as well as
a prokaryotic antibiotic resistance gene for antibiotics such as
kanamycin, streptomycin, spectinomycin or chloramphenicol (Comai et al.,
1983; Paskowski et al., 1984; Hain et al., 1985; Potrykus et al., 1985;
Rogers et al., 1988). Generally other genes including a selectable marker
such as kanamycin (NPTII gene), hygromycin (hph gene), or bialophos (bar
gene) and/or reporter genes such as -glucuronidase (uidA gene), or
chloramphenicol acetyl transferase (cat gene) are also included for
selection of transformed plants or cells.
Many of the plasmids constructed today are integrative intermediary
plasmids that contain pBR322 sequences. Autonomously replicating vectors
are constructed similarly to integrative vectors but contain an additional
wide host range function and thus do not require a co-integrate formation
for stable maintenance in Agrobacterium and subsequent transfer to plant
cells (Hoekema et al., 1983; Bevan, 1984; Rogers et al., 1988).
Direct Gene Transfer Using Particle Bombardment
One of the most popular methods for gene transfer today is particle
acceleration (bombardment) In this method, the DNA to be transferred is
carried through the cell wall and into the cytoplasm on the surface of
small (0.5 to 5 Am) metal (usually tungsten or gold) particles which have
been accelerated to speeds of one to several meters per second (Klein et
al, 1987). These particles are capable of penetrating up to several
layers of cells and allow for the transformation of cells within tissue
explants. This method is very popular for the transformation of many
cereals in which protoplast culture is difficult, as well as dicots which
can be recalcitrant to Agrobacterium infection.
Particle bombardment has several advantages over Agrobacterium-
mediated transformation. First, nonhosts of Agrobacterium, such as
monocots, can be transformed. Second, plasmid construction does not
require insertion of the sequences essential for T-DNA replication and
transfer in Agrobacterium. Third, the introduction of multiple plasmids,
referred to as cotransformation, is possible with particle bombardment.
Fourth, false positives resulting from growth of the Agrobacterium in host
cells are eliminated. Finally, the transformation protocols are
simplified by eliminating the complex bacteria-plant interrelationships
present in Agrobacterium systems (Gray and Finer, 1993).
Virtually every type of cell or tissue has been used as a target for
gene transfer by particle bombardment (See Table 2-1). Particle
bombardment has been especially valuable for the transient assay of gene
constructs in specialized cells or tissues (Goldfarb et al., 1991; Lee et
al., 1991a; Seki et al., 1991). On the negative side, plants regenerated
from bombarded plant tissues are usually chimeric in terms of introduced
foreign genes due to random bombardment of a small number of cells in a
multiple cell system (Sanford, 1990). This requires an additional
selection using selectable markers to sort out transformants that are
stabilized in their progenies (McCabe et al., 1988; Finer and McMullen,
1990; Fromm et al., 1990; Gordon-Kamm et al., 1990; Tomes et al., 1990;
Christou et al., 1991; McCown et al., 1991; Lowe et al., 1995). In
addition, the success of particle bombardment transformation to produce
stable transgenic plants is partly dependent on having an efficient
multiple shoot regeneration or embryogenic system. Further, the failure
Table 2-1: Types of cells or tissues used for particle bombardment, and
whether or not transgenic plants were obtained.
Tissue used
Mendel et al., 1989
Gordon-Kamm et al., 1990
Wang et al., 1988
Wang et al., 1988
Fitch et al., 1990
Iida et al., 1990
Finer and McMullen, 1990
Hebert, et al., 1993
Fromm et al., 1990
Board et al., 1990
Daniell et al., 1991
Ye et al., 1994
Embryos Black Spruce no Bommineni et al., 1994
Barley no Kartha et al., 1989
Corn no Klein et al., 1989
Pearl Millet no Taylor and Vasil, 1991
Wheat no Lonsdale et al., 1990
Rice yes Christou et al., 1991
Peach no Ye et al., 1994
Leaf Tobacco yes Tomes et al., 1990
Wheat no Daniell et al., 1991
Peach no Ye et al., 1994
Sugarcane no Gallo-Meagher and Irvine, 1993
Meristems Soybean yes McCabe et al., 1988
Soybean Yes Christou et al., 1989
Nodules/Stems Poplar yes McCown et al., 1991
to regenerate transgenic plants after particle bombardment of either
embryogenic suspensions or immature scutelli may be due to the limited
amount of DNA carried into the cells by particles or in the efficiency
with which the DNA disassociates from the particle. As a result, there is
a low potential for integration even in cells containing the particles.
This low frequency has to be multiplied by the low frequency of competent
cells. Thus the production of transgenics by particle bombardment can be
expected at a low frequency (Potrykus, 1990).
The first report of microprojectile bombardment was made using a
gunpowder-based acceleration system to propel tungsten particles carrying
DNA through an evacuated chamber and into the cells (Klein et al., 1987).
Later modifications were made to optimize the gun parameters and
evaluation of gene expression (Russell et al., 1992; Morrish et al.,
1993). Modifications of the gunpowder version of particle bombardment
system used high pressure helium as a means to accelerate the
microprojectiles (Russell-Kikkert, 1993). This design is commercially
available from DuPont. A second design was a homemade version based on
the DuPont airgun which utilized accelerated tungsten particles to
transform monocot cells (ard et al., 1990; Oard, 1993). A third concept
utilized a stream of helium to deliver DNA (Takeuchi et al., 1992). This
concept was further refined to become the commercially available particle
inflow gun (PIG) (Finer et al., 1992).
Particle bombardment has made it possible to transfer foreign DNA
into organelles. Daniell et al. (1990) reported on the introduction and
transient expression of foreign genes in suspension cell-derived
chloroplasts of tobacco. Daniell et al. (1991) also reported the
successful transient expression of -glucuronidase in chloroplasts
isolated from wheat leaf and callus and transformed using particle
bombardment. Stable gene replacement at a very low frequency (a total of
five transformants from 245 particle bombardment events) has been reported
for tobacco (Svab et al., 1990; Staub and Maliga, 1992).
Gene Transfer By Direct DNA Uptake
In species with a reliable protoplast regeneration system, direct
gene transfer by either chemical (polyethylene glycol (PEG)- mediated), or
electroporation, or a combination of the two techniques has been quite
popular. Protoplasts are in principle ideal cells for the delivery of DNA
and selection of transgenic events. Removal of the cell wall eliminates
one of the major barriers to the delivery of DNA. Further, the efficiency
for recovery of transgenic events is higher because cross feeding and
chimerism between transgenic and wild-type cells is minimized in
comparison to transformation systems based on multicellular tissues
(Songstad et al., 1995). Because plants regenerated from protoplasts come
from a single cell, all cells in the transgenic plant should contain the
genes of interest that were inserted. It has been shown that foreign
genes integrate predominantly at a single locus, are inherited as single
mendelian traits, and can be as stable as the wild-type genes (Potrykus et
al., 1985a).
The most commonly used procedure for introduction of DNA into
protoplasts involves treatment with PEG, much the same as in somatic
hybridization. PEG alters the plasma membrane properties by causing a
reversible permeability that enable exogenous macro-molecules to enter the
cytoplasm. The exact mechanism of PEG-mediated membrane permeabilization,
and thus DNA delivery, is not completely understood (Songstad et al.,
The first report of direct DNA uptake and stable transformation
involved the transfer and expression of Agrobacterium tumefaciens T-DNA
into tobacco protoplasts using PEG (Draper et al., 1982; Krens et al.,
1982). The first transgenic plants obtained in this manner were reported
by Paszkowski et al. (1984). The keys to being able to accomplish this
were optimization of the protoplast to plant regeneration system,
development of the aminoglycoside phosphotransferase gene II from E. coli
transposon Tn5 as a selectable marker conferring plant cell resistance to
kanamycin, and optimization of the DNA delivery frequency (Potrykus et
al., 1985a).
PEG-mediated transformation is performed in much the same way as
protoplast fusion but with the second donor cell being the plasmid as
opposed to a second plant protoplast cell. Protoplasts are isolated the
same as for fusion, and 3-15 ug of plasmid DNA per ml of protoplasts, is
added (Krens et al., 1982; Saul et al., 1988; Paszkowski and Saul, 1988).
A variety of different procedures have been used depending on the species
being transformed. A number of different parameters have been looked at
in order to optimize the systems.
Many early experiments included calf thymus DNA in the protocol
(Krens et al., 1982; Krens et al., 1985; Shillito et al., 1985; Peerbolte
et al., 1985; Uchimaya et al., 1986), usually at concentrations of 5 times
the amount of calf thymus DNA as plasmid DNA. The calf thymus DNA acts as
a carrier in the transformation and has been shown to appear in
transformed plants when the transformation frequency is high (Krens et
al., 1985; Peerbolte et al., 1985); but has also been reported to not be
expressed in those plants (Krens et al., 1985). In many of the early
experiments in transformation of protoplasts, calf thymus DNA was reported
to be essential for transformation (Krens et al., 1982; Shillito et al.,
1985; Negrutiu et al., 1987). However this may not always be the case.
Successful transformation has been obtained without using calf thymus DNA
or other carrier DNA (Hain et al., 1985; Damm et al., 1989; Tautorus et
al., 1989).
Optimization of PEG-mediated transformation is more than likely
dependent on a number of factors other than just the presence of carrier
DNA. Shillito et al., (1985) demonstrated that, for electroporation (to
be discussed later), a PEG concentration of 7.8% w/v was optimum for
transformation, particularly when the protoplasts were heat shocked at 45
C for 5 minutes prior to the addition of DNA and PEG. More commonly,
particularly for PEG-mediated transformation, an optimum of 20% PEG has
been shown (Saul et al., 1988; Maas and Werr, 1989; Armstrong et al.,
1990). Damm et al. (1989) showed that with a final PEG concentration of
10-20% the transformation frequency was 3 to 5 times higher than at lower
concentrations, but at a PEG concentration of 30 % the frequency was
virtually the same. Others have reported success with PEG concentrations
of 10% (Tautorus et al., 1989) or 25 % (Antonelli and Stadler, 1989).
Divalent cations are also reported to play a major role in the
success of transforming protoplasts. Calcium has been shown to aid in the
uptake of DNA by protoplasts. It was thought that calcium phosphate co-
precipitates the DNA, which is then taken up by endocytosis (Hain et al.,
1985). This was supported by later research which showed that DNA
precipitation is more efficient in the presence of divalent cations (Ca2,
Mg2. and Mn ), and efficient DNA incorporation required both PEG and a
divalent cation (Maas and Werr, 1989). Ca2' and Mg2 were more efficient
than Mn2 Armstrong et al. (1990) reported higher transformation
frequencies with Ca2 than with MJ +. Other groups have reported higher
transformation frequencies with Mg2' than with Ca2+ (Negrutiu et al., 1987;
Vasil et al., 1988). This difference may be due to additional factors
including methods used, the species being transformed, and genotypic
Also reported to play a major role in the efficient transformation
of protoplasts is the pH of the PEG solution (Maas and Werr, 1989). This
is probably due to the fact that at higher pH (9.0) values large
aggregates of DNA are formed that can be taken up less efficiently than
the smaller DNA aggregates formed at lower pH (6.0) values.
As mentioned previously, heat shock treatment at 45 C for 5 minutes
prior to transformation may increase transformation frequency as in maize
(Zea mays) (Shillito et al., 1985), sunflower (Helianthus annuus) (Moyne
et al., 1989), black spruce (Pinus mariana), and jack pine (Pinus
banksiana) (Tautorius et al., 1989). Tyagi et al (1989) found little
effect from a heat shock treatment on tobacco (Nicotiana tabacum).
Electroporation is often used either alone or in combination with
PEG. Electroporation involves the subjecting of protoplasts to
electrical pulses of AC and DC current to cause reversible
permeabilization of the plasma membrane enabling macromolecule delivery.
Isolated protoplasts and the plasmid are mixed prior to electroporation.
An AC field giving 25 volts (peak-to-peak) at 500 KHz which aligns the
protoplasts in chains of varying lengths is applied. Excessively long
chains are avoided by using a voltage strong enough to bring about
alignment, but applied at a slow rate of increase over a five minute
period. A DC pulse of 2 milliseconds is given and a few seconds later the
AC field is progressively reduced to zero. (Power et al., 1988). Many of
the earlier successes in stably transforming tobacco protoplasts involved
combinations of PEG and electroporation treatments (Shillito, 1985; Fromm
et al., 1986; Negrutiu et al., 1987; Saul et al., 1988). Electroporation
is commonly used for transient gene expression studies in both mammalian
and plant protoplast systems (Songstad et al., 1995).
Other Methods of Transformation
Microinjection is performed under the microscope with the DNA
injected directly into individual cells. Microinjection has the advantage
in that the amount of DNA injected into a cell is not limited, the
delivery is precise, and cells with high regeneration capacity can be
targeted. Microinjection is, however, very labor intensive and requires a
highly skilled individual to perform the injection (Potrykus, 1990). In
haploid canola, microspore-derived embryoids microinjected at the 12 cell
stage of development resulted in the recovery of transformed plants, but
they were chimeric and required recovery by secondary embryogenesis
(Neuhaus et al., 1987). Protoplasts have been shown to survive
microinjection (Griesbach, 1985; Lawrence and Davies, 1985; Morikawa and
Yamada, 1985 a,b; Reich et al., 1986a,b; Steinbiss and Stabel, 1983), as
have pollen grains (Crossway et al., 1986; Kranz and Lorz, 1990) and onion
epidermal cells (Bradley, 1979). However, transformation by
microinjection of protoplasts has not been reported (Songstad et al.,
Liposome Mediated Transformation.
Liposomes entrap a large variety of macromolecules such as enzymes
or nucleic acids and can be used to deliver them into cells by fusion with
the plasma membrane or by endocytosis. The choice of the proper lipid
composition is very important to the system. Most of the successes using
liposomes has been with animals (Gad et al., 1990). The mode of action
of DNA delivery is unclear, but there is evidence that it is by cellular
uptake (endocytosis) as determined by electron microscopy (Fukunga et al.,
1983; Nagata, 1984; Guerineau and Tailliez, 1986) Negatively charged
liposomes work better than positively charged liposomes (Gad et al.,
1986). The genetic material encapsulated in negatively charged liposomes
is inserted into the cells usually by the addition of fusogens to the
reaction mixture. Divalent cations such as Ca2* promote liposome fusion
(Gad et al., 1988).
Protoplasts appear to be best suited for receiving exogenous DNA
from liposomes. Both transient and stable gene expression has been
obtained in tobacco protoplasts using liposome-encapsulated DNA (Rosenberg
et al., 1988). The traits transformed to the zygotic offspring were shown
to be inherited as one dominant gene (Deshayes et al., 1985).
Transformation Using Silicon Carbide Fibers.
DNA delivery into plant cells using silicon carbide fibers was first
reported by Kaepler et al. (1990). This simple method involves vortexing
a mixture of plant cells, plasmid DNA and silicon carbide fibers. Silicon
carbide fibers likely mediate DNA delivery because of their shape, size,
strength, and chemical composition. However, silicon carbide fibers pose
a lung disease and carcinogenic danger similar to asbestos, if inhaled
(Songstad et al., 1995).
Cross Protection
Cross protection can be defined as the use of a mild or attenuated
strain of a virus to prevent further infection from more severe strains of
a virus or to other closely related viruses (Sequiera, 1984; Fulton, 1986;
Palukaitis and Zaitlin, 1984; Wilson, 1993). A variety of terms have been
applied to this phenomenon including acquired immunity, antagonism, cross
immunization, dominance, interference, premunity, induced immunity,
prophylactic inoculation and protective inoculation (Fulton, 1986).
The first report of cross protection was made in 1929 when McKinney,
working with tobacco mosaic virus, reported the ability of one strain of
the virus to protect against later infections by a second strain of the
virus (McKinney, 1929). Later Salaman (1933) found that tobacco plants
infected by a mild strain of potato virus X were protected against other
strains of this virus. Salaman proposed that this cross protection could
have economic importance, and since then there have been numerous attempts
to use avirulent strains to improve the yield of many crops, but with
limited success (Fletcher, 1978). However, for a number of viruses this
approach has been successfully applied. These include tomato mosaic virus
(Broadbent, 1976), papaya ringspot virus (Yeh et al., 1988), tobacco
mosaic virus (Rast, 1972), and citrus tristeza virus (Muller and Costa,
1966, 1969, 1977; Costa and Muller, 1980).
The mechanisms by which cross protection works are not well
understood (Gadani et al., 1990). Earlier theories on the mechanism of
action of cross protection centered around four general categories. First
was the theory of precursor exhaustion in which the inducing virus has
exhausted one or more of the essential materials required for the
replication of the challenging virus. The second theory is that the
inducing virus produces substances which specifically inhibit related
virus, but not unrelated viruses. It would be possible for this specific
inhibitor to bind to the incoming challenging virus. A possibility for
such a specific inhibitor would be the viral replicase. A third theory is
that the inducing virus has altered the metabolism of the host to such an
extent as to render it insusceptible to a challenging virus. Finally, a
fourth theory centers around coat protein sequestration. The coat protein
of the inducing virus encapsidates the nucleic acid of the related
challenging virus, and the virions that are then produced are not in an
environment of the cell enabling their nucleic acid to be released and
replication to begin. (Sherwood and Fulton, 1982; Palukaitis and Zaitlin,
1984). However, none of these theories can be shown to be correct for all
viruses, and in fact the true mechanism may be a combination of these or
an as yet undescribed mechanism.
Cross protection of CTV in Citrus
In order to have effective cross protection the mild isolate used
must be mild in all citrus cultivars and susceptible hosts grown in a
given area. In addition, the virus used must be genetically stable with
little or no chance of reverting to a severe form. Artificially
attenuated or mutated viruses might be more likely to revert back to the
severe form. Finally, the slight reduction in yield and performance of
the host due to the effects of the mild strain should be economically
acceptable (Lee and Rocha-Pena, 1992).
The first report that cross protection could be possible in citrus
was made in 1951 (Grant and Costa, 1951) Most early work on cross
protection of citrus was done to protect against the stem pitting isolates
of CTV. Consequently, a great deal of work has been done in countries
where severe stem pitting isolates exist. By 1980 over 15 million 'Pera'
sweet orange trees were inoculated for protection against CTV-induced stem
pitting in Brazil (Costa and Muller, 1980). Mild strain cross protection
has been used on grapefruit in Japan (Sasaki, 1979; Leki, 1989; Koizumi et
al, 1991), Australia (Stubbs, 1964; Cox et al., 1976; Thornton et al.,
1980), South Africa (De Lange et al., 1981) and Venezuela (Romero et al.,
1991). Also reported was cross protection of naval orange in Japan
(Koizumi, 1991), and limes in India (Balaraman and Ramakrishnan, 1980) and
Venezuela (Romero, et al., 1991).
Because most countries abandon sour orange as a rootstock when CTV-
induced quick decline occurs, relatively little research has been done on
cross protection against quick decline. However, because of its excellent
horticultural characteristics, there has been recent interest in
evaluating whether mild strain cross protection would work to prevent
quick decline of trees on sour orange rootstock. Thornton and Stubbs
(1976) reported a delay in the onset of tristeza induced quick decline of
'Marsh' grapefruit on sour orange. However, by 1980 they were reporting
some breakdown in the immunity of these trees (Thornton et al., 1980).
Wallace and Drake (1976) also reported a delay in symptoms of 'Valencia'
sweet orange on sour orange after preimmunization. Van Vuuren et al.
(1991) evaluated 11 mild CTV isolates from Florida, Israel, and South
Africa for cross protection of 'Valencia' sweet orange on sour orange in
South Africa. Quick decline symptoms appeared after 4 years for some of
the isolates. Only one isolate appeared to have potential commercial
value under the South African climatic conditions. In a similar study in
Florida, 'Hamlin' sweet orange and 'Redblush' grapefruit trees on sour
orange rootstock were preinfected with 14 different mild strain isolates
of CTV. These trees were exposed to natural challenge in an area where
severe decline strains were prevalent. The CTV decline was more prevalent
in the 'Hamlin' trees than in the 'Redblush' Grapefruit (Yokomi, et al.,
1991). Another field trial by Rocha-Pena et al. (1991) showed little or
no decline after four years for 'Hamlin' and Grapefruit on sour Orange,
suggesting that with proper selection of mild CTV isolates, cross
protection against quick decline on sour orange is possible.
There are, however, several problems involved in the use of cross
protection as a control strategy against severe strains of citrus tristeza
virus (Lee and Rocha-Pena, 1992). First, the budwood source trees have to
be biologically indexed on a frequent basis to ensure that a severe strain
has not been introduced (De Graca et al., 1982). The erratic distribution
of CTV within the scion can result in as much as 50% of the buds being
propagated that should contain the mild strain isolate, actually being
virus free (Lee et al., 1987). Hot weather can induce a kind of
thermotherapy which often prevents the spread of the mild strain isolates,
thus leaving parts of the tree virus free and exposed to infection from
severe isolates, breaking down the cross protection (Lee et al., 1988).
Selection of the mild isolates to use can be very difficult and
inefficient (Costa and Muller, 1980). This may be partly due to the fact
that many isolates which show cross protection in one environment may
express severe symptoms or at least fail to cross protect in other
environments (Lee and Rocha-Pena, 1992). It is these problems which have
contributed to the idea of genetically engineering the cross protection
into citrus.
Genetic Engineering for Virus Resistance
With the dramatic advances in plant transformation technology there
has been a tremendous interest in introducing virus resistance by genetic
engineering. With a few exceptions, the genes used to induce this virus
resistance have come from the genome of the virus. This is partly due to
the difficulties still inherent in isolating plant resistance genes from
the relatively large plant genome. In contrast, plant viruses contain
relatively few genes which can be more readily identified and cloned. A
majority of the examples of genetically engineered viral resistance have
involved the use of the coat protein gene but other genes have been used
to obtain virus resistance as well.
Replicase Gene
The first report of replicase mediated resistance was demonstrated
with tobacco mosaic virus (Golemboski et al, 1990; Carr et al., 1992).
While attempting to determine the role of a putative 54-kDa protein
encoded within the replicase of TMV, they found that the plants expressing
the 54-kDa protein were completely resistant to TMV, even at inoculum
levels up to 1000 fold higher than conferred by the TMV coat protein gene.
Donson et al., (1993) demonstrated no resistance to TMV in tobacco plants
transformed with the full length (183 kDa) replicase gene, but in
transgenic plants with an additional insertion which would terminate
translation in the middle of the 183 kDa gene, a high level of resistance
to systemic infection by TMV and other tobamoviruses was noted. Similar
results were obtained with cucumber mosaic virus (CMV) in tobacco
(Anderson et al., 1992), potato virus X (PVX) in tobacco (Braun and
Hemenway, 1992; Longstaff et al., 1993) pea early browning virus (PEBV) in
Nicotiana benthamiana (MacFarlane and Davies, 1993), potato virus Y (PVY)
in tobacco (Audy et al., 1994), and cymbidium ringspot virus (CyRSV) in N.
benthamiana (Rubino et al., 1993). Rubino and Russo (1995) later reported
that the transgenic N. benthamiana plants containing the CyRSV full-length
replicase gene were not resistant to the closely related artichoke mottled
crinkle tombusvirus (AMCV) and carnation italian ringspot tombus virus
(CIRV), indicating that the resistance was probably RNA-mediated
resistance. In most of these cases the resistance of the most resistant
replicase expressing lines was greater than for the coat protein
expressing lines. No field testing of the replicase expressing transgenic
plants has yet been reported.
Movement Protein
Tobacco plants expressing a defective movement protein of TMV or
transformed with the movement protein from brome mosaic virus (BrMV),
which cannot spread in tobacco, were shown to be protected from TMV
(Malyshenko et al., 1993). The authors theorized that the presence of
either of these movement proteins before viral inoculation inhibited the
ability of the incoming functional viral movement protein from TMV to
successfully interact with the plasmadesmata.
The potyvirus genome codes for a single large polypeptide that is
subsequently cleaved into individual mature viral proteins through the
action of three viral encoded proteases (Dougherty and Carrington, 1988).
One of these genes which encodes the NIa protein was used to transform
tobacco (Vardi et al., 1993). Of the 50 transgenic plants produced, 2
were found to be resistant to PVY. However the authors believe that this
resistance may be due to accidental alterations which may have occurred
during the cloning of the gene which may have resulted in a dysfunctional
protein. Nevertheless, this may lead to a possibility of intentionally
modifying the protease genes of viruses which produce polyproteins to
obtain virus resistance.
Antisense and Sense-defective RNAs
There have been several reports using antisense or sense-defective
RNAs to engineer virus resistance. It is thought that the interaction
between complementary transgene and viral encoded gene sequences
interferes with translation, replication or viral nucleic acid stability
(Grumet, 1995). Cuozzo et al. (1988) transformed tobacco plants with the
cauliflower mosaic virus (CMV) antisense coat protein (CP). They found
resistance only at low inoculum levels indicating that antisense CP-RNA
resistance was less effective than the coat protein mediated resistance.
Similar results were shown for PVX (Hemenway et al., 1988), TMV (Powell-
Abel et al., 1989), and zucchini yellow mosaic virus (ZyMV) (Fang and
Grumet, 1993). The lack of protection at higher inoculum levels may be
due to a gene dosage effect (Gadani et al., 1990). The mechanism of
action of viral antisense RNA may be due to an inhibition of coat protein
synthesis by the formation of an antisense-sense RNA hybrid, prevention of
replication by binding of the antisense RNA to the origin of replication
or by competition with the viral negative strand for viral or host
components necessary for replication (Gadani et al., 1990).
There are, however, examples where RNA mediated protection was much
more effective, most notably in the luteovirus and potyvirus groups.
Lindbo and Dougherty (1992a,b) demonstrated that both sense and antisense
forms of the coat protein gene of Tobacco Etch Virus (TEV) conferred a
high level of resistance to TEV. Hammond and Kamo (1993) demonstrated
varying degrees of resisance in their N. benthamiana plants transformed
with the antisense coat protein of bean yellow mosaic potyvirus (BYMV).
Kawchuk et al., (1991) showed that both the sense and antisense RNA of the
coat protein gene of potato leaf roll luteovirus (PLRV) showed a high
level of resistance to PLRV infection.
Coat Protein
Since the phenomenon of cross protection was first observed over 65
years ago by McKinney (1929) there has been considerable interest in the
development of plants protected from severe virus infection. There have
been several studies which have indicated that the coat protein plays a
major role in cross protection (DeZoeten and Fulton, 1975; Sherwood and
Fulton, 1982; DeZoeten and Gaard, 1984; Wilson and Watkins, 1986; Zinnen
and Fulton, 1986). In order to test this hypothesis, Powel-Abel and her
colleagues introduced the capsid protein gene of tobacco mosaic virus into
tobacco plants by using a chimeric gene construct containing a cDNA from
the coat protein coding sequence, with the CaMV 35S promoter and the
polyadenylation seguence from the Agrobacterium nopaline synthase gene.
After Agrobacterium-mediated transformation and subsequent plant
regeneration, these plants were tested for virus resistance. They
determined that high levels of accumulation of coat protein corresponded
to high levels of resistance to virus infection, and a corresponding delay
in symptom development in the progeny of self-fertilized transgenic
plants. The protection was overcome by inoculation of the transgenic
seedlings with unencapsidated viral RNA and was less effective when a high
virus concentration was used (Powel-Abel et al., 1986) This mimics what
happens with classical cross protection with TMV. Since this first
successful demonstration of coat protein-mediated resistance (CP-mediated
resistance), there has been a considerable interest in using this approach
in a variety of plant species (See Table 2-2).
There is no consensus on how CP-mediated resistance works. In fact,
the mechanism may be dependent on the virus-plant-coat protein system and
the exact nature of how the protection works may depend on the viral gene
construct used, transgene position effects, copy number, transcriptional
activity, the extent and site of uninhibited virus replication and spread
within the host plant, and secondary effects of transgenesis on host cell
metabolism and general stress anddisease resistance responses (Wilson,
1993). For example, in the case of TMV (Powel-Abel et al., 1986), alfalfa
mosaic virus (AlMV) (Loesch-Fries et al., 1987), and potato virus X
(PVX) (Hemenway, 1988) the level of resistance correlated positively with
the levels of coat protein in the transgenic plants. Plants that only
accumulated the coat protein transcript of TMV or AIMV and not the coat
protein itself were not resistant. However, in plants transformed with
the coat protein gene of potato virus Y (PVY) (Farinelli and Malnoe, 1993)
and potato leaf roll virus (PLRV) (Barker et al., 1993), resistance
correlated with the levels of coat protein transcripts and not with the
levels of coat protein. Moreover, in the case of PVY and PLRV some virus
resistant transgenic lines were identified in which the coat protein was
not detectable. To further complicate things, tobacco transformed with
the coat protein gene of tomato spotted wilt virus (TSWV) were resistant
when an intact coat protein gene was used, as well as when the transgene
was rendered untranslateable through the removal of the initiating start
codon (DeHaan et al., 1992). This would indicate that expression of the
coat protein is not necessarily required for resistance.
Table 2-2. Examples of coat protein-mediated resistance.
Mosaic Virus
Alfalfa Mosaic Virus
Tobacco Rattle Virus
Tobacco Streak Virus
Cucumber Mosaic Virus
Tomato Spotted Wilt Virus
Potato Virus Y
Lettuce Mosaic Virus
Papaya Ringspot Virus
to other Tobamo-also
yes (tobacco also)
yes- Pea Early Browning
Virus also
field tested resistance
not tested
Powel-Abel et al., 1986
Nejidat and Beachy, 1990
Nelson et al., 1988
Halk et al., 1989
Loesch-Fries et al., 1987
Tumer et al., 1987
van Dun and Bol, 1988
van Dun et al., 1987
Gonsalves et al., 1991a
Gonsalves et al., 1992
Cuozzo et al., 1988
Gonsalves et al., 1991b;
Gonsalves et al., 1994
MacKenzie and Ellis, 1992
Ultzen et al., 1995
Yepes et al., 1995
Lawson et al., 1990
Dinant et al., 1993
Fitch et al., 1992
Scorza et al., 1991
Virus Plant Resistance Reference
Bean Yellow Mosaic Virus tobacco Hammond and Kamo, 1991
Plum Pox Virus plum yes Scorza et al., 1994
tobacco variable Ravelenandro et al., 1993
Tobacco Vein Mottling Virus tobacco Main et al., 1991
Soybean Mosaic Virus tobacco broad spectrum Stark and Beachy, 1989
Potato Leaf Roll Virus tobacco yes Kawchuck et al., 1990
potato +/- sense Kawchuck et al., 1991
Potato Virus X tobacco +/- sense Hemenway et al., 1988
potato yes Hoekema et al., 1989
potato yes- field tested Jongdijk et al., 1992
Cassava Mosaic Virus tobacco Fauquet et al., 1991
Potato Virus S tobacco Mackenzie and Tremaine, 1990
Rice Stripe Virus rice yes Hayakawa et al., 1993
Grapevine Fan Leaf Virus grape yes Bardonnet et al., 1994
grape variable Krastanova et al., 1995
Maize Dwarf Mosaic Virus maize yes- also MCMV" Murry et al., 1993
Tomato Yellow Leaf Curl tomato yes Kunik et al., 1994
Arabis Mosaic Virus tobacco yes Bertioli et al., 1992
MCMV is Maize Chlorotic Mottle Virus
Table 2-2 (continued). Examples of coat protein-mediated resistance.
In the potyviruses (PVY, TEV, ZYMV), and luteoviruses (PLRV), when
intentionally truncated antisense or nonexpressing (with no start codon) coat
protein genes were transformed into plants, there was a measurable or even
complete protection against the parent virus (Kawchuck et al., 1991; van der Wilk
et al., 1991; Lindbo and Dougherty, 1992a,b; DeHaan et al., 1992; van der Vlugt
et al., 1992; Fang and Grumet, 1993; Farinelli and Malnoe, 1993; Lindbo et al.,
1993). In all these cases the resistance may be due to some direct form of RNA-
RNA interference between the transgene transcript and challenge virus. In TEV
with a truncated CP gene, it was common for the inoculated leaves to develop
symptoms and virus titer which was similar to that of control plants, but for the
plants to outgrow the infection, suggesting some interference with virus spread
(Lindbo and Dougherty, 1992a).
Recently, Dougherty and his colleagues have studied in detail coat protein-
mediated resistance using TEV. Their results confirm that transgene-derived RNA
may be either translatable or untranslatable indicating that a product of the
transcript is not involved in eliciting the resistance. That is, plants
displaying the virus resistance phenotype transcribe the transgene at a high
rate, yet accumulate the transcript at a low level (Dougherty et al., 1994; Smith
et al., 1994; Mueller et al., 1995). In plants which exhibit these
characteristics, the cytoplasmically located virus does not replicate to a
detectable level. It has been suggested that a post-transcriptional RNA
targeting system could be activated, resulting in the elimination of both
transgene and viral RNA from the cytoplasm (Lindbo et al, 1993; Dougherty and
Parks, 1995).
Dougherty and his colleagues (Smith et al., 1994) have further suggested
that there is a threshold level of transgene-derived transcripts that must be
exceded in order to activate the cytoplasmic post-transcriptional RNA degradation
process and thus elicit virus resistance. This suggests that the number of
transgenes and their level of expression are important in determining the
activation state of the cytoplasmic system. To further explore this RNA-mediated
virus resistance Dougherty and his colleagues (Goodwin et al., 1996) undertook
a study to characterize the biochemical and genetic processes of the resistance.
Using a set of plants that contained zero, one, two, or three copies of the
transgene in both a homozygous and heterozygous condition, it was determined that
three or more transgenes were necessary to establish the highly resistant state.
One or two transgene copies resulted in an inducible form of resistance. The
steady state RNA levels and the rates of transcription of the transgenes in the
plants in this study supported a post-transcriptional RNA degradation process as
the underlying mechanism for transgene transcript reduction and virus resistance.
Resistance appears to be initiated by a cleavage of specific sites within the
target RNA sequence. Further experiments are being conducted to see if this
really is the case.
Transformation of Citrus
The first successful transformation of Citrus was reported by Vardi et al.
(1990) using PEG-mediated direct DNA uptake. They were able to successfully
transform Rough Lemon (Citrus jambhiri) protoplasts. The first successful
transformation of Citrus by Agrobacterium tumefaciens was reported by Moore et
al. (1992). Transgenic Carrizo Citrange (C. sinensis X Poncirus trifoliata) were
produced by co-culturing with Agrobacterium tumefaciens. Transformation and
stable integration in regenerated plants was confirmed by PCR and Southern
analyses. Hidaka et al. (1990) reported the recovery of transgenic 'Washington
Navel' sweet orange (C. sinensis) from Agrobacterium-mediated transformation.
Kobayashi and Uchimaya (1989) reported the transfer of foreign DNA into sweet
orange protoplasts by direct DNA uptake but since no plants were regenerated,
stable transformation can not be claimed. Schell (1991) reported the
transformation of 'Hamlin' sweet orange protoplasts and the regeneration of
transgenic plants as demonstrated by histochemical assaying for GUS and PCR
analysis, but not by genomic Southern analysis. Pena et al. (1995) reported the
transformation and subsequent regeneration of transgenic plants from sweet orange
by Agrobacterium mediated transformation. Integration of the UidA (GUS) gene was
confirmed by Southern analysis.
Attempts are now being made by several labs to insert the coat protein gene
from citrus tristeza closterovirus (CTV) into citrus. It is hoped that this
genetically engineered cross protection will result in the production of trees
resistant to the tristeza virus, which is one of the most economical damaging
pests of Citrus. Espinosa (1995) successfully introduced the coat protein gene
of CTV into 'Carrizo' citrange (C. sinensis X Poncirus trifoliata) and sour
orange. These results have been confirmed by Southern and western immunoblot
analyses. These are the only results so far confirmed.
In addition, Febres-Rodriguez (1995) has characterized the genes for three
other open reading frames (P18, P20, P27) and has attempted to transform Citrus
with these genes. He showed the production of plants which were GUS positive and
positive by PCR but not confirmed by Southern analysis.
Plasmid Preparation
The two plasmids used in this research, were supplied by the
laboratory of Dr. C.L. Niblett. The two plasmids used were pMONI0098
(provided by Dr David Stark, Monsanto Corp. to Dr. Niblett) and pBPFQ-7
(provided by Dr. G. Selman, Institute for Genetic Engineering (Havana,
Cuba, to Dr. Niblett). The coat protein genes from Florida isolate T30 (a
mild isolate) of CTV, and Florida isolate T36 (a severe decline isolate)
of CTV were inserted into each of the two plasmids. The only difference
in the transformations into Escherichia coli for the two plasmids was that
pMONI0098 was selected for using spectinomycin (75Al/ml), and pBPFQ-7 was
selected for using ampicillin (50Ag/ml). The plasmid pMONI0098 contains
an enhanced CaMV 35 S promoter, nos terminator, and the NPTII gene. The
plasmid pBPFQ-7 contains two CaMV 35 S promoters in tandem, plus the 71
base pair non-coding 5' leader sequence of TMV (TMV Q) attached to the 3'
end of the nos terminator, in pUCI8 (See Figure 3-1). A detailed map of
the pUCl8 plasmid can be found in Sambrook, et al. (1989). Neither
plasmid contained a scoreable marker such as the uidA gene for -
EcoRI Pui
BglI 4 E9 -3
CTY CP ge ne r'. r h
M Py u I
P- e355left bo rde r--
right border i
0 ri -Y
pMON1 0098- CP
-9.3 kb
Pst I "o ri-322
Figure 3-1a. Map of pMON0098. Spc/str is the Spectinomycin
and streptomycin resistance gene. P-e35S is the enhanced CaMV
35S promoter. CP is the coat protein gene. E9 3' is the
terminator sequence. P-35S is the CaMV 35S promoter. Kan is
the Kanamycin resistance gene (equivalent to the NPTII gene).
Nos-3' is the Nopaline synthase terminator. ori-V is the
origin of replication. R is the right border and L is the
left border. The -> or <- is the direction of translation.
The Kan gene confers resistance in the plant cells. The
spc/str gene allows for selection in E. coli. The rest of the
plasmid that is not shown is pBR322 (See Sambrook et al., 1989
for the map of pBR322).
Q leader seq
CTV CP aer e
1 e ft t O
Sall Bgltl
Figure 3-1b. Map of pBPFa-7. amp is the ampicillin gene used
for bacterial selection. P-35S is the enhanced CaMV 35S
promoter which consists of two CaMV 35S promoters in tandem.
CP is the coat protein gene. n is the 71 base pair 5' leader
sequence of TMV. The rest of the plasmid is pUC18 (See
Sambrook et al., 1989 for the map of pUC18).
Transformation of E. coli (Sambrook et al., 1989; E. Anderson, pers.
Before a large scale plasmid preparation can be performed, the
plasmid must first be transformed into competent E. coli cells. Competent
cells were prepared using the following procedure (E. Anderson, pers.
comm.). An microfuge tube containing 1.5 ml of 2xYT medium (16 g/l
bactotryptone, 10 g/l yeast extract, 5 g/l NaCI) was inoculated with E.
coli strain HB101 and incubated overnight on a shaker at 225 RPM at 370C.
To 50 ml of 2xYT in a 125 ml Erlenmeyer flask was added 125 Al of the
bacterial culture. The bacterial culture was then incubated at 370C at on
a shaker at 225 RPM. After 2 hours 10 ml of this bacterial culture was
removed and placed in a prechilled Falcon 2056 tube and left on ice for 15
minutes. The sample was then centrifuged for 10 minutes at 5000 RPM in a
J-20 rotor. The supernatant was decanted off and the pellet resuspended
in 5 ml of 50 mM CaCl2 (sterile), vortexed, and incubated on ice for 15
minutes. The sample was again centrifuged at 5000 RPM for 10 minutes in
a J-20 rotor and the supernatant decanted off. The pellet was then
resuspended in 500 Al of 50 mM CaCl2 by gently tapping the sides of the
tube and placed back on ice for at least two hours. The cells were then
ready to be used or could be stored at -800C (with 50% glycerol added)
until needed for transformation with the plasmid.
To a prechilled Falcon 2056 tube was added 200 Al of competent cells
along with 1-2 il of plasmid. The cells and plasmid were gently mixed by
tapping the sides of the tube and the sample left on ice for 30 minutes.
The sample was then heat shocked for 2 minutes at 420C and immediately
placed back on ice for 5 minutes. To this sample was then added 1 ml of
2xYT and the sample gently mixed by tapping the sides of the tube, and the
samples incubated for 1 hour at 370C shaking at 225 RPM. After one hour
100 Al of sample was plated onto LB solid media (10 g/l bactotryptone, 5
g/l yeast extract, 10 g/l NaCl, 15 g.l agar) containing the appropriate
antibiotic, and incubated at 370C inverted in a 370C incubator.
The surviving colonies were selected for further use by transferring
cells from the colony with a sterile toothpick which was then swirled in
an microfuge tube containing 1 ml of 2XYT supplemented with 100 Ag/ml of
either spectinomycin or ampicillin (depending on the plasmid). These
cells were then grown overnight and tested for presence of the plasmid by
doing a plasmid miniprep, and then stored at -20 for future use.
Ouick Miniprep Plasmid Purification. (from E.J. Anderson, pers. comm.)
Plasmid DNA was extracted from bacterial increases of several of the
colonies and compared to the DNA from the known plasmid stock by
restriction analysis using agarose gel electrophoresis in the following
Single colonies were selected from the plated cultures using a
sterile toothpick to transfer cells from the individual colonies to an
microfuge tube containing 1.5 ml of 2xYT media and the appropriate
antibiotic, and grown overnight at 37 C, shaken at 225 RPM. The samples
were pelleted by centifuging in a microfuge for 2 minutes. The
supernatant was discarded and the tube shaken vigorously to remove as much
of the media as possible, and set upside down on a kimwipe to dry briefly.
The cells were then resuspended by gentle vortexing after adding 250 Y1 of
SucTET (8.8 g Sucrose, 1 ml of 1M Tris-HCl pH 8.0, 10 ml of 0.5 M EDTA,
500 Al Triton X100, volume adjusted to 100 ml) plus 15 y1 of lysozyme
(from 10 mg/ml stock), and placed on ice for 10 minutes. The cells were
then boiled for 50 seconds and placed on ice for another 10 minutes. The
cellular debris was pelleted by centrifuging for 10 minutes in a
microfuge. The debris was then removed using a sterile toothpick. To the
remaining liquid was added 65 Al of 7.5M ammonium acetate, and the samples
incubated for 10 minutes on ice. The samples were centrifuged for 5
minutes and the supernatant transferred to a fresh tube. To each sample
was added 200 ,I of isopropanol, the sample mixed by inversion, kept on
ice for 20 minutes, and then centrifuged for 10 minutes. The supernatant
was poured off, the pellet gently washed with 70% ethanol, and dryed under
vacuum. The pellet of plasmid DNA was then reuspended in 50 A1 of TE
A restriction digest was then performed to test for the presence of
the coat protein gene. First, 5 A1 of the isolated plasmid (plus a
control of the original plasmid) was removed and placed in an microfuge
tube. To this was added 5 A1 of 1OX of the restriction enzyme buffer H
(Promega), 1.0 Al of EcoRI (Promega), and 39A1 of distilled water, and the
restriction digest allowed to proceed by incubating for at least 60
minutes at 370C. The plasmid was then ethanol precipitated by adding 5 gi
3 M sodium acetate and 200 g1 of 95% ethanol (cold) and centrifuging for
15 minutes. The supernatant was discarded and the pellet washed by
carefully adding 250 Al of 70% ethanol (cold) and centrifuging for another
5 minutes. The supernatant was then discarded and the pellet dried under
vacuum. The pellet was then resuspended in 44 Al of sterile ddH2o and 5
gi of restriction enzyme buffer D (Promega) and 1.0 Al of BglII was added.
The samples were placed at 370C for 1 hour and ethanol precipitated as
before. The dried sample was then resuspended in 10 Al of TE buffer.
Meanwhile, an agarose gel (1% agarose in TBE (see Appendix B)) was
prepared. Ten minutes before loading the gel 0.5 ml of RNase (10mg/ml)
was added to each tube. Indicator dye (3 Al of 6X loading buffer) was
added and the gel was loaded. The gel was run for 1 to 2 hours at 100
Volts. The gel was then removed and stained with 10 Al of ethidium
bromide (10 mg/ml) in 100 ml of distilled water with gentle shaking for 30
minutes or longer. The gel was de-stained in distilled water (approx. 100
ml) for at least 5 to 10 minutes, then observed and photographed on a UV
light box. Samples which contained the band corresponding to the band for
the coat protein gene (as observed in the control) were saved to use for
the large scale isolations.
Large Scale Isolation and Preparation of Plasmid (Sambrook et al., 1989)
The day before plasmid isolation, four 1-liter flasks containing 500
ml of Luria Broth plus the appropriate antibiotic were inoculated with the
transformed E. coli and the cells were incubated overnight at 370C on a
shaker at 225 rpm.
The next morning the bacterial cultures were dispensed into 500 ml
centrifuge bottles and centrifuged at 5000 rpm in a JA-10 rotor. The
supernatant was discarded and the bacterial pellet resuspended in 20 ml of
solution I (50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, with
5 mg/ml lysozyme added just before use). The solution was allowed to
stand at room temperature for 5 minutes and then 40 ml of freshly prepared
Solution II (0.2 M NaOH, 1% SDS) was added. The solution was then gently
mixed by inverting the bottles gently several times and allowed to set at
room temperature for 10 minutes.
The contents of each bottle were then split between two 50 ml
centrifuge tubes. To each tube was added 10 ml of ice-cold Solution III
(60% 5 M potassium acetate, 11.5% glacial acetic acid, 28.5% distilled
H20). The tubes were covered with a cap, mixed by shaking the tubes
several times, and placed on ice for 10 minutes. At this time a
flocculent white precipitate formed. The samples were then centrifuged at
10,000 rpm (JA-20 rotor) for 10 minutes at 4C and the rotor allowed to
stop without braking.
The supernatant from all eight tubes was filtered through four
layers of cheesecloth and into a 500 ml centrifuge bottle. The solution
was then equally divided between two 500 ml centrifuge bottles. To each
bottle, 100 ml of isopropanol was added and the bottles were placed at
room temperature for 30 minutes or longer.
The nucleic acids were recovered by centrifuging at 5000 rpm (JA-10
rotor) for 30 minutes at 40C. The supernatant was decanted off carefully
and discarded. The pellet was washed with 200 ml of cold 70% ethanol and
centrifuged again for 10 minutes at 5000 rpm at 40C. The alcohol was
poured off carefully and the pellet allowed to air dry. After air drying
the pellet was resuspended in 10 ml of TE buffer (see Appendix B) and
allowed to set at room temperature for at least an hour to allow for as
much of the plasmid as possible to go into solution. The solution was
transferred to a 50 ml centrifuge tube and 5 ml of chloroform-isoamyl
alcohol (24:1) and 5 ml of tris-equilibrated phenol (Sambrook et al. 1992)
was added. After thoroughly mixing, the solution was centrifuged at 5000
rpm (JA-20 rotor) for 10 minutes to separate the phases. The aqueous
(upper) phase containing the plasmid was removed and placed in another 50
ml centrifuge tube. The plasmid was ethanol precipitated by adding 3.5 ml
of sodium acetate and 35 ml of ethanol and placed in the refrigerator at
40C overnight, followed by centrifuging for 30 minutes at 10,000 rpm (JA-
20 rotor) at 40C.
Immediately after centrifuging the supernatant was decanted, 10 ml
of cold 70% ethanol added, and the tubes were recentrifuged for 10 minutes
at 10,000 rpm at 40C. The supernatant was immediately decanted and the
pellet dried under vacuum. The pellet was then resuspended in 10 ml of TE
buffer. To determine the concentration of plasmid DNA, an absorbance
reading at an O.D.260 was obtained. An absorbance reading of 1.0
corresponds to 50 Ag of plasmid DNA per ml. The plasmid was ethanol
precipitated as before and resuspended at 1 jig/gl in BH3 (See Appendix A).
Establishment and Maintainance of Suspension Cultures
The Citrus suspension cultures used for protoplast isolation were
obtained from ovule-derived callus of 'Nova' tangelo ('Clementine'
mandarin (Citrus reticulata Blanco) X 'Orlando' tangelo (C. reticulata X
C. paradisi Macf)) or 'Rohde Red Valencia' sweet orange (C. sinensis
L(Osbeck)). The suspensions were maintained in 40-50 ml of liquid 1/2-1/2
medium (see Appendix A) in 125 ml erlenmeyer flasks covered with aluminum
foil and sealed with masking tape. Cultures were maintained on a shaker
at 140 rpm with no supplemental lighting. These suspensions were
subcultured every two weeks by splitting the contents between two sterile
flasks and adding a half volume (25 ml) of fresh culture medium to each
flask. The cells were used for protoplast isolation 5-14 days after
Isolation and Transformation of Citrus Protoplasts
Isolation of protoplasts from suspension cultures was performed as
follows (Grosser and Gmitter, 1990, as modified by Schell (1991)). First,
one ml of suspension culture was placed in a 10 mm X 60 mm petri plate
(usually 2 to 3 plates for each suspension used). Three ml of 0.7 M BH3
was added followed by 1 ml of enzyme solution (see Appendix A). Plates
were wrapped with Nesco film and placed on a shaker at 50 rpm inside an
incubator at 280C overnight (16 hours).
Protoplasts were collected by passing the above mixture through a
sterile 0.45 micron stainless steel screen (which had been adhered to the
top section of a 30 cc disposable syringe) and into a 50 ml collection
tube. This mixture was then transferred into a sterile 15 ml glass
conical centrifuge tube with a screw cap. The cells were then pelleted by
centrifuging for 5 minutes at 500 rpm (75 x g).
The supernatant was removed using a sterile pasteur pipet and the
pellet was resuspended in 4 pipets (approx. 6-7 ml) of CPW25SUC (see
Appendix A). Then the solution was overlayed with 5 pipets (approx 7-8
ml) of CPW13MAN (See Appendix A). The CPW13MAN was added slowly, and the
tube was slanted so that the two solutions were not mixed. At this point
there were two distinct layers visible- a lower layer containing the
protoplasts and debris, and an upper clear layer. They were then
centrifuged for 10 minutes at 500 rpm (75 x g).
After centrifugation a distinct band was visible at the interphase
of the two solutions, a pellet at the bottom of the tube, and a cloudy
appearance between the two. The band at the interphase layer contained
the viable protoplasts, and the other two layers contained the non-viable
protoplasts and debris. The top layer of CPW13MAN which remained clear
and did not contain any cellular debris or protoplasts was carefully
removed without disturbing the band (removing the top layer of CPW13MAN
prior to removal of the band minimizes the amount of debris removed with
the band) and placed in a second sterile tube. The band was then
carefully removed from the top, being careful to avoid taking any of the
cloudy layer of debris and transferred to the tube containing the CPW13MAN
and gently resuspended (Frearson et al., 1973; Schell, 1991). The
protoplasts were pelleted by centrufuging for 5 minutes at 500 rpm (75 x
g) and the supernatant removed. The cells were then resuspended to a
volume of approximately 10 times the volume of the pellet with 0.7 M BH3.
This corresponds to a density of approximately 1 X 106 protoplasts/ml and
can be confirmed by counting using a haemocytometer.
At this point the protoplasts were ready to be used for the
transformation experiments. First, 10-40 plates (10mm X 60mm) were laid
out and 3 drops of the protoplasts mixture was placed in the center of
each plate. Of these plates, 3-4 were used as control and are treated the
same as the other plates except no plasmid or other DNA was added. To the
other plates, 10-20 kil (10-20 jig) of plasmid DNA was added to the
protoplast drop. To this was added 4 drops of PEG solution (see Appendix
A), and the cells were allowed to stand for 10 minutes. To this mixture
was then added 4 drops of a 9:1 v:v solution of Solution A:Solution B (see
Appendix A) around the edge, and the cells incubated for another 20
minutes. Finally 15 drops of BH3 were added and the cells then incubated
another 10 minutes.
The liquid was then pipetted off very carefully, another 15 drops of
BH3 added, the cells allowed to stand another 5 minutes and the liquid
again removed carefully. This was repeated twice more to ensure that the
cells were adequately washed and the PEG removed. The cells were then
suspended in 1 ml of a 1:1 mixture of 0.6 M BH3 and 0.6 M EME (see
Appendix) and swirled to spread the medium thinly across the whole plate.
The plates were then wrapped with a double layer of Nesco film, placed in
a cardboard box, and incubated in the dark at 280C.
Culturing and Regeneration of Treated Protoplasts
Regeneration of plants from treated protoplasts requires close
observation as the cells grow. Not doing this may result in the cells
growing too fast and losing their embryogenic capacity. If this happens
it is virtually impossible to recover any plants, as rapid cell growth
inhibits somatic embryo induction. The procedure for culturing and
regenerating the treated protoplasts was as described in Schell (1991).
Testing for Transformed Plants
Polymerase Chain Reaction for Detection of Transformants
The polymerase chain reaction (PCR) was used to initially screen the
regenerated 'Nova' tangelo and 'Rohde Red Valencia' sweet orange plants.
Since the procedure used was directly related to the results obtained, the
discussion of the procedures, primers used, PCR profile and other elements
of the PCR procedure will be discussed in the Results and Discussion
Testing for Transformed 'Rohde Red Valencia' Sweet Orange Using Dot Blot
Isolation of DNA from Citrus Leaves for Dot Blot Analysis (Dellaporta et
al., 1983, as modified by R. Durham, pers. comm.; Sambrook et al., 1989;
Manning, 1991)
Approximately 0.75 to 1.00 g of young leaf tissue was ground in
liquid nitrogen using a mortar and pestle. The powder was then
transferred to a 50 ml Oak Ridge centrifuge tube and placed on ice. After
the nitrogen had evaporated, 15 ml of ice cold DNA extraction buffer (100
mM Tris-HCl pH 8.0, 50 mM Na2EDTA pH 8.0, 500 mM NaCI, 10mM Beta-
mercaptoethanol) and 2 ml of 10% SDS were added. The tube was capped and
shaken vigorously, then placed in a water bath at 650C for 10 minutes.
The tubes were then removed from the water bath and 5 ml of 3 M
potassium acetate solution (60 ml/100 ml 5 M potassium acetate, ll.5ml/100
ml glacial acetic acid, 28.5 ml/100 ml H20) were added. The tubes were
again capped, shaken vigorously, and incubated on ice for 20 minutes. The
samples were then centrifuged at 25,000 X g for 30 minutes. The
supernatant was filtered through a miracloth filter and into a clean 50 ml
Oak Ridge tube. To this was added 10 ml of isopropanol and the samples
allowed to sit at room temperature for 30 minutes or longer. At this
point a gelatinous polysaccharide material precipitated with the DNA.
This polysaccharide material did not appear to inhibit enzyme digestion
but made the final DNA solution very viscous. This could be partially
overcome by heating the DNA to 65C prior to pipetting the samples for
The DNA was pelleted by centrifugation at 20,000 X g for 30 minutes.
The supernatant was poured off and the pellet lightly dried by inverting
the tubes on a paper towel for 10 to 15 minutes. The DNA was then
resuspended in 0.7 ml of Buffer B (50 mM Tris-HCl pH 8.0, 10 mM Na2EDTA pH
8.0) and transferred to a 1.5 ml microfuge tube, then centrifuged for 10
minutes in a microfuge to remove any debris. The supernatant was then
transferred to another 1.5 ml microfuge tube.
To the DNA solution was then added 0.7 ml of Tris-equilibrated
phenol, and the solutions mixed by inverting the tube several times. The
samples were then microfuged for two minutes followed by extracting the
aqueous upper phase and transferring it to a clean microfuge tube. This
was repeated using 0.7 ml of chloroform/iso-amyl alcohol (24:1).
The resulting DNA extract contains a large amount of polysaccharides
which can be a problem when resuspending the DNA after ethanol
precipitation in a small quantity of TE buffer or water. The extract can
be very viscous and difficult to pipet unless diluted. To remove the
polysaccharides so as to sufficiently concentrate the DNA for further uses
a differential precipitation with 2-butoxyethanol (ethylene glycol
monobutyl ether) was performed (Manning, 1991). The solution obtained
after phenol/chloroform extraction was supplemented with 100 il of a 1.0
M acetate solution (pH 6.5) and 0.4 volumes (250 Al) of 2-Butoxyethanol.
The samples were vortexed and placed on ice for 30 minutes (The
polysaccharides are visible as a whitish cloud). The samples were then
centrifuged in a microfuge for 30 minutes. The supernatant was then
removed and transferred to a fresh tube (the polysaccharides remain as a
pellet in the bottom of the tube). The volume was then brought up to one
volume of 2-butoxyethanol by the addition of another 400 jil 2-
butoxyethanol, vortexed and again placed on ice for 30 minutes. The
samples were again microfuged for 30 minutes, then the supernatant was
decanted off. The resulting DNA pellet was then washed by the addition of
500 Al of 95% ethanol. The samples were allowed to stand at -200C for at
least 30 minutes. At this point the DNA pellet becomes visible as a white
pellet in the bottom of the tube. The samples were then centrifuged for
10 minutes and the supernatant decanted off. A final wash with the
addition of 500 Al of 70% ethanol followed by microfuging for 5 minutes
was then performed. The supernatant was decanted off, the tubes inverted
on a kimwipe to remove excess alcohol, and then vacuum dried in a
dessicator. The pellet was then suspended in 50 pI of TE buffer and
quantified by spectrophometry. An O.D. at 260nm of 1.0 is equivalent to
50 Ag/ml of DNA. The quality of the DNA was checked by determining the
O.D. 260 to O.D. 280 ratio. DNA should have a ratio of 1.8, RNA a ration
of 2.0 (Sambrook et al., 1989). Alternatively the DNA was quantified by
agarose gel electrophoresis using lambda DNA of known quantities and
visually estimating DNA content in the samples. The DNA was then placed
in the -800C freezer until needed and thawed on ice prior to use.
Preliminary Screening Using DNA Dot Blots (from instructions supplied with
the Amersham Hybond N+ nylon membrane)
Five Mi of the sample from the DNA extraction just described was
removed, placed in a 1.5 ml microfuge tube, and placed on ice. The
samples were boiled for 10 minutes and quick chilled on ice to denature
the DNA. To each tube was added 5 il of 20X SSC (see Appendix B), the
samples spun briefly in the microfuge to collect all liquid at the bottom,
and placed back on ice. A nylon membrane was wet thoroughly in water,
then soaked in 6 X SSC and allowed to dry. The entire contents of each
sample were then spotted on the membranes 2.5 gl at a time. The DNA was
then cross linked using a DNA cross linker (1200 joules). The membrane
was then used for hybridization using radioactive probes as described
Confirmation of Transformation
Isolation of DNA for Southern Blot Hybridization (Duran-Vila et al.,1986;
Marais et al., 1995)
Prior to isolation of nucleic acids, all labware was sterilized by
autoclaving for one hour at 15 psi and dried in the drying oven. All
buffers and solutions were sterilized by autoclaving. Gloves were used to
handle all glassware during isolation.
For each sample, 5 g of tissue were ground in liquid nitrogen and
transferred to a labeled 100 ml beaker containing 10 mls of extraction
buffer (0.4 M Tris-HCl (pH 8.9), 1 % SDS, 0.005 M EDTA (pH 8.0)) and 10
mls of phenol-Chloroform-isoamyl alcohol (12:12:1) and stirred on a stir
plate. After all samples were ground they were stirred for an additional
20 minutes, transferred to a 50 ml centrifuge tube, capped and centrifuged
at 12,000 RPM for 20 minutes in a JA-20 rotor. The top layer of liquid
was removed and placed in another 50 ml centrifuge tube and 3 mls of 3 M
sodium acetate (pH 5.5) and 30 mls of cold 95% ethanol added. Samples
were incubated for 30 minutes at -80'C and then centrifuged as before.
The supernatant was decanted and the tube containing the pellet air dried
inverted on several layers of paper towels. The pellet was resuspended in
3 mls of TKM buffer (0.01 M Tris-HCl (pH 7.4), 0.01 M KCl, 0.0001 M MgCl2).
The samples were then placed in dialysis tubing (6000-8000 MWCO) which had
been boiled in 1 %SDS, 10mM EDTA (pH 8.0) and dialyzed overnight in 1 L of
TKM buffer on a magnetic stirrer with gentle stirring at 40C. The samples
were then removed from the dialysis tubing using a pasteur pipet,
transferred to 50 ml centrifuge tubes and 3 mls of 4 M LiCL added.
Samples were gently mixed and incubated for 4 hours to overnight at 40C.
Samples were then centrifuged for 20 minutes at 12,000 RPM in a JA-20
rotor. The supernatant which contains the DNA was transferred to another
50 ml centrifuge tube and 18 mls of cold 95% ethanol added. Samples were
placed at -800C for 30 minutes and centrifuged as before. The supernatant
was discarded, the pellet air dried and resuspended in 100 Al of sterile
ddH20. The pellet from the LiCl precipitation, which contains ssRNA, was
dried and resuspended in 100 Al of sterile double distilled H20 (ddH20).
The samples (2 Al of each, with 8 Al H20 and 3 pi of loading buffer (40%
sucrose, 0.25% bromphenol blue) were then ran on a 1% agarose gel in TBE
(see Appendix B) at 100V for 1 hour to quantify the DNA (against Lambda
DNA of known concentrations), and to confirm that the desired nucleic
acids were in the proper fraction.
Southern Transfer of DNA Isolated from Transformed Plants (Sambrook et
al., 1989; R.F. Lee, pers. comm.).
Southern analysis is routinely used to prove that the target DNA has
been incorporated into the plant genome. The following procedures were
used to test plants which were positive in the preliminary testing by dot
blot or PCR assay.
Transfer under neutral conditions.
DNA was isolated from plants to be tested by the methods described
previously. At least one non-transformed (never exposed to the plasmid or
virus) plant was used as a control in every analysis. A sample of each
DNA to be analyzed (around 10-20 pg as determined by quantification) was
removed and placed in a separate 0.5 ml microfuge tube. The DNA was then
restriction digested by adding 2 jil/i0 pi of 10 X OPA enzyme buffer
(supplied by Pharmacia with the restriction enzymes) and 10-15
units/sample of restriction enzyme(s) (BglII with or without EcoRl) and
incubated for 3 hours to overnight at 370C. The samples were run on a gel
containing 0.5 X TBE (see Appendix B) and 1% agarose in 0.5 X TBE buffer.
The gel was then stained with 10 Al of ethidium bromide solution (10
mg/ml) in approximately 100 ml of distilled H20 for a minimum of 30
minutes, and then destained for 10 minutes in distilled H20. The gel was
then observed on the UV light box, photographed (if desired), and the
unused portions of the gel trimmed away.
The DNA was nicked by depurination in several volumes of 0.2 N HCl
for 10 minutes, followed by a brief rinse in distilled H20. The gel was
denatured by soaking the gel in several volumes of denaturation solution
(1.5 M NaCl, 0.5 M NaOH) for 45 minutes with constant gentle agitation on
a shaker. The gel was then rinsed for a few minutes in distilled H20. The
gel was neutralized by soaking under gentle agitation in several volumes
of neutralizing solution (1 M Tris pH 8.0, 1.5 M NaCI) for 15 minutes. The
neutralizing solution was then discarded and replaced with fresh
denaturing solution and allowed to soak under gentle agitation for another
15 minutes. This was repeated twice more.
While the gel was in the neutralizing solution a piece of Whatman
3MM paper was wrapped around a piece of plexiglass or a stack of glass
plates to form a support that was longer and wider than the gel. This was
placed in a large baking dish. The dish was then filled to just below the
rim of the top support with 10 X SSC (see Appendix B) After the paper
was thoroughly wetted, the air bubbles were smoothed out using a glass
rod. A piece of nylon membrane (Hybond N+) was cut to the size of the gel
and placed in a dish of distilled water until the membrane was wetted
completely. The membrane was then immersed in transfer buffer (10 X SSC)
for at least five minutes.
After neutralization was complete, the gel was removed, inverted so
that the gel was upside down and placed in the center of the wet filter
paper. All air bubbles were carefully removed. The wet nylon membrane
was than carefully placed on top of the gel and one corner cut to use as
a reference point. Once the membrane was placed on the gel it was not
moved. All air bubbles were again carefully removed so as not to
interfere with DNA transfer. Two pieces of 3MM paper were then cut to the
same size as the gel, wet with 2 X SSC, and placed on top of the nylon
membrane. The air bubbles were once again removed.
A stack of newspaper 3-5 cm high were cut just smaller than the 3MM
papers and placed on top of the 3MM papers. A glass plate was placed on
top of the stack and weighed down with a 500 g weight. The objective was
to set up a flow of liquid from the reservoir up through the gel and the
membrane and into the stack of newspaper. The DNA fragments should be
eluted from the gel and deposited onto the membrane. The transfer was
allowed to proceed overnight (8-24 hours).
After the transfer was completed, the newspaper and the 3MM papers
above the gel were removed. The gel, with the membrane still in place,
was turned over onto a dry sheet of 3MM paper and the positions of the gel
slots marked with a very soft lead pencil. The gel was then peeled away
and discarded. The membrane was soaked in 6 X SSC for 5-15 minutes with
gentle agitation to remove any pieces of agarose sticking to the membrane.
The membrane was removed and dried on a fresh piece of 3MM paper for 30-60
minutes. The DNA was then ready to be hybridized with a labeled probe.
Transfer under alkaline conditions,
The DNA extraction through electrophoresis and photographing of the
gel was performed in the same manner as for transfer under neutral
conditions. After electrophoresis the DNA in the gel was nicked by
placing the gel in 0.25 N HC1 for 15-20 minutes. The gel was then soaked
in 0.4 N NaOH for 30-45 minutes. At the same time the transfer system was
set up as for transfer using neutral conditions, but with the 1oX SSC
replaced by 0.4 N NaOH. The transfer was allowed to continue overnight.
The membrane was removed and the lanes marked with a red pen. The
membrane was soaked with gentle agitation in 5X SSC to neutralize, then
air dried for a few minutes. Many protocols call for baking the membrane
at 80'C for two hours or UV cross-linking to fix the DNA to the membrane,
but this is not necessary for positively charged membranes. The membrane
was then placed in a plastic bag and was ready for hybridization.
Preparation of Radioactive Probes by Random Primer for Southern Blot
The probe used for Southern blot hybridization was prepared using
PCR. The coat protein gene of T30 and T36 were amplified using primers
C104 (CCTACTCGAGATTAACGTGTGTTGAATTTCC, 3' neg. sense) and C118
(AACTGCAGCGGCCGCCGACGACGAAACAAAG,5' plus sense)(obtained from J.
Castillo). The PCR reaction was performed in capillary tubes using a
profile with an initial denaturation at 940C for 15 sec. followed by 30
cycles of denaturation at 940C for 5 sec., annealing at 500C for 10 sec.,
extension at 73C for 20 sec., followed by a final extension at 72C for
5 minutes. Samples were then ran on a 1 % agarose gel (using low melting
point agarose in 0.5 X TBE), and the band of the appropriate size (approx.
670 bp) was excised from the gel and placed in a 1.5 ml microfuge tube.
Five volumes of TE buffer were added and the samples incubated at 650C for
ten minutes or until the agarose was fully melted. If the volume was more
than 750 il, the samples were subdivided and then an equal volume of tris-
equilibrated phenol was added. Samples were vortexed, than centrifuged
for 3 minutes. The top layer containing the DNA was removed and placed in
another microfuge tube. To the sample was then added 0.5 volumes of Tris-
equilibrated phenol and 0.5 volumes of chloroform/iso-amyl alcohol (24:1)
and the samples vortexed and centrifuged for 3 minutes. The top layer was
again removed and transferred to a separate tube and an equal volume of
chlorform/iso-amyl alcohol (24:1) was added. Samples were vortexed and
centrifuged for 3 minutes and the top layer again removed and transferred
to a separate tube. The DNA was ethanol precipitated by the addition of
0.1 volumes on 3M sodium acetate and 3 volumes of 95 % ethanol followed by
centrifugation for 15 minutes. The supernatant was decanted and the DNA
pellet was then washed by the addition of one volume of 70% ethanol
followed by centrifugation for 5 minutes. The supernatant was again
decanted and the tube inverted on a Kim Wipe for a few minutes, then
placed in a vaccuum dessicator and the DNA dried under vacuum. The DNA
was then resuspended in sterile distilled water at an approximate
concentration of 25 ng/pl.
The probe was prepared by random primer labeling using the Prime-a
Gene Labeling System (Promega Corp.) as per the instructions provided.
For each labeling reaction 25 ng of probe DNA was denatured by boiling for
5 minutes and then quick chilling on ice. The DNA was usually placed in
nuclease free water to bring the final volume of the reaction up to 50 P1
prior to boiling. After denaturation, 10 Al of 5 X buffer, 2 Al of the
unlabeled dNTP mix (dATP, dCTP, dGTP), 2A1 nuclease free BSA, 5P1 [a-32p]
dCTP (50 ACI, 3000Ci/mmol), and 1 pl (5 units) DNA Polymerase (Large
(Klenow) fragment) were added. The reaction was allowed to proceed for 3
or more hours and the reaction stopped by the addition 2.5 Al of 0.5 M
EDTA (pH 8.0), followed by boiling for two minutes and quick chilling
prior to use.
Hybridization of DNA With a Labelled Probe. (Sambrook et al., 1989; T.
Kamps, pers.comm.)
The nylon membrane prepared previously for either the dot blot
analysis or Southern blot hybridization, was placed in a plastic bag and
prehybridization solution was added. The prehybridization solution
consisted of 6 X SSC, 5 X Denhardts reagent, 0.5 % SDS, and 100 Ag/ml
salmon testes DNA (boiled 10 minutes and quick chilled). The bag was
carefully sealed and placed inside a ziploc bag and incubated for 1-2
hours to overnight (16 hours) in a water bath at 680C. the probe prepared
by Random Priming was added at 5 X l05 to 2 X 106 CPM after boiling for 3
minutes and quick chilling on ice. The bag was resealed, placed in a
ziploc bag, and incubated overnight (16-24 hours) in a water bath at 650C.
The next morning the hybridization solution was disposed of in the
radioactive waste and the membrane was rinsed twice in wash solution (100
ml/l 20 X SSC, 10 ml/l 10% SDS, 200 ml/l of 0.1 M NaPO4, pH 7.2) for 15
minutes each at 65'C with occasional gentle agitation. The membrane was
placed in a plastic bag and approximately 50 mls of stringency buffer (5
ml/l 20 X SSC, 10 ml/l 10% SDS, 20 ml/l of Na P04 pH 7.2) was added, the
bag sealed and incubated in the water bath for 15 minutes at 650C. The
solution was discarded and fresh stringency buffer added and the membrane
was incubated in the water bath at 65C for another 15 minutes.
The membrane was dried on a piece of 3MM paper for a few minutes,
then placed on a fresh piece of 3 MM paper. The corners of the membrane
were taped down and then wrapped in plastic wrap. The membrane was placed
face down on a piece of X-Ray film in a film cassette with an intensifier
screen. The cassette was sealed, wrapped in aluminum foil, and placed in
the -800C freezer for 1 to 7 days (depending on the half-life status of
the isotope and the quality of the random priming). The film was
developed according to the directions provided with the developer.
Southern Blot Hybridization Using the GENIUS Non-radioactive
Labeling System (Boehringer-Mannheim)
Prior to hybridization, the labeled DNA to be used as a probe needed
to be prepared. The plasmid DNA which was to be used for the probe was
first denatured by boiling for 10 minutes followed by quick chilling in
ice/alcohol. An aliquot of 1 Ag of freshly denatured DNA was removed and
placed in a microfuge tube on ice and to this was added 2 pl of the
supplied hexanucleotide mixture, and 2 Al of the supplied dNTP labeling
mixture. The volume was then brought up to 19 Al with sterile distilled
H20, 1 Al of Klenow enzyme added and the mixture was incubated for 60
minutes or more at 37C. The reaction was stopped by adding 2 Al of EDTA
solution (0.2 M, pH 8.0).
The labeled DNA was then precipitated by adding 2 Al of LiCl (4M)
and 60 yi of cold 100% ethanol. The DNA was left for at least 30 minutes
at -70 C, or at least 2 hours at -20 C, and then centrifuged at 12,000 X
g for 10 minutes. The supernatant was then poured off and the pellet
washed with cold 70% ethanol and recentrifuged at 12,000 X g for 10
minutes. The alcohol was then poured off and the pellet dried under
vacuum. Finally, the DNA was dissolved in 50 il of TE buffer and stored
at -20'C until needed.
Approximately one hour before starting the hybridization, the
hybridization solution was prepared (the blocking reagent does not
dissolve very rapidly, thus the need to prepare in advance). The
hybridization solution consisted of 5X SSC (see Appendix B), 0.5% blocking
reagent (supplied), 0.1% N-laurylsarcosine (Na-salt), and 0.02% SDS. It
was heated in a 70'C water bath in order to facilitate the blocking
reagent going into solution.
The membranes which had been previously prepared (by Southern
transfer or dot blotting) were wet briefly with 5X SSC and then placed in
a plastic sealable bag. Hybridization solution (20 ml) was added, the bag
sealed, and prehybridization carried out at 68C for 4 hours. The
solution was replaced with 25 ml of hybridization solution and 50 Al of
labeled DNA. The membranes were incubated overnight (18 hours) at 680C.
The membranes were washed twice, 5 minutes each, with at least 100 ml of
wash buffer (2X SSC, 0.1% SDS), followed by two washes of 15 minutes each
at 68C in stringency buffer (0.1X SSC, 0.1% SDS). The membranes were air
dried and stored protected until used for the immunological detection
using the labeled DNA probe.
The membranes were first washed for 1 minute in buffer 1 (Tris-HCl,
100 mM, NaCl, 150 mM, pH 7.5). Buffer 1 was poured off and 100 ml of
buffer 2 (0.5% blocking reagent in Buffer 1) was added and the membranes
incubated for 30 minutes. The membranes were again washed for 1 minute
with buffer 1 and placed in a plastic sealable bag. Antibody-conjugate to
the labeled probe was diluted to 150 mU/ml (1:5000) in buffer 1, and added
to the membranes and incubated another 30 minutes. Unbound antibody-
conjugate was removed by washing twice (15 minutes each) with buffer 1.
The membranes were equilibrated for 2 minutes in 20 ml of Buffer 3 (Tris-
HCI, 100 mM, NaCl, 100 mM, MgCl2, 50 mM, pH 9.5) Freshly prepared color
solution (45 p1i NBT solution, 35 Al x-phosphate solution in 10 ml of
buffer 3) was added and the color precipitate allowed to form overnight in
the dark. The reaction was stopped by washing the membranes overnight in
TE buffer. The membranes were stored in TE Buffer until they were needed.
Alternatively, the membranes could be stored dry, however the color may
fade. The color could be enhanced by placing the dry membrane back in TE
Western Immunoblot analysis for Detection of the Coat Protein
Leaves were collected and kept in the cold room or in the -20'C
freezer until needed. The leaves were then ground in liquid nitrogen and
the powder transferred to a 1.5 ml microfuge tube. Immediately, 1 ml of
1X treatment buffer (60 mM Tris, 2% SDS, and 1% 2-mercaptoethanol) was
added and the sample vortexed. This was repeated until all samples were
prepared. The samples were allowed to set for 15-30 minutes, then
centrifuged in the microfuge at 10,000 RPM for 10 minutes to pellet the
debris. The supernatant was removed and placed in another sterile 1.5 ml
microfuge tube. A 100 Al aliquot of the sample was removed and placed in
1.5 ml microfuge tube and frozen until used. The rest of the sample was
concentrated using an RC10 concentrator/evaporator (Jouan, Inc) to
approximately 1/5 the original volume. The heat setting of around 3.5 was
used in order to keep the samples from freezing. Concentration usually
was finished in around 2 hours. The volume of the samples was then
brought to approximately one fifth the original volume by the addition of
additional ix treatment buffer (all samples were not concentrated equally
so the volumes need to be made equal for all the samples) and the samples
placed at -200C until needed.
The proteins were separated on a 14% polyacrylamide gel on a BioRad
minigel apparatus. The gel was prepared by adding 4 ml/gel of running gel
stock (see Appendix B), 53 Al/gel of 10% TEMED, and 13 p1/gel of 10%
ammonium persulfate. The gel was then loaded to a depth of 5 cm using a
transfer pipet. This layer was then overlayed with Stacking gel
containing 2 ml/gel stacking gel stock (see Appendix B), 40 pi/gel TEMED,
and 10 pl/gel of 10% ammonium persulfate. A gel comb was then place in
the gel and the gel allowed to polymerize for at least one hour.
While the gel was polymerizing, the protein samples were aliquoted
into an microfuge tube and boiled for 10 minutes. After the gel was
polymerized the samples (30 Al) were loaded on the gel and the gel was run
at 40mA/gel for 105 minutes in SDS tank buffer (ig/l SDS, 14.5 g/l
Glycine, 3g/l Tris).
After the gel was finished running, the gel was removed and placed
in protein transfer buffer (25 mL Tris-HCl pH 8.3, 192 mM Glycine, 20 %
Methanol) for 15-20 minutes. The buffer was then removed and two pieces
of Whatmann filter paper cut to the size of the gel was wet with protein
transfer buffer and placed on top of the gel. The gel and filter paper
were carefully removed from the tray and trimmed to size. A nylon
membrane (Immobilon P from Millipore Corp.) was wet briefly in methanol,
the methanol removed by placing the membrane in H20 until submerged, and
soaked briefly in protein transfer buffer. (Alternatively, a
nitrocellulose filter wet in transfer buffer can be used.) The membrane
was placed on top of the gel and an additional two pieces of Whatmann
filter paper wet with protein transfer buffer placed on the membrane, and
again trimmed to size. The whole sandwich was placed with the membrane
side down on a semidry electroblotter and the proteins transferred by
running at 0.4A/cm2 for 2 hours.
The membrane was removed and placed in 20 ml TTBS (0.5M Tris-HCl pH
7.5, 2M NaCl, 0.0005% Tween-20) containing either 3% bovine serum albumin
(BSA) or 1% gelatin as a blocking agent, and placed on a shaker at 75RPM
for at least 20 minutes. The desired primary antibody was added at a
1:10000 v:v dilution and placed back on a shaker for 30-45 minutes (or
overnight). The membrane was then washed with TTBS at least 3 times for
10 minutes on a shaker. Fresh TTBS (20 ml) with 1% gelatin or 3% BSA was
added along with the appropriate secondary antibody at a dilution of
1:10000 v:v and again placed on shaker for 4-6 hours (or overnight). The
membranes were again washed at least 3 times for 10 minutes on shaker with
TTBS. The membranes were developed by the addition of a thin layer
(approx. 20 ml ) of NBT-BCIP substrate solution (12 g/l Tris, 5.8 g/l
NaCl, 1 g/l MgCl2, 80 mg/l BCIP (disodium salt), 160 mg/l NBT, pH 9.5).
Modifications of this procedure were used and are described as appropriate
in the results and discussion.
Plasmid Preparation
The coat protein genes for CTV isolates T30 and T36 were inserted
into the plasmids pMONI0098 and pBPFQ-7 by Dr. Niblett's laboratory and
supplied in E. coli strain DH5. These plasmids were designated
pMONI0098-T30, pMONI0098-T36, pBPFQ7-T30, and pBPF07-T36. (Figure 3-3
shows a map of these plasmids.) Testing for correct orientation and coat
protein expression were performed in Dr. Niblett's laboratory prior to
sending the plasmids. The bacterial cultures were replated and single
colonies selected to begin large scale plasmid preparation as described in
the materials and methods.
Transformation of Citrus Protoplasts and Plant Regeneration
The success or failure of isolation and transformation of Citrus
protoplasts and the ultimate regeneration of plants is
contingent on the quality of the starting material and quality of the
protoplasts after isolation (high yields of viable protoplasts with little
debris). Citrus suspension cultures
used were chosen because they were exhibiting good regeneration capacity
at the time. The best protoplasts were obtained from suspensions 5 to 12
days after subculture when isolated as described in the materials and
methods. Many isolation procedures do not use the sucrose/mannitol
gradient for purification. However using the gradient allows for the
removal of the non-viable protoplasts and cellular debris which could
interfere with the uptake of the plasmid by the viable protoplasts,
possibly increasing efficiency.
After the protoplasts had been collected from the gradient, they
were resuspended in 0.6 M BH3 at a density of approximately 1 X 106/ml as
determined by visual observation of the pellet. This would usually yield
a final protoplast density after the transformation experiments of 5 X l05.
Transformation experiments and plant regeneration were performed as
described in the materials and methods. For each transformation
experiment attempted, 20 to 30 plates were used. Cell wall regeneration
was observed within 24 to 48 hours, and cell divisions began after
approximately three days. Colonies (microcalli) begin to form in as
little as two to three weeks. It was found to be critical to not let the
colonies develop too fast prior to osmoticum reduction and the eventual
plating of the developing microcolonies onto solid EME medium (see
Appendix A). With extremely rapid colony development, microcalli tended
to lose their embryogenic capacity, thereby inhibiting embryo and plant
Generally, the colonies were ready to be transferred to solid EME in
4 to 8 weeks. Shortly thereafter embryos began to form. These embryos
were removed and transferred to another fresh plate containing EME, and
allowed to proliferate. Embryos were then transferred to 1500 medium (see
Appendix A) for further growth, and finally to B+ medium (see Appendix A)
for germination. After the formation of plantlets, the plantlets were
removed and placed on rooting medium (see Appendix A) until roots formed
and then subsequently transferred to soil.
The time from initial protoplast isolation to plant establishment in
soil was quite variable. Several factors influence this chain of events.
Delays in the osmoticum reduction process and in transferring slowed down
the growth. In some cases the developing embryos could be transferred
directly from EME to B+ medium, skipping the transfer to 1500 medium. At
other times they may need an additional pass on EME, 1500, or B+ media.
However, if all went smoothly plantlets were established in soil in as
little as 5 to 6 months.
Over 40 transformation experiments were attempted over the duration of
this project. These experiments were done with a variety of scion
cultures using all four of the plasmids. The reasons for the lack of
plants regenerated from many of the experiments can generally be traced to
problems related to the culturing of the protoplasts, or to the condition
of the suspension cultures used for protoplast isolation. If large
numbers of microcalli are produced (i.e. high plating efficiency) there is
a tendency for callus to be formed at the expense of the production of
embryos. If embryos do form there is often an overgrowth of these embryos
by the callus and the result is no further development of the embryos.
This is the biggest factor contributing to the lack of plant regeneration
in many of the transformation experiments. Other reasons for the failure
of plant regeneration include contamination of the media, the condition of
the protoplasts after isolation and transformation, and other undetermined
factors. For a detailed description and photograph of the developmental
sequence for protoplasts see Schell (1991).
As a result of these transformation experiments numerous regenerated
plants were obtained. Using the plasmid pMONI0098-T30, 59 'Nova' tangelo
plants were obtained. Using the plasmid pMONI0098-T36, 12 'Valencia'
sweet orange plants were obtained. Using the plasmid pBPF07-T36, 500
'Rohde Red Valencia' sweet orange plants were obtained. Several other
attempts were made to transform these and other scion varieties, with the
four plasmids but were unsuccessful due to difficulties in isolation,
culture, and regeneration. These difficulties are the limiting factors in
transformation by direct DNA uptake.
The number of plants recovered per embryo was not tracked due to the
amount of time necessary to do this. As a result, there is a possibility
that some of the individual plants may have come from the same embryo.
Multiple shoots from a single embryo were observed in these experiments.
Screening for Transformants
Selection During the Early Stages of Protoplast Growth and Microcalli
The stage at which selection is performed is dependent on the method
of transformation and the plasmid used. Using Agrobacterium as a vector
for transformation of plant tissues, selection is commonly done
immediately after co-cultivation utilizing the antibiotic resistance
marker contained in the plasmid. This has also been done with protoplasts
transformed by direct DNA uptake (Davey et al., 1989). However, there are
problems with immediate selection for transformed protoplasts using the
antibiotic resistance conferred by the plasmid. First, plating density
plays a major role in regeneration and survival of protoplasts. Kao and
Michayluk (1975) developed a medium (K8P) for low density plating of Vicia
hajastana protoplasts which has been commonly used in other systems. This
is the medium from which the BH3 medium used in this study was derived
(Grosser, 1994). However no studies have been performed to determine the
lowest concentration at which Citrus protoplasts can survive. Some labs
have attempted to circumvent this problem by delaying the placement of the
protoplasts on antibiotic-containing medium for a period of time
(Paskowski and Saul, 1988) Secondly, if selection is done at the
protoplast level the effect of the dead protoplasts has to be considered.
If transformation efficiency is as good as 1%, then 99% of the protoplasts
would die under selection. These dying protoplasts could release many
toxic compounds into the medium which would adversely effect the
development and survival of those that are antibiotic resistant (or
putative transformants). Therefore selection at the protoplast level was
not a practical idea for Citrus.
The second most likely stage for selection is after the formation of
microcalli or calli. Experiments were conducted to test whether or not
the microcalli could survive on medium containing the antibiotic
geneticin. Resistance to geneticin is controlled by the NPTII gene
contained in both plasmids. Microcalli from transformation experiments
using 'Rohde Red Valencia' sweet orange, 'Nova' tangelo and 'Valencia'
sweet orange were placed on EME media containing 0, 5, 10, 20 and 50 Ag/ml
of geneticin. All of the microcalli died on levels of 20 and 50 Ag/ml and
little or no death was noted in the lower two levels of 0 and 5 Ag/ml.
The reaction at 10 gg/ml was mixed with either the whole plate of
microcalli dying or the whole plate of microcalli surviving. No selection
seemed to occur at any antibiotic concentration. No embryos developed
from any of the surviving microcalli. It appeared that some sort of
interaction between the microcolonies may have been taking place which was
interfering with any selection processes that may have been occurring.
Repeated experiments yielded the same results. The fact that the
antibiotic was in the medium may also have been playing a role. To test
this, experiments were also conducted placing the geneticin at levels of
0, 2.5, 5 and 10 Ag/ml in liquid EME, which was layered over the solid EME
at the rate of 1 ml/ plate (60mm X 10mm). Microcalli were placed in this
liquid EME. Death occurred at 10 Ag/ml, some of the plates showed no
effect at 5 pg/ml while others showed total death of the microcalli.
There seemed to be variable survival in some of the plates at 2.5 jg/ml,
ranging from death of the microcalli to total survival. However none of
the surviving microcalli were able to form embryos. (It should be noted
that these microcalli were from the same transformation experiments that
eventually gave rise to the transgenics reported herein) Because of the
wide variation in callus survival rates, and the lack of repeatability, it
was concluded that selection with geneticin at this stage was not the best
selection procedure.
It is quite common to use a scoreable marker such as the
histochemical GUS assay for the detection of the 1-glucuronidase product
of the uidA (GusA) gene (Jefferson et al., 1986; Jefferson, 1987;
Jefferson et al., 1987; Jefferson, 1989). This scoreable marker can be
used for selection by performing the assay on a sample of plant tissues
such as a leaf piece. However, the plasmids that were supplied did not
contain the uidA gene, thus selection by this method was not an option.
Selection using neomycin phosphotransferase (NPTII), was not performed as
the assays are tedious and relatively difficult, in addition to being
susceptible to various endogenous activities in the plant cells, which
limits the sensitivity (Reiss et al., 1984). Recently, easier and cheaper
ELISA procedures for NPTII selection have become available from many
commercial companies, but these were not available at the time these
experiments were performed.
Polymerase Chain Reaction for Selection of Transformants
Testing of 'Nova' tangelo and 'Valencia' sweet orange regenerants
The first plants regenerated were 59 'Nova' tangelo from experiments
using plasmid pMONI0098-T30, and 12 'Valencia' sweet orange from
experiments using plasmid pMONI0098-T36. Since the objective of this
study was the production of plants containing the coat protein gene, and
not in the development of an improved transformation procedure, data was
not taken to determine the percentage of protoplasts regenerated into
plants. It was determined that time was better spent in obtaining the
largest number of plants possible, rather then in the tedious chore of
cataloging the development of individual protoplasts and microcolonies.
These plants were tested by using a pooled polymerase chain reaction
(PCR) in which the samples were pooled together in groups of 5 (and one of
6) prior to DNA isolation. Isolation of DNA (see materials and methods)
is the most time consuming step in the PCR process and pooling the samples
in the initial screening process allows the test to be performed much
quicker. Also, the cost of all reagents can be greatly reduced. The
members of each positive pool could then be retested individually and
sorted out from the pool. The PCR reactions were carried out using a
Precision Scientific GTC1 Thermal Cycler. This is a water cooled (as
opposed to Peltier-effect cooled) machine. The PCR profile was an initial
2 minute denaturing at 940C, followed by 35 cycles of 1 minute
denaturation at 940C, 1 minute annealing at 550C, 1 minute extension at
72C, and then ending with a 10 minute final extension at 72'C. The
reaction was carried out in 1X Promega Taq Polymerase reaction buffer,
25kim MgCl2, 200 nmol of each primer and 1 unit of Taq polymerase per
sample. The T30 infected control also contained 0.001M DTT, 1 p1 RNasin,
and 1 unit AMV reverse transcriptase and was incubated at 42 C for 45
minutes prior to amplification. Final reaction volume was 50 Al, and the
samples were overlayed with 2 drops of sterile mineral oil. The primers
were JC-10 (5'-GAATTCGCGGCCGCTCAACGTGTGTTAAATTTCC) and JC-11 (5'-
AGATCTACCATGGACGACGAAACAAAG), specific to the coat protein gene of
isolates T36 and T30. The final PCR product was run on a 1% Agarose Gel
in TBE.
After performing the PCR on the pooled samples from the 59 'Nova'
tangelos and 12 'Valencia' sweet orange regenerants, one pool was
identified as containing at least one positive sample. DNA from the
individual plants represented in this pool (containing samples N29, N30,
N32, N33, and N34 where the N signifies a 'Nova' tangelo plant) was then
isolated and the PCR repeated as before. From this PCR, 3 plants (N32,
N33, N34) were identified as being PCR positive for the coat protein gene
(Figure 4-1). This result was repeatable. These plants were repotted and
placed in the greenhouse to allow for adequate leaf growth for Southern
Testing of the 'Rohde Red Valencia' sweet orange regenerants
The 'Rohde Red Valencia' sweet orange plants were produced later
than the other two varieties described in the previous section. A new MJ
Research PTC-100 thermal cycler was purchased and used for testing these
plants. This machine is cooled by the Peltier effect and reactions are
carried out in 96 well plates, as opposed to the 0.5 ml tubes used with
the old machine. Initial experiments were performed using the method
described in the previous section except that the Idaho Technology medium
buffer was substituted for the Promega buffer A, and the final reaction
volume was 25 il/reaction. The PCR profile was as follows: an initial 2
minute denaturation at 930C; 35 cycles of 1 minute denaturation at 930C,
30 seconds annealing at 55C, 30 seconds extension at 72C; and a 5 minute
final extension at 72C.
While screening these plants 'false positives' began to be
produced. These 'false positives' would appear in all samples including
the non-transformed controls and sometimes the buffer controls. The bands
were of the same size (approx. 700 bp) as expected for the CTV coat
protein gene which we were trying to detect. The appearance of the bands,
however, differed in that the band was diffuse rather than a clear
distinct band as should be obtained. Even more problematic, these false
1 2 3 4 5 6 7 8 9 10 11 12
Figure 4-1. Results of the PCR reaction of the samples contained in the
positive pool. Lanes 1 and 12 are Lambda HindIII markers. The arrow
points to the marker for 564 bp. Lane 10 is blank, Lane 11-- buffer
control. Lane 2-- N29, Lane 3-- N30, Lane 4-- N32, Lane 5-- N33, Lane 6--
N34, Lane 7-- untransformed control plant, Lane 8-- a T30 infected plant,
and Lane 9-- the plasmid control.
positives did not manifest themselves all the time, particularly in the
buffer control. As a result, we had to take a step backwards and try to
determine the source of this problem, and how to alleviate it.
The initial inclination was to blame the problem on the primers,
specifically the primers JC-10 and JC-1l. These primers were originally
used by H. R. Pappu (Dr. Niblett's Lab) to isolate and clone the CTV coat
protein gene from isolate T36, which was then inserted into the plasmids
that were used for transformation. Closer examination of the primers
revealed the possibility of a hairpin structure that could occur with
primer JC-10 (Figure 4-2) because there is a sequence of 4 bases at the
ends of the primer which can align with each other and anneal. This
hairpin structure could contribute to mispriming and result in false
positives. Onesolution obtained from the Molecular Biology usergroup on
the Internet was to denature the primers by boiling and quick chilling
them prior to adding them to the reaction mix. This was attempted and
seemed to work the first time; however, in subsequent experiments, the
same variability was evident. Consequently, new primers were obtained or
designed in an attempt to circumvent this problem (Figure 4-3). The
primers 728 and 729 are used to amplify a portion of the CaMV 35 S
promoter, which should have been inserted into the genome of the
transformants along with the coat protein gene. The other primers were
specific to various segments of the coat protein gene. Despite using
different primers, 'false positives' continued to be an intermittent
problem. Occasionally bands would be found for only a few of the samples,
indicating a true result ( that is-- no 'false positives'), but the
results were not repeatable.
Figure 4-2. A Schematic diagram of how the Primer JC-10 can form a
secondary hairpin structure.
C-10 (equivalent to CN120 of C. L. Niblett)
C-Il (equivalent to CN119 of C. L. Niblett)
Eli-i (CTV-CP,
Eli-2 (CTV-CP,
nt 116-139,
nt 577-600,
seq. from isolate T-36)
seq. from isolate T-36)
internal primer)
internal primer)
rTA CCA ATA CCC TTA G -3'
728 (CaMV 35 S PROMOTER)
729 (CaMV 35
Figure 4-3. PCR primers used for testing regenerated plants for
integration of the coat protein gene or CaMV 35 S promoter gene.
Other parameters were looked at as the source of the problem. These
included changing the reaction buffer, magnesium chloride concentration,
addition of bovine serum albumin, and lowering the DNA concentration. The
same variable results were obtained. Similarly, experiments with E. S.
Louzada looking at whether the concentration of the primers was
responsible for the 'false positive' results were also inconclusive. In
addition, changes in the PCR reaction profile involving the annealing
temperature were also unsuccessful.
As a result of these experiments the conclusion was drawn that these
'false positives' were the result of contamination in the laboratory
and/or equipment. Without significant expenditure to purchase PCR
dedicated equipment to use throughout the procedure this problem could not
be overcome. Because of this, screening by PCR was not a reliable
technique to screen for transformants, and another more reliable technique
had to be found.
Screening For Transformants Using Dot Blot Analysis
Although there were no recent reports in the literature of the use
of a pooled dot blot analysis for the detection of transformants, the
decision was made to see if it would be possible to select transformants
in this manner. The approach used was similar to that used with the PCR
screening, using pools of five samples in the initial screen.
DNA isolation and dot blot analysis were carried out as described in
the materials and methods section. From the 90 pools tested, 23 pools
showed a postive or inconclusive result. DNA from the members of these
pools was isolated and the procedure was repeated with these individuals.
From these blots 22 positive individuals were identified, representing 11
of the original 23 pools (figure 4-4) These positive plants were
identified as R73, R82, R83, R121, R124, R129, R140, R142, R146, R147,
R149, R150, R152, R153, R268, R269, R270,R271, R271b, R272, R276, R328,
and R368. The plant R368 died shortly after the Southern dot blot
analysis. The large size of the plasmid control blots is due to the
amount of sample in the blot. All trees testing positive were repotted
and saved for further analysis.
Confirmation of Transformation
Southern Hybridization Analysis Using the Non-radioactive GENIUS System
In order to confirm transformation, Southern hybridization analysis
of the PCR product of the 'Nova' tangelos was performed. This analysis
was performed using the GENIUS non-radioactive system as described in the
materials and methods. Using this method, all 3 'Nova'tangelos were shown
to be transformed. Figure 4-5 shows the results of the Southern
hybridization analysis experiment. In this experiment the lower band
corresponds to the coat protein gene, while the higher molecular weight
bands are probably the result of duplexes being amplified in the PCR
Figure 4-4. Dot blot hybridization screening for transformed 'Rohde Red
Valencia' sweet orange. Arrows point to the samples identified as
positive. Starting from the upper right hand corner of the top
autoradiograph, the positive plants are R146, R147, R149, R150, R152,
R153, R276, R328, R124, R129, R140, R142, and R271b. From the upper right
corner of the bottom autoradiograph the samples are R121, R73, R82, R83,
R268, R269, R270, R271, R272, and R368. The overexposed dark dots on the
bottom of each blot are plasmid controls.
Figure 4-5. Southern hybridization using the GENIUS Non-Radioactive
System with 'Nova' tangelo plants found to be transformed by PCR analysis.
Lane 1 is a non-transformed control plant Lanes 2-4 are transformants N32,
N33 and N34 respectively. Lane 5 is a confirmed transformed Carrizo (from
G. Moore). Lane 6 is a T30 infected plant. Lane 7 is the plasmid
control. The probe was made to the plasmid pMON10098-T30.
Southern Hybridization Analysis Using Radioactive Probes
Initial experiments using DNA isolated by the method used for dot
blot analysis were not successful. Efficient cutting of the DNA was
difficult to achieve. Changing from the Promega restriction enzymes to
enzymes purchased from Pharmacia Co. improved the efficiency, but the
digestions were still not complete. Consequently, it was concluded that
the problem was possibly due to the quality of the DNA isolated. To
improve the DNA quality and subsequent restriction digest, a new DNA
isolation procedure was used (see materials and methods) This procedure
was initially developed to isolate viroids from infected tissue (Marais et
al., 1995), but it was also efficient at isolating total genomic DNA from
the plant tissue. The method achieved the isolation of high yields of
high molecular weight DNA (up to 10 Ag/g of fresh leaf tissue). This DNA
could be very efficiently digested with the Pharmacia Co. restriction
enzymes. Using DNA isolated in this manner, Southern hybridization
analysis was performed on the putative transformants. The genomic DNA was
restricted with the restriction enzymes EcoRI and BglII to cut out the
coat protein gene. The results obtained with the 'Nova' tangelo plants
are shown in figure 4-6. The DNA was probed using a 32p labeled probe to
the plasmid pMONI0098-T30. The expected band of 670 bp for the coat
protein gene was obtained. The Southern hybridization analysis confirms
that the 'Nova' tangelo plants N32, N33, and N34 were transformed with the
coat protein gene of isolate T30.
Southern hybridization analysis was also performed on the healthiest
of the 'Rohde Red Valencia' sweet orange plants which were positive by the
1 2 3
5 67
Figure 4-6. Southern hybridization of the 'Nova' tangelo transformants
using a radioactive probe to the coat protein gene that was prepared by
PCR (see text). Lane 1 is a nontransformed 'Nova' tangelo. Lane 2 is
N32, lane 3 is N33, and Lane 4 is N34. Lanes 5-7 are plasmid controls of
0.00001, 0.0001, and 0.001 jg. The arrows point to the coat protein gene
band. The bands for N32 and N34 are very faint and may not be visible in
the photograph, but are visible on the original autoradiograph. The top
band in the plasmid lanes is undigested plasmid and the visible smear in
the other lanes is the result on incomplete digestion of the DNA.
Figure 4-7. Southern hybridization using a radioactive probe of the
transformed 'Rohde Red Valencia' sweet orange. In the upper
autoradiograph, lanes 1-8 are the transformants R121, R124, R129, R140,
R146, R150, R152, and R153. Lanes 9 and 10 are nontransformed 'Valencia'
controls. Lanes 11-13 are plasmid controls of 0.00001, 0.0001, and 0.001
Ag. In the lower autoradiograph lanes 108 are the transformants R268,
R269, R270, R271, R271 (second sample), R271b, R272, and R276. Lanes 9 and
10 are nontransformed 'Valencia' controls. Lanes 11-13 are plasmid
controls of 0.00001, 0.0001, and 0.001 Ag. The background is due to
incomplete cutting of the genomic DNA and the large amount of DNA present.
12345678 vv pp p
vv pp p
123 4 567 8
dot blot hybridization (figure 4-7). Not all of the plants could be
analyzed due to their poor growth and lack of leaf material.
This lack of growth could be due to a positional effect in regards
to the location of insertion of the coat protein gene in the DNA of the
plant. The large amount of background in the Valencia nontransformed
controls is due to the accidental loading of twice the quantity of sample
as for the putative transformants. Despite the increased amount of
sample, no band appeared corresponding to the coat protein gene.
Variability in the intensity of the bands in the transformants is due to
the variation in amount of DNA used, efficiency of restriction digestion,
and possibly due to copy number, although copy number was not determined.
The background at the top of the lanes is the result of undigested DNA and
background due to large amount of DNA present. Variation in the amount of
DNA used is due to the limits of quantification using gel electrophoresis
of a small quantity of the isolated DNA. Sensitivity could be increased
by using a higher percentage of the isolated DNA, but would compromise the
amount available for the Southern hybridization. Spectrophotometry for
quantifying the DNA was found to be more variable than gel
electrophoresis. Southern analysis was successfully repeated for all
samples shown.
Western Analysis
Western analysis of transformants can be difficult because
transformed plants often express the protein in very low quantities.
Initial experiments were performed using 12 il of sample removed from a
stock supply of approximately 1 cm2 of leaf material in 1000 il of protein
extraction buffer. No coat protein was detected in the transformants
although the coat protein could be detected in control plants inoculated
with CTV isolates T30 or T36. Western analysis was performed on the
'Nova' tangelo plants as described in the materials and methods. On one
occasion a very weak band was detected in N32 and N34 but not in N33, but
this was not repeatable.
It is often necessary to concentrate low abundance proteins in order
to detect them on a western blot. An experiment was performed to test
whether the coat protein gene could be detected after concentration using
a Jouan RC10 Concentrator/Evaporator. Duplicate samples were isolated
from the three 'Nova' tangelo plants, plus a healthy and CTV infected
control. One set of samples was boiled prior to concentration while the
other set was not. The samples were then concentrated to approximately
1/5 volume. Prior to loading the polyacrylamide gel both sets of samples
were then boiled and the western blotting performed as described in the
materials and methods. The antibody used for detection was the polyclonal
1053 antibody provided by Dr. R. F. Lee. The results using the samples
that were boiled prior to concentration were all negative. However, the
results from the samples which were not boiled were as expected and are
shown in figure 4-8.
Shortly after achieving the above results the Jouan RC10
concentrator/evaporator suffered a major breakdown, so a new way of
increasing the protein concentration in the samples for western
hybridizations. The same amount of leaf material (approx. 10 cm2) was
ground as before but resuspended in 700 i of protein extraction buffer.
Instead of using 12 pl of sample, 30 gi of sample was used. The exception
Figure 4-8. Western immunoblot of the 'Nova' tangelo transformants.
Samples were prepared as described in the text. Lane 1 is transformant
N32, lane 2 is transformant N33 lane 4 is transformant N34, lane 4 is a
T30 infected plant and lane 5 is a non-transformed 'Nova' tangelo control.
was the T36 or T30 positive controls for which only 5 pl of sample was
used. Also, the antibodies were diluted at 1:20000 v/v in order to
decrease the background (R.F. Lee, pers. comm.). The primary antibody
used for 'Nova' tangelo was G604 (from M. Keremane) and for 'Rohde Red
Valencia' sweet orange the primary antibody used was MCAl3. G604 will
detect isolate T30 while MCA13 is a monoclonal antibody specific to
decline isolates, including T36. The results for the 'Nova' tangelo are
shown in figure 4-9, and the results for 'Rohde Red Valencia' sweet orange
are shown in figure 4-10. Migration of the proteins in the transformant
samples was slowed because of the amount of sample loaded. Similarly,
partly due to the large amount of protein loaded, there was a significant
background for the high molecular weight, abundant proteins. These are
evident in the upper portion of the blots. Confirmation that these bands
are associated with the coat protein can be inferred based on the lack of
these bands in the non-infected controls. However, the quality of the
western blots suggests that the protein may be expressed in quantities too
low to be detected by western immunoblot analysis, and no definite
confirmation of protein expression can be made based on these results. It
is possible that proteins may be detectable using procedures to seperate
the proteins, but these experiments were not conducted.
Figure 4-9. Western immunoblot of transformed 'Nova' tangelo using
primary antibody G604. Lanes 1-3 -- transformants N32, N33, N34
respectively, lanes 4-7 -- non infected, non transformed Mexican Lime
plants, lane 8 -- a T30 infected control plant, and lane 9 -- molecular
weight markers. Arrow points to the band corresponding to the coat
protein. The Lane marked T36-8 is mislabeled and should be T30.
a). lane 1 -- R73, lane 2 -- R82, lane 3 -- R121, lane 4 -- R129,
lane 5 -- R140, lane 6 -- R142, lane 7 -- 'valencia' nontransformed
control, lane 8 -- a T36 infected plant, lane 9 -- molecular weight
I ilsil ill
b). lane 1 -- R146, lane 2 -- R147, lane 3 -- RI50, lane 4 -- R152,
lane 5 -- R269, lane 6 -- R270, lane 7 -- 'Valencia' nontransformed
control, lane 8 -- T36 infected plant and lane 9 -- molecular weight
Figure 4-10. Western immunoblot analysis of the 'Rhode Red Valencia'
sweet orange transgenic plants using MCA13 antibody.
c). lane 1 -- R83, lane 2 -- R124, lane 3 -- R149, lane 4 -- R153,
lane 5 -- R268, lane 7 -- 'valencia' nontransformed control, lane 8
-- T36 infected plant, lane 9 -- molecular weight markers.
ilu x.
d) lane 1 --R271, lane 2 --R27lb, lane 3 --R272, lane 4 --R276,
lane 5 -- R328, lane 7 -- 'Valencia' nontransformed control, lane 8
-- T36 infected plant, and lane 9 -- molecular weight markers.
Figure 4-10- -continued
The successful transformation of Citrus scions with the coat protein
gene of Citrus Tristeza Virus using PEG-mediated direct DNA uptake was
achieved. Three 'Nova' tangelo transformed with the coat protein gene
from mild isolate T30 (from 59 total regenerants), and 22 'Rohde Red
Valencia' sweet orange transformed with the coat protein gene from florida
severe isolate T36 (from 500 total regenerants) were obtained. No
transformants were obtained from the 12 'Valencia' sweet orange
regenerants. It is important to note that these transgenic plants were
obtained without selection at the cellular level. Identification was made
using the polymerase chain reaction for the 'Nova' tangelos, and Dot Blot
hybridization for the 'Rohde Red Valencia' sweet orange plants. The time
required to select after regeneration of plants was not significantly
different than the time required for selection at the cellular level.
Confirmation of stable transformation was shown by Southern hybridization
and western analysis. Western analysis suggested a low level of
expression of the coat protein gene in the transgenic plants.
The results also showed that pooled PCR can be used for selection of
transgenics, as was demonstrated for the 'Nova' tangelos, but PCR is also
very susceptible to outside interference. In order to reliably use PCR
for screening, measures need to be taken to guard against contamination
from outside sources. Screening by pooled Southern dot blot hybridization
can also be used successfully, as was demonstrated with the 'Rohde Red
Valencia' sweet orange. Southern dot blot hybridization was found to be
as easy and fast as PCR, and by using the DNA isolation procedure that was
used for later experiments, efficiency could be even better. These
results are summarized in Figure 5-1.
Evidence of somaclonal variation was difficult to ascertain. There
were differences in growth rate, particularly for the 'Rohde Red Valencia'
sweet orange, but the reasons for this are difficult to determine. The
stunting of the plants could be due to a positional effect of the inserted
gene, or the effect of the coat protein expression, or a combination of
these, and a number of other unknown effects.
The regenerated transgenic plants are now ready to be tested by
challenge inoculation with different severe quick decline and stem-pitting
isolates of CTV in the greenhouse. The results of these tests will help
to determine whether these plants have acquired a greater level of
resistance against infection by CTV. If these tests are positive, field
testing can then be carried out. Selection will indirectly be made at
this point for other important horticultural traits, and thus for the
possible somaclonal variants exhibiting normal or improved horticultural
traits. Positive results in the field will mean that tristeza resistant
Citrus scions have been achieved.
N32 Normal yes yes
N33 Normal yes yes
N34 Normal yes yes
R73 somewhat small plant No yes
R82 Small, w/ elongate leaves no yes
R83 Slow growing no yes
R121 Normal yes yes
R124 Normal yes yes
R129 Normal yes yes
R140 Normal yes yes
R142 Leaves slightly smaller no yes
R146 Normal yes yes
R147 Normal no yes
R149 Very small plant no yes
R150 Normal yes yes
R152 Normal yes yes
R153 Normal yes yes
R268 somewhat small plant yes yes
R269 Normal yes yes
R270 Normal ??? yes
R271 Normal yes yes
R271b Normal yes yes
R272 Normal yes yes
R276 Normal yes yes
R328 Normal yes yes
Table 5-1. Summary of the transgenic plants regenerated in this
experiment, and whether confirmed by Southern and/or Western analysis.