DEVELOPMENT OF DIPLOID STRAWBERRY Fragaria vesca...
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1 DEVELOPMENT OF DIPLOID STRAWBERRY Fragaria vesca GENOTYPE 5AF7 AS A FUNCTIONAL GENOMICS RESOURCE By MOHAMAD FADHLI MAD’ ATARI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010
Transcript of DEVELOPMENT OF DIPLOID STRAWBERRY Fragaria vesca...
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DEVELOPMENT OF DIPLOID STRAWBERRY Fragaria vesca GENOTYPE 5AF7 AS A FUNCTIONAL GENOMICS RESOURCE
By
MOHAMAD FADHLI MAD’ ATARI
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2010
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© 2010 Mohamad Fadhli Mad' Atari
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To my mother Arbaayah Isa, and father, Mad' Atari Muhamad Sanif who always inspired and encouraged me since I came to the United State to pursue a master's degree, also to my advisor, Kevin, Folta Lab colleagues and friends that help me to achieve all that I
have to this day
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ACKNOWLEDGMENTS
I would like to express my gratitude to my chair, Dr. Kevin M Folta, and committee
members, Dr Michael E Kane and Dr Jose Xavier Chaparro for helping me through
these studies. Dr Folta always inspired and motivated me with his brilliant ideas, past
fruitful comments and suggestions. I also thank my committee members, Dr Michael E
Kane and Dr Jose Xavier Chaparro for their valuable contributions toward this work both
during in committee meetings and outside.
I would like to thank Maureen Clancy, Dr Mithu Chatterjee, Dr Asha Brunings,
Dave Salama, Jiao Wu, Kyle Schmitt, Sasha Ricaurte and other Folta lab members their
advice and suggestions that helped me to complete this research. The first time going
abroad was a very difficult experience for me and my mother. Fortunately with
technology the distance feels much closer. Her daily encouragement during my studies
has kept me strong even though we haven't meet for almost 2 years. I also would like
to thank my other family members in Malaysia for keeping in touch with me via the
internet.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 10
ABSTRACT ................................................................................................................... 12
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 14
1.1 Cultivated Strawberry ..................................................................................... 14 1.2 Benefits to Health ........................................................................................... 15 1.3 History and Genetics ...................................................................................... 16 1.4 Plant Media Used in Propagation and Regeneration ..................................... 17 1.5 Plant Tissue Culture Micropropagation .......................................................... 18
1.5.1 Selection of Donor Plant and Explant Types ....................................... 19 1.5.2 Surface Sterilization and Pre-Treatment Media ................................... 20 1.5.3 Shoot Proliferation/ Multiplication ........................................................ 21 1.5.4 In vitro and Ex vitro Rooting ................................................................ 22 1.5.5 Acclimatization ..................................................................................... 23
1.6 Regeneration and Transformation Efficiency ................................................. 24 1.6.1 Transformation .................................................................................... 24 1.6.2 Selection Agents .................................................................................. 25
1.7 Reverse and Forward Genetics ...................................................................... 27 1.8 Research Problem.......................................................................................... 29 1.9 Research Objectives ...................................................................................... 29
2 IN VITRO YW5AF7 SEEDLINGS GROWN ON DIFFERENT MEDIA TYPES ........ 35
2.1 Introduction .................................................................................................... 35 2.2 Materials and Methods ................................................................................... 37
2.2.1 Plant Material ....................................................................................... 37 2.2.2 Seed Sterilization ................................................................................. 37 2.2.3 In vitro Media Types and Sucrose Concentration Experiment ............. 37
2.3 Results ........................................................................................................... 38 2.4 Discussion ...................................................................................................... 39
3 REGENERATION AND TRANSFORMATION OF YW5AF7 ................................... 47
3.1 Introduction .................................................................................................... 47
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3.2 Materials and Methods ................................................................................... 48 3.2.1 Plant Material ....................................................................................... 48 3.2.2 Preliminary Auxin and Cytokinin Experiment ....................................... 48 3.2.3 Plant Growth Regulators (PGRs) Optimization .................................... 49 3.2.4 Comparison Between 5AF7 Medium & Hawaii-4 medium ................... 49 3.2.5 Regeneration of Six Cultivars of Diploid F. vesca on 5AF7 Medium .... 50 3.2.6 Transformation .................................................................................... 50
3.3 Results ........................................................................................................... 52 3.3.1 Preliminary Shoot Regeneration Experiment ....................................... 52 3.3.2 Optimization of 5AF7 Medium Candidates .......................................... 53 3.3.3 Agrobacterium-Mediated Transformation ............................................ 54
3.4 Discussion ...................................................................................................... 55
4 MUTAGENESIS WITH TNT1 RETROTRANSPOSON ........................................... 63
4.1 Introduction .................................................................................................... 63 4.2 Materials and Methods ................................................................................... 65
4.2.1 Plant Material ....................................................................................... 65 4.2.2 Polymerase Chain Reaction (PCR) Analysis ....................................... 65 4.2.3 Electrophoresis .................................................................................... 66
4.3 Results ........................................................................................................... 66 4.4 Discussion ...................................................................................................... 67
4.4.1 Understanding Tnt1 Retrotransposons ................................................ 67 4.4.2 Strawberry Retrotransposons .............................................................. 68 4.4.3 Experiment Observations .................................................................... 68 4.4.4 Future Suggestions ............................................................................. 69
LIST OF REFERENCES ............................................................................................... 75
BIOGRAPHICAL SKETCH ............................................................................................ 84
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LIST OF TABLES
Table page 1-1 Approximate nutritional value for raw portions of strawberry (100 grams)
(USDA, 2009). .................................................................................................... 30
1-2 Donor plant selection and surface sterilization procedure on different types of explant. ............................................................................................................... 31
1-3 Pretreatment medium on explant after surface sterilization and before transfer to shoot proliferation medium stage. ..................................................... 31
1-4 Combinations of auxin and cytokinin used in media for regeneration for Fragaria x ananassa octoploid and Fragaria vesca diploid strawberry. .............. 32
1-5 Compositions for rooting medium. ...................................................................... 33
1-6 Explant types, plant growth regulators and selective agents used in shoot induction medium of transgenic plants. .............................................................. 34
2-1 Media types tested, A; MS mineral salts with MS vitamins, B; MS mineral salts with B5 vitamins and C; an octoploid strawberry formulation with 1 % and 3 % of sucrose concentration on Yellow Wonder 5AF7 seedlings.. ............. 43
2-2 Three different media types treated with 1% sucrose concentration on Yellow wonder 5AF7 seedlings.. .................................................................................... 44
2-3 Average of leaf number and average of fresh weight of YW5AF7 seedlings on 3 different media types; MS mineral salts with MS vitamins, MS mineral salts with B5 vitamins and Gamborg mineral salts with B5 vitamins. .................. 44
3-1 Auxin and cytokinin group PGRs combinations were presented in groups. List of sections contained in the template. .......................................................... 57
3-2 Media compositions used in transformation procedure of Tnt1 retrotransposon YW5AF7. .................................................................................. 58
3-3 Adventitious shoot regeneration from leaves explant following treated with combinations of two different auxins types with BA on respective concentrations. ................................................................................................... 58
3-4 Adventitious shoot regeneration from leaves explant following treated with combinations of three different auxins types with TDZ on respective concentrations .................................................................................................... 59
3-5 Adventitious shoot regeneration from YW5AF7 leaves explant following treated with 5 different combinations and concentrations of PGRs. ................... 61
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3-6 Average number of shoots per explant on YW5AF7 leaf disks on 5AF7 medium and Hawaii-4 medium after 8 weeks of treatments. .............................. 62
3-7 Average shoot number per explant for six F. vesca accessions on 5AF7 medium.. ............................................................................................................. 62
4-1 PCR mixture for 1X reaction. .............................................................................. 71
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LIST OF FIGURES
Figure page 2-1 Plant health score experiment on (A) MS mineral salts with MS vitamins, (B)
MS mineral salts with B5 vitamins, and an (C) octoploid strawberry formulation media ............................................................................................... 45
2-2 The morphology of 5AF7 seedlings on different media type after 6 weeks. ....... 46
3-1 Regeneration of YW5AF7 leaf disks and petiole on 5AF7 medium and Hawaii-4 medium. ............................................................................................... 60
3-2 Callus formation was observed about 5 days after transformation. .................... 61
4-1 A schematic representation of the tnk23 T-DNA region showing Hinc II restriction enzyme sites, RB, Right border; LB, Left border redrawn from Mazier et al. (2007). ............................................................................................ 71
4-2 Agarose gel size fractionation of PCR amplification products from DNA extracts isolated from kanamycin resistant plants. ............................................. 72
4-3 Four DNA extracts of kanamycin resistant plant with Tnt1 primers. Lanes; 1) 2-Log DNA ladder, 2) plant A, 3) plant B, 4) plant C, 5) plant D, 6) Tnt1 plasmid (positive control), 7) negative control (no DNA template). ..................... 73
4-4 Four DNA extracts of kanamycin resistant plant with F-box protein primers. Lanes; 1) 2-Log DNA ladder, 2) plant A, 3) plant B, 4) plant C, 5) plant D, 6) YW5AF7 plant (positive control), 7) negative control (no template). ................... 74
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LIST OF ABBREVIATIONS
BA benzylaminopurine
BSAA benzo[b]selenienyl acetic acid
F. × ananassa Fragaria × ananassa
F. vesca Fragaria vesca
g grams
g/L grams per liter
GA3 Gibberellic Acid
hyg hygromicin
HgCl2 mercuric chlorite
IBA Indole-3-butyric acid
IAA Indole acetic acid
kan kanamycin
mg/L milligrams per liter
ml/L milliliter per liter
min minutes
mg milligrams
NAA 1-naphthaleneacetic acid
PGRs Plant growth regulators
TDZ Thiadazuron
s seconds
v/v volumes per volume
% percent
2,4-D 2,4-Dichlorophenoxyacetic acid
µg/ml micrograms per milileter
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µl microliter
µM micromolar
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
DEVELOPMENT OF DIPLOID STRAWBERRY Fragaria vesca GENOTYPE 5AF7 AS A
FUNCTIONAL GENOMICS RESOURCE
By
Mohammad Fadhli Mad' Atari
August 2010
Chair: Kevin M Folta Major: Horticultural Sciences
Cultivated strawberry (Fragaria ×ananassa Duch.) is one of the major crops in
United States and Florida with a substantial high contribution to the economy. Many
studies focus on the cultivated strawberry which has an octoploid genome, making
genetic and genomic analyses complicated. An alternative is to investigate strawberry
biology using diploid strawberry, which shares a common ancestor with the cultivated
strawberry. Unlike octoploid strawberry, diploid strawberry grows quickly from seed to
seed and has a simple and remarkably small genome. Diploid strawberry has become
an attractive system for studies in all rosaceous crops.
As the interest in diploid strawberry as a model system grows, various labs are
performing tests in different accessions. The main genotype used is called F. vesca
semperflorens Hawaii 4 (H4; PI551572). H4 has been sequenced and is readily
transformable. The problem with H4 is that it is not homozygous, leading to a range of
phenotypes among plants for most responses studied. One solution is to perform such
studies in an inbred line. The Yellow Wonder F. vesca genotype 5AF7 (YW5AF7) is a
seven- generation inbred diploid strawberry. Its phenotypes are static, making this plant
line a potential candidate for functional-genomic research.
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This thesis documents the optimization of in vitro growth seedlings and leaf disks
regeneration of YW5AF7. It was determined that MS media with B5 vitamins and 1%
sucrose supported healthy in vitro plant growth after two months. Optimization on
various combinations of plant growth regulators (PGRs) and media types was
conducted to obtain robust, high regeneration efficiency. A combination of 1.5 µM IBA
with 15 µM BA gave the highest percentage of shoots, (about 70 % of explants) and 5
shoots per explant within the same period. These concentrations of plant growth
regulators were selected after a comprehensive test with three different types of
cytokinins and auxins over a range of concentrations.
In the final section of the thesis, the use of the Tnt1 retrotransposons as a
mutagenesis tool is probed. While the YW5AF7 line is readily transformable, we were
unable to obtain transgenic shoots with evidence of the Tnt1 retroransposon. Future
experiments on optimization of variables such as pre- and post transformation
treatments, kanamycin concentrations for selection agent, Agrobacterium
concentrations and ability of Tnt1 insert to be transformed in established diploid F.vesca
genotype Hawaii 4 may lead to transformation success and use of this tool in the near
future.
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CHAPTER 1 LITERATURE REVIEW
1.1 Cultivated Strawberry
The genus Fragaria belongs to the Rosaceae family, subfamily Rosoidae, tribe
Potentilleae and includes many fruit tree crops, ornamental plants and herbaceous fruit
plants, such as rose, hawthorn, strawberry, blackberry, nut, apple, peach, raspberry,
and ornamentals (Folta and Davis, 2006; Lunkenbein et al., 2006; Slovin et al., 2009)
The family is comprised of 100 genera and 300 species, with 23 species at different
ploidy levels (Folta and Davis, 2006), and collectively ranked as the third most important
crops in temperate regions. This includes fruit (apple, strawberry), forest (mazzard),
ornamental species (rose), and genus Prunus (peach, sour cherry, apricot, almond,
European plum, myrobalan plum and sweet cherry) (Dirlewanger et al., 2002; Shulaev
et al., 2008).
Strawberry is a major crop in United States and Florida with a value of more than
USD 2 billion in year 2009 (Lunkenbein et al., 2006; USDA, 2009). Cultivated strawberry
(Fragaria ×ananassa Duch.) is an economically valuable crop that is farmed widely in
Florida and California (Folta and Davis, 2006). Fragaria ×ananassa is an octoploid
strawberry (8x=2n=56) resulting from a cross between F. chiloensis and F. virginiana,
which are native to the west coast of North and South America, and to eastern North
America (Darrow, 1966; Peres et al., 2010), respectively.
Eight patented Florida varieties have been released; 'Sweet Charlie', 'Rosa
Linda', 'Earlibrite', 'Strawberry Festival', 'Carmine', 'Florida Radiance', 'Florida Elyana'
and 'Winter Dawn' (FSGA, 2010). Nine varieties of strawberries are routinely grown in
Florida, five of which come from the University of Florida (UF) breeding program, three
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from the University of California-Davis (UC-Davis) and one variety from JP Research
(Peres et al., 2010). The varieties from UF are 'Camine', 'Gaviota', 'Strawberry Festival',
'Sweet Charlie' and 'Winter Dawn'. UC-Davis varieties are 'Camarosa', 'Camino Real',
'Ventana' and one variety from JP Research is 'Treasure' (Peres et al., 2010).
1.2 Benefits to Health
Strawberry is beneficial to human health, providing a high amount of nutrients
(Table 1-1) which makes it a good choice as part of a balanced diet. At least five
servings of fresh fruits and vegetable daily are recommended by the USDA, and adding
strawberry to the diet (Pajk et al., 2006), is a great way to add flavor and provide
nutrients. Antioxidant levels vary between cultivars but phenolic compounds are not
significantly different between cultivars (Meyers et al., 2003). Pajk et al. (2006) used
pigs as a model to demonstrate that apple and strawberry contributed the most
antioxidants to the diet.
Strawberry contains high levels of antioxidants associated with the potential to
reduce the risk of cardiovascular disease, DNA damage, several common cancers, and
other oxidative stress related diseases (Cao et al., 1998; Meyers et al., 2003; Pajk et al.,
2006). In addition, strawberry, spinach and red wine significantly increase the serum
antioxidant capacity on elderly women that helps to decrease oxidative activity by
decreasing malondialdehyde (MDA) formation and decrease cell damage during
oxidation by protecting mononuclear blood cells, thus decreasing the free radical attack
on cellular DNA (Cao et al., 1998; Pajk et al., 2006). Vitamin C, the most abundant
vitamin present in strawberry is an antioxidant enhancing the overall antioxidant status
in the serum (Cao et al., 1998). Based on several studies, strawberries are superior
compared to apples and tomatoes with respect to in vitro antioxidant capacity and result
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in a higher level of water soluble antioxidant in blood plasma (Pajk et al., 2006). The
blood plasma is used for analysis of MDA concentrations (Pajk et al., 2006), while
whole serum is used to determine glucose, protein, ureate, bilirubin and vitamin C
concentrations (Cao et al., 1998).
1.3 History and Genetics
Historical accounts show that octoploid strawberry was introduced to the Old
World from the Americas at least as far back as the 1500s through 1700s (Folta and
Davis, 2006). Since then there have been many breeding efforts to improve strawberry
quality. The cultivated strawberry is octoploid, which makes inheritance studies very
difficult. Commercial strawberries grown today are bred by cross pollination to produce
highly-variable progenies from which to choose desirable traits and eventually develop
new cultivars. The abilities for mass propagation and gene manipulation offer an
alternative to the cost, time, labor, involved in selecting and pollination associated with
traditional breeding (El-Mansouri et al., 1996). To elucidate gene function, attention has
been turned to the diploid strawberry Fragaria vesca. F. vesca is sometimes referred to
as the “woodland strawberry” or “alpine strawberry” depending on where it originates
and its photoperiod sensitivity. More importantly, F. vesca is thought to share a common
ancestor with cultivated strawberry. Other potential shared ancestors might include F.
iinumae, F. nubicola, F. mandshurica, F. nilgerrensis, and F. viridis, although it is almost
certain that ancestors of F. vesca and F. iinumae are genome donors (Rousseau-
Gueutin et al., 2009) to at least a subset of octoploid lines.
Polyploid genomes can originate as alloploids or autopolyploids depending upon
the origin of the polyploidization event when rare mitotic or meiotic events cause the
formation of gametes that have additional sets of chromosomes (Comai, 2005).
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Strawberries range from dioecious to hermaphroditic, and exist naturally as
diploids, tetraploids, hexaploids, octoploids and decaploids. Darrow (1966) mentioned
that there three known tetraploids are F. orientalis, F. corymbosa, and F. moupinensis.
Two of them, F. orientalis and F. moupinensis are hermaphrodites, staminate and
pistillate while F. corymbosa is a male strawberry. F. moschata a hexaploid species, is
known for its perfect flowers and musky flavor when cultivated (Darrow, 1966). The
parents of F. × ananassa (F. chiloensis and F. virgianana) are both octoploid (2n=56)
(Darrow, 1966; Folta and Davis, 2006). Both likely have several diploid species
represented in their subgenomes that are then combined to produce the modern
cultivated strawberry (Folta and Davis, 2006).
Over the last century several models of octoploid genomes organization have
been proposed. The AABBBBCC model (Fedorova, 1946) based on cytological
observations, was reviewed by Senayake and Brighurst (1967) who suggested an
alternative AAA'A'BBBB configuration (Senayake and Bringhurst, 1967). Senayake and
Brighurst (1967) also proposed that the AA genome is consistent with the presence of
F. vesca as a constituent of the cultivated strawberry. Years later, after observing
chromosomes during meiotic stages, AAA’A’BBB’B’ was suggested as a new model for
the subgenome organization of octoploid strawberries (Bringhurst, 1990). This is
accepted by most strawberry researchers today (Folta and Davis, 2006). An
understanding of the basic genome composition is important because it helps guide
better decisions about the genetic lines chosen for study in the laboratory.
1.4 Plant Media Used in Propagation and Regeneration
Common media used in plant tissue culture and propagation are Murashige &
Skoog and Gamborg media. Murashige & Skoog (MS) media is used widely in plant
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tissue culture. It was optimized for tobacco with different components of macronutrients,
micronutrients, and vitamins added to customize the mixture. Gamborg medium is used
primarily for strawberry cell suspension. There is substantial evidence in the literature
that supports the use of either media type. Reports describing the differences in
properties with respect to macronutrient and micronutrient composition and
concentration are well established and exhaustively tested (Murashige and Skoog,
1962; Gamborg et al., 1968).
1.5 Plant Tissue Culture Micropropagation
Plant tissue culture can be divided into five stages, which are; selection of donor
plant, establishment of in vitro culture, shoot proliferation, rooting and acclimatization
(Mohamed, 2007; M. Kane personal communication). In vitro micropropagation is an
improvement tool in plant breeding, and is used widely by including exogenous PGRs to
induce somaclonal variation in bananas, apple, peach and many other crops (Biswas et
al., 2009).
In strawberry, combinations of auxin and cytokinin promote higher adventitious
shoot formation via the shoot organogenesis pathway similar to many woody plants
species (Kane et al., 1994; Folta et al., 2006; Landi and Mezzetti, 2006). Common
auxins used in strawberry tissue culture are Indole-3-butyric acid (IBA), indole acetic
acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 1-naphthaleneacetic acid (NAA),
while the cytokinins are benzylaminopurine (BA), thiadazuron (TDZ) and kinetin (Table
1-4). The source of the explant will determine specific requirements for optimum plant
growth regulators needed (Table 1-4 and Table 1-3). The regeneration of strawberry
depends on the genotype, type of explant and culture conditions (Passey et al., 2003)
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and it is not necessarily the same or even similar between different genotypes (Folta
and Dhingra, 2006).
1.5.1 Selection of Donor Plant and Explant Types
Strawberry offers the advantage of having many tissues that are appropriate for
plant tissue culture and micropropagation (Table 1-2, Table 1-4). The selection of plant
material is very important for the effectiveness of surface sterilization when using ex-
vitro explants, reducing the risk of contamination, and increasing the regeneration rates.
Common explants used for strawberry tissue culture are lateral buds (Landi and
Mezzetti, 2006), runner tips (Passey et al., 2003; Yonghua et al., 2005; Biswas et al.,
2009), nodal segments (Sakila et al., 2007), leaf disks (Barcelo et al., 1998; Mohamed
et al., 2007), shoot tips (Mohamed, 2007) and vegetative buds (Kaur et al., 2005) (Table
1-4). All explants listed except leaf disks potentially have pre-existing meristems. Shoot
organogenesis was performed by Mohamed et al. (2007) and Barcelo et al. (1998)
using leaf disks which did not have pre-existing meristems by applying certain
hormones in certain concentrations to help inducing the explant's competence for cell
division and cell differentiation.
Information on the origin of plant material is very important to in helping choosing
an effective protocol for surface sterilization (Table 1-2). A donor plant can originate
from the field, greenhouse, growth chamber or in vitro culture (Passey et al., 2003;
Mohamed, 2007; Sakila et al., 2007). Explants derived from field and greenhouse-grown
plants may respond differently from those grown in growth chamber or in vitro
environments. The choices of explant types: petioles, leaves, root, or stipules can effect
regeneration and transformation efficiency, depending on the cultivar (Folta and
Dhingra, 2006).
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Folta and Dhingra (2006) assayed the effect of explant type on regeneration of
'Laboratory Festival #9' (LF9), a unique genotype that allows progression from explant
to rooted plant in fewer than 60 days. The study demonstrated that the young petioles
adjacent to the leaf blades are better explants than leaves or other petiole segments.
1.5.2 Surface Sterilization and Pre-Treatment Media
Surface sterilization is necessary to decrease the microbial population on explants.
Plants grown in the field and greenhouse are exposed to bacteria, fungi, and pests. An
effective and safe disinfectant used in many plant tissue culture labs today is 0.5-0.6 %
of sodium hypochlorite (Table-1.2). Some labs still use highly toxic compounds such as
mercuric chlorite (HgCl2) for this procedure (Kaur et al., 2005; Yonghua et al., 2005;
Sakila et al., 2007) (Table 1-2). This toxic compound is very dangerous for the aquatic
environment and can cause harm directly or indirectly to human health (Clarkson,
1992).
Explants from the ex vitro environment are rinsed under tap water to remove
foreign particles from their surface (Table 1-2). Then, simple alcohol such as 70%
ethanol is applied for few seconds to remove surface bacteria and fungi, as well as
serve as a wetting agent, allowing more coverage from subsequent solutions of sodium
hypochlorite. As for strawberry petioles and leaves, the pubescent surface can be
sterilized effectively by applying tap water, simple alcohol and sodium hypochlorite for
respective time (Table 1-2). As the concentrations of sodium hypochlorite increase, the
number of rinses also should increase (Table 1-2). The number of rinses works to
decrease the effects of residual bleach on the pH of culture media.
Pre-treatment on basal medium with 2 to 3% sucrose after surface sterilization
help the adjustment of explant to the in vitro environment (Table 1-3). Treatment with
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PGRs can possibly enhance the genetic variability among regenerated shoots (Cassells
and Curry, 2004; Landi and Mezzetti, 2006; Mohamed et al., 2007). Gibberellic Acid
(GA3) in pretreatment medium (Table 1-3) helps to promote accumulation of
carbohydrates in foliage and in high concentration, GA3 promotes vegetative growth in
strawberry plants (El-Shabasi et al., 2009).
1.5.3 Shoot Proliferation/ Multiplication
This stage allows shoot proliferation from explants with the aim to maximize the
number of shoots produced in the presence of exogenous PGRs. In strawberry, callus
initiation after application with exogenous PGRs is robust (Slovin et al., 2009). Auxins
not only induce rooting but also play an important role in shoot proliferation along with
cytokinin since no regeneration is observed in the absence of this growth regulator on
the cultivar 'Chandler' (Barcelo et al., 1998).
TDZ has both cytokinin and auxin-like effects on plant tissues and has been
observed in many experiments to give the highest number of shoot regeneration events
for woody species, including octoploid and diploid strawberry and others normally
considered recalcitrant (Passey et al., 2003; Folta and Dhingra, 2006). This compound
has been used widely in octoploid strawberry and generated 100 % shoot regeneration
when applied in high concentration (60-80 µM) (Passey et al., 2003; Landi and Mezzetti,
2006; Mohamed et al., 2007). Although TDZ is a potent inducer of cell differentiation, it
inhibits shoot elongation and highly induced somaclonal variation on regenerated in
many crops (Cassells and Curry, 2004; Folta and Dhingra, 2006). Other problems such
as hyperhydricity symptoms were observed in 'Toyonoka' lines when treat with TDZ,
thus TDZ application is not recommended for other diploid strawberry (Yonghua et al.,
2005). Hyperhydricity is a problem that occurs in woody plants that show translucent
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stems with thick, brittle, water-soaked elongated and curled leaves which lack a
differentiated palisade layer (Zimmerman et al., 1991; Cassells and Curry, 2004).
Combinations of BA and 2,4-D observed by Passey et al. (2003) gave the highest
shoot regeneration on six different commercial strawberry cultivars. Different
combinations of BA, 2,4-D, NAA and TDZ demonstrated that regeneration efficiency
depends on explant type and cultivar (Passey et al., 2003). Passey et al. (2003)
observed different responses for shoot regeneration in seven commercial strawberries.
The study used leaf disks of 'Calypso', 'Emily', 'Bolero' and 'Elsanta' with combinations
of TDZ and 2,4-D. Regeneration was more efficient using BA, TDZ and 2,4-D in other
commercial cultivars, 'Pegasus' and 'Tango'. Combinations of BA and 2,4-D were also
found to induce callus formation on young leaves without causing browning on
commercial strawberry accessions (Nehra et al., 1990).
1.5.4 In vitro and Ex vitro Rooting
Plantlets need to be rooted before they can be acclimatized to increase the
chances of survival and enhance the vigor (Kaity et al., 2009). Plants that root easily in
vivo have a good chance to regenerate roots ex vitro (Borkowska, 2001). After
treatment on shoot proliferation media, plantlets, shoot clusters or microcuttings can be
moved to basal medium to induce root initiation before transferring them to rooting
media (Passey et al., 2003). Low concentrations of auxin are added in rooting media to
promote rooting. (Table 1-5). Application of BA in rooting media (Table 1-5) helps to
decrease rooted shoots and obtain a shorter, thicker, and more abundant root system to
avoid injury during acclimatization state (El-Mansouri et al., 1996; Passey et al., 2003).
As for ex vitro rooting, the shoots were treated as soft cuttings & place in multiplates
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filled with rock wool for 4 weeks. Mohamed et al. (2007) used in vitro leaf disk explants
transferred on rooting media (Table 1-5).
1.5.5 Acclimatization
Acclimatization is a critical stage for the in vitro grown plant enabling their survival
to ex vitro conditions. Based on different cultivars, Biswas et al. (2009) concluded that
the leaves obtained from the greenhouse gave more robust regeneration compared to
in vitro tissue. This has also been observed in our laboratory (K. Folta, unpublished
obs.). However, Mohamed et al. (2007) observed the highest regeneration efficiency
among in vitro tissue compared to greenhouse tissue for leaf explants from cultivar
'Sweet Charlie'. The advantage of using explants from the in vitro environment is the
increased vigor observed when plants do not have to undergo harsh surface
sterilization.
Mohamed (2007) washed the rooted plantlets under tap water thoroughly after
removing them from rooting medium. Then the plantlets were transplanted into 6 cm
plastic pots with a soil mixture of 2 peat moss: 1 sand (v/v). To minimize water loss, the
pots were enclosed in polyethylene bags and gradually introduced to green house
conditions. Borkowska (2001) removed multiple shoot clusters together with all
substrate surrounding the plantlet's root. Then the plantlets then transplanted into small
pots containing a mixture of peat and water before being transferred to the greenhouse.
Mohamed et al. (2007) put the rooted plantlets into small pots with a mixture of 1 peat
moss: 1 vermiculite (v/v) then applied intermittent mist as they gradually transferred to
the greenhouse over 3 months. Kaur et al. (2005) washed the rooted plantlets
thoroughly with running tap water and transferred to plastic pots with a preautoclaved
mixture of sand: soil : farmyard manure (FYM, 1:1:1).
24
1.6 Regeneration and Transformation Efficiency
The regeneration efficiency is assessed during the shoot proliferation stage under
conditions when the explants are treated with PGRs to initiate shoot formation.
Exogenous PGRs play an important role in both plant tissue culture regeneration and
transformation (Passey et al., 2003). Transformation efficiency is described by Folta et
al. (2006b) as the percentage of explants that produces a transgenic shoot. James et al.
(1990) defined transformation efficiency as transgenic shoots per total number of
explants capable of regenerating. Transformation efficiency greater than 100% was
observed as transformation in diploid and octoploid strawberries become more efficient
indicating that each explant produces at least one transgenic shoot (Folta and Dhingra,
2006).
1.6.1 Transformation
The use of Agrobacterium for transformation in many horticultural and agronomical
important crops continues to increase (Gelvin, 2003). Agrobacterium has a broad host
range among eudicot and monocot angiosperm species (Anderson and Moore, 1979;
Gelvin, 2003). Agrobacterium tumifaciens is a naturally soil-borne bacterium that causes
crown gall disease in a large number of dicot species. The bacterium harbors a Ti
plasmid (tumor inducing) which enables the successful transformation of the infected
plant cell when the plant is wounded (de Mesa et al., 2000). There are several reviews
available describing Agrobacterium biological properties in details (Zupan et al., 2000;
Gelvin, 2003). Octoploid strawberry has also been transformed using particle
bombardment (Folta and Dhingra, 2006).
de Mesa et al. (2000) combined gold particles with transformed Agrobacterium
cells and tissues were bombarded. An optimization led to a transformation rate of
25
20.7%. Standard co-cultivation techniques, which normally have an efficiency of 7% in
their hands are exceeded by this method (de Mesa et al., 2000). Explant types,
combinations of PGRs and selection agents used in strawberry transformation are
summarized in Table 1-6.
1.6.2 Selection Agents
Transgenic plants are often created with a functional gene under the control of
the 35S promoter from Cauliflower mosaic virus (CaMV). Zhao et al. (2004)
demonstrated the recovery of six representative transgenic lines from the regeneration
and rooting medium with kanamycin selection (Table 1.3). Southern blot analysis can
be used to verify the transgenic status of regenerated shoots and can also provide a
means to investigate potential genetic rearrangements or variation in copy number
(James et al., 1990; Zhao et al., 2004). Regardless of the explant, increasing
kanamycin exposure time up to 60 days reduced the transformation frequency. The
presence of kanamycin during that period increased nopaline synthase (NOS) activity
in plant tissues and in vitro rooting (James et al., 1990). Kanamycin (Alsheikh et al.,
2002), geneticin (Mathews et al., 1995), phosphinothricin (Folta et al., 2006) and
hygromicin (Mathews et al., 1995; Oosumi et al., 2006; Slovin et al., 2009) have been
successfully used for selection of transgenic shoots.
1.6.3 Antibiotics
After transformation, the next critical step is the elimination of the A. tumifaciens
(Cheng et al., 1998; Teixeira da Silva and Fukai, 2001). Common antibiotics used for
this procedure are timentin, carbenicillin, vancomycin and cefotaxime. The antibiotics for
selection can be used alone or in combinations with other antibiotics to effectively
eliminate the Agrobacterium (Cheng et al., 1998; Teixeira da Silva and Fukai, 2001).
26
Timentin was introduced as an alternative for costly carbernicilin, and cefotaxime
(Cheng et al., 1998). Timentin is a mixture of clavulanic acid and ticarcilin, ratios of
clavulanic: ticarcilin acid of 1 :50 and 1:100 have little effect on shoot regeneration of
tobacco (Cheng et al., 1998). The combinations of ticarcilin and clavulanic acid forms a
broad spectrum treatment (Cheng et al., 1998). Timentin remained stable and effective
for approximately 70 days in solid agar medium but was unstable in mixed solutions or
as separate clavulanic acid and ticarcilin stock solutions at -20ºC and -80ºC freezer
after 4 weeks. Cheng et al. (1998) also observed a delay of about 1 week in tobacco
shoot regeneration, and the callus turned light yellow color when treated with timentin.
There are no negative effects of high concentration of Timentin reported by Teixeira da
Silva (2001) on vegetative and flowering morphology after chrysanthemum plants were
transferred to the greenhouse.
Carbenicillin and cefotaxime are known for activity against a wide spectrum of
gram positive and gram negative organisms through inhibition of of peptidoglican cross-
linking by binding and inactivating of transpeptidase, blocking cell wall mucopeptide
biosynthesis (Teixeira da Silva and Fukai, 2001). Phytotoxic effects were reported at
500 µg/ml carbenicillin on tobacco and chrysanthemum. The higher the carbenicillin,
cefotaxime or hygromycin concentration, the greater the morphogenic capability and
callus regeneration in tobacco, attributed to an increase in methylation of DNA (Schmitt
et al., 1997). Al sheikh et al.(2002) compared the effects three antibiotics types which
are carbenicillin, cefotaxime and cefotixin on diploid strawberry and concluded that
carbenicillin has the least impact on shoot regeneration compared to cefotixin and
cefotaxime.
27
1.7 Reverse and Forward Genetics
Arabidopsis thaliana was proposed as a functional genomics tool for higher plant
model systems especially after the completion of Arabidopsis genome in the year 2001
(Bouchez and Höfte, 1998; Sessions et al., 2002; Rosso et al., 2003). Functional
genomics is defined as the "development and application of global (genome-wide or
system -wide) experimental approaches to assess gene function by making use of the
transformation and reagents provided by structural genomics" (Bouchez and Höfte,
1998). High-throughput approaches to identify gene function are developing rapidly as
the need to associate -genes to traits of interest grows (Ajjawi et al., 2010). There are
many ways to establish genomics resources for plants that have not been completely
sequenced, which may include Expressed Sequence Tags (EST) (Folta et al., 2005;
Zhao et al., 2006), physical and genetic maps (Zhang and Wing, 1997), chromosome
libraries, molecular markers (Cabrera et al., 2009), DNA microarray technology
(Boonham et al., 2003), metabolite profiling (Feihn et al., 2000), genetic transformation
protocols and bioinformatics tools for sequence sampling or whole genome sequencing.
A forward genetics approach starts with the observation of specific mutant
phenotype resulting from DNA sequence change (Peters et al., 2003). Mutant
phenotype if resulting from a T-DNA or transposon insertions (Ajjawi et al., 2010), can
be identified rapidly by sequencing and the gene affected the mutation are analyze by
map-based cloning (MBC) (Peters et al., 2003). Conversely, a reverse genetics strategy
starts with alterations in the gene of interest and observation of phenotypes in the
resulting plants (Sessions et al., 2002). Reverse genetics can lead to large insertions
collections (Ostergaard and Yanofsky, 2004) and can be identified by PCR screening of
pooled mutant populations (Xin et al., 2003).
28
Agrobacterium-mediated transformation is one of the effective forward and reverse
genetics approaches for generating mutant phenotypes by inactivation genes by
inserting foreign DNA (Cheng et al., 1998; Gelvin, 2003; Oosumi et al., 2006; Husaini,
2010). Loss-of function phenotypes may be produced by T-DNA insertion. Gain-of-
function mutants also have been observed through activation tagging in Arabidopsis
(Kardailsky et al., 1999; Weigel et al., 2000). Weigel et al. (2000) demonstrated the
integration of transcriptional enhancers (35S enhancer) into the Agrobacterium
mediated transformation, which greatly increases the basal transcription rates of genes
near the insertion event.
F. vesca is an excellent candidate to generate a library of T-DNA or transposon
insertion or activation tagged mutants (Folta and Davis, 2006; Oosumi et al., 2006;
Shulaev et al., 2008; Oosumi et al., 2010; Ruiz-Rojas et al., 2010). As for translational
studies for genetic resources in the Rosaceae family, diploid strawberry gives
advantages comparable to Arabidopsis thaliana. F. vesca has a small genome,
approximately 200-Mb (Davis et al., 2010; Shulaev et al., 2010), with a gene content
and distribution similar to Arabidopsis. Currently, a collection comprised of AcDs
activation tag lines and T-DNA mutation insertion mutants, representing 1000
independent regenerated lines is under development and being characterized at
Virginia Polytechnic State University (Virginia Tech) (Shulaev et al., 2008). Resources
for both forward and reverse genetics and future mapping efforts can be gained by
sequencing flanking regions adjacent to T-DNA integrations, which can lead to
information of interesting and important genes affecting valuable phenotypes
(Kardailsky et al., 1999; Weigel et al., 2000; Oosumi et al., 2010). Currently, the biggest
29
achievement of reverse genetic approaches with T-DNA integration in rosaceous
species is the analysis of genomic DNA flanking the T-DNA insertions in F. vesca
genotype Hawaii 4 (Oosumi et al., 2010).
1.8 Research Problem
How can we test gene function in planta? It is not practical in cultivated strawberry
because of its polyploid genome. The complexity of the genome and the fact that
various subgenomes are present (and not necessarily understood) suggests that it
would be difficult to certainly suppress transcript accumulation with RNAi for instance. In
this case a diploid system would be much more amenable to research in functional
genomics.
1.9 Research Objectives
The research described here has three goals. The first is to optimize the in vitro
seedling growth of the 5AF7 diploid strawberry. Different media types and sucrose
concentrations were tested to achieve this goal. The second is to enhance
transformation and regeneration efficiency for this genotype. A combination of three
different auxins and three different cytokinins at five and three different concentrations,
respectively, were tested to measure the regeneration efficiency using in vitro and ex
vitro explant materials. The third objective is to introduce the Tnt1 retrotransposon into
genotype YW5AF7 as a potential tool for rapid mutagenesis for future forward and
reverse genetics research.
30
Table 1-1. Approximate nutritional value for raw portions of strawberry (100 grams) (USDA, 2009).
Nutrient Value per 100 grams. Nutrient Value per 100 grams. Protein 0.67 g Selenium 0.4 mg Total lipid (fat) 0.30 g Vitamins C, total
ascorbic acid 58.8 mg
Carbohydrate 7.68 g Thiamine 0.024 mg Sugars 4.89 g Riboflavin 0.022 mg Calcium 16 mg Niacin 0.386 mg Iron 0.41 mg Panthothenic acid 0.125 mg Magnesium 13 mg Vitamin B-6 0.047 mg Phosphorus 24 mg Folate, total 24 mg Potassium 153 mg Vitamin A, RAE 1.0 mg Sodium 1.0 mg Vitamin A, IU 12 mg Zinc 0.14 mg Vitamin K
(phylloquinone) 2.2 mg
Copper 0.048 mg Vitamin E (alpha-tocopherol)
0.29 mg
Manganese 0.386 mg Amino acids 0.563 mg Fluoride 4.4 mg
31
Table 1-2. Donor plant selection and surface sterilization procedure on different types of explant.
Donor explant
Tap water
70% ethanol
Surface sterile Rinse
Landi and Mezzetti (2006)
lateral buds - - chlorine-active solution, 20 min
4-5 times
Biswas et al. (2009) Passey et al. (2003)
runner tips 30 min -
0.525 % sodium hypochlorite, 20 min
6 times,
Yonghua et al. (2005) runner tips Yes - 0.1 % HgCl2, 8 min 3 times
Sakila et al. (2007) nodal segments
Yes 10 s 0.1 % HgCl2, 5 min 4 times
Mohamed et al. (2007)
runner tips, leaves
Yes - 0.605 % sodium hypochlorite, 3 min
3 times
Mohamed (2007) shoot tips - - 3.25 % sodium hypochlorite,15 min
4 times
Kaur et al. (2005) vegetative buds
- - 0.1 % HgCl2, 2-3 min 5 min
Table 1-3. Pretreatment medium on explant after surface sterilization and before transfer to shoot proliferation medium stage.
Explant IBA (µM)
BA (µM)
GA3 (mg/L)
Agar (%)
Sucrose (%)
Landi and Mezzetti (2006)
leaf lamina - - - 0.75 3.0
Biswas et al. (2009) runner tips - - 0.5 0.8 3.0
Yonghua et al. (2005) runner tips 0.89 0.04 0.1 - -
Mohamed et al. (2007) leaf disk - - - 0.7 3.0 Mohamed (2007) meristem
tips - 0.44 - 0.7 3.0
32
Table 1-4. Combinations of auxin and cytokinin used in media for regeneration for Fragaria x ananassa octoploid and Fragaria vesca diploid strawberry.
Auxin (µM) Cytokinin (µM) Explant IBA IAA 2,4-D Others BA TDZ Kin Octoploid Passey et al. (2003) leaf disk
petioles, root, stipules
- -
- -
0.90 0.90
- -
8.88 8.88
4.54 2.27
- -
- - - 1.07 NAA
4.44 2.27 -
Landi and Mezzetti (2006)
leaf lamina
0.98
-
0.90
0.84 BSAA
- 4.54 0.98
Mohamed et al. (2007) meristem tips, leaf
- 2.07
- - - - -
Barcelo et al. (1998) leaf disk - 2.46 - - 8.88 - - Sakila et al. (2007) nodal
segment - - - - 6.66 - 2.32
Biswas et al. (2009) runner tips
- - - - 8.88 - 2.32
Yonghua et al. (2005) runner tips
- - - 0.16 NAA
8.88 - -
Borkowska (2001) - 0.41 - - - 8.88 - - Mohamed (2007) meristem
tips - -
- -
- -
- -
1.33 2.22
- -
- -
Kaur et al. (2005) buds - - - - 4.44 - 2.32
Diploids Landi and Mezzetti
(2006) leaf lamina
0.98
-
0.90
0.84 BSAA
- 4.54
33
Table 1-5. Compositions for rooting medium. MS medium
strength (x) IBA (µM)
BA (µM)
Sucrose (%)
Passey et al. (2003) 1/2 - 13.32 3.0 Mohamed (2007) 1 4.14 - 3.0 Mohamed et al. (2007) 1 4.14 - - Sakila et al. (2007) 1 0.41 - 3.0 Borkowska (2001) 1 0.41 - - Kaur et al. (2005) 1/4 4.14 -
34
Table 1-6. Explant types, plant growth regulators and selective agents used in shoot induction medium of transgenic plants.
Auxin(µM) Cytokinin (µM) Selection mgl-1 Explant IBA 2,4-D others BA TDZ Octoploids. Barcelo et al. (1998) leaf disk 2.46 - - 8.88 - Kan 25 Zhao et al. (2004) petioles
sections, leaf segments.
1.50 - - - 10.00 Kan 50
Folta et al. (2006) leaf disk, petioles
- 0.05 - 0.5 4.54 Kan 2.5, then Kan 5.0
Mathews et al. (1995)
leaf disk 0.49 - - 0.88 -22.2 - Kan 25 -120
Mathews (1998) leaf lamina 0.49 - - 0.08 -222.2 - Kan 25 James et al. (1990) petioles 2.46 - - 2.20 - Kan 25 de Mesa et al.
(2000) leaf - - 2.21 Kin - 4.56 Kan 25
Monttironi (2009) in vitro Leaf - - - 1.11 - Kan 25
Jin (2009) leaf disk - - 0.57 IAA 13.32 - Kan 25
Diploids
Oosumi et al. (2006) leaves 0.83 - - 13.32 - Hyg 4
Alsheikh et al. (2002) leaf disk petioles
1.04 - - 13.32 - Kan 25
Zhao et al. (2004) petioles sections, leaf segments.
1.50 - - - 10.00 Kan 50
El-Mansouri et al. (1996)
leaves 1.04 - - 17.75 - Kan 25
Haymes (1998) runners 10.00 - 1.0 NAA
- - Kan 50
35
CHAPTER 2 IN VITRO YW5AF7 SEEDLINGS GROWN ON DIFFERENT MEDIA TYPES
2.1 Introduction
The diploid strawberry Fragaria vesca is widely used in many laboratories around
the world for studies ranging from plant physiology to biochemistry of flavor to large-
scale genomics efforts. F. vesca is thought to share a common ancestor with octoploid
strawberries, as been demonstrated by early cytological studies (Ichijima, 1926; Yarnell,
1931). These findings suggested that F. vesca would be an ideal candidate for studies
in its simple genetic background that then could be applied to the complex cultivated
octoploid. The F. vesca genotype Hawaii 4 (H4; PI551572) has been studied specifically
because of its ease of transformation (Oosumi et al. 2005) and it is also the genotype
being sequenced (Shulaev et al., 2010).
However, the Hawaii 4 genotype is not nearly as homozygous as anticipated,
leading to considerable variability in outcomes when performing physiological tests in
this background (Slovin et al., 2009). Even sequencing with Next Generation technology
was problematic, as assembly was hindered by the large amount of heterozygousity (K.
Folta, unpublished). Another potential genotype for transgenic analysis was introduced
by Janet Slovin (2009) known as Yellow Wonder Fragaria vesca genotype 5AF7
(YWFAF7). The YW5AF7 genotype is a seven-generation inbred line that is easy to
grow, germinates quickly, does not runner and has a well characterized phenotype. The
phenotypic variance among plants is minimal, consistent with the hypothesis that the
inbred line is more homozygous than F. vesca genotype Hawaii 4. Thus, YW5AF7 is a
potential candidate for testing functional genomics (Slovin et al., 2009).
36
F. vesca is known for rapid regeneration and can be transformed using A.
tumefaciens (Oosumi et al., 2006). Seed to seed time takes less than four months,
plants can be grown in green house or lighted laboratory benches in 10 cm pots. This
plant is day neutral, they do not have runners and they produce yellow fruit. It also listed
as a non commercial cultivar by United States National Germplasm Repository
(http://www.ars.usda.gov/Main/docs.htm?docid=11324).
YW5AF7 will flower approximately 8 weeks after sowing. The leaves are thin and
have the typical morphology of F. vesca. The leaf color is light medium green, with both
sides pubescent. The largest leaves are 13 cm width and 8 cm length if grown under
greenhouse conditions. Petioles are long, pubescent, have a distinct adaxial groove,
with straight, unbranched hairs. Stipules are red (Slovin et al., 2009).
The flower is typical to F. vesca with five petals, a calyx consisting of 2 whorls of
five sepals, and 20 stamens (Slovin et al., 2009). It has primary flower, about 1.5-2.0 cm
in diameter and secondary flower which is smaller. The seeds germination is uniform
after cold treatment of moist seed, with 87% germination 21 days after sowing. At the
early stages of development, YW5AF7 seedlings showed no resistance to powdery
mildew, thrips, two spotted mites and aphids (Slovin et al., 2009). YW5AF7 is also
runnerless, which make the maintenance in the greenhouse a lot easier. Plus, -Slovin
(2009) demonstrated by not having the runners, it can eliminate one of source of
contamination or germplasm mis-identification. The goal of this chapter is to investigate
the effect of three media types in strawberry tissue culture and two different sucrose
concentrations on the vigor and growth of YW5AF7 seedlings.
37
2.2 Materials and Methods
2.2.1 Plant Material
The plant material used in this study were seeds of diploid strawberry YW Fragaria
vesca genotype 5AF7 supplied by Janet Slovin (personal communication).
2.2.2 Seed Sterilization
One-hundred seeds of 5AF7 were placed in 17 ml round-bottomed test tubes and
soaked in nanopure water overnight. The following day, the seeds were washed in
water (1 h) and immersed in 95% ethanol (5 min). After the ethanol was poured off,
seed were soaked in 33% aqueous bleach (1.98% sodium hypochlorite) for 30 minutes.
After each treatment, the seeds were rinsed thoroughly five times with sterile water.
The water was removed from the tubes and the seeds were mixed with melted <55°C
0.7% agarose with no mineral salts and then spread on 9 cm Petri dishes. The Petri
dishes were kept in dark for 1 week at 4°C. Finally, the plates were kept under light to
germinate them for 3 weeks under 16/8 h light/ dark cycle at 25± 2 ºC.
2.2.3 In vitro Media Types and Sucrose Concentration Experiment
The goal of this work was to compare the effects of commercially-available media
on the in vitro growth of YW5AF7. Commercial preparations are preferred to custom
prepared mixes because they are readily available and standardized. The media types
used in this experiment was in powder form by Research Product International Corp
(RPI), Illinois, USA. These 3 week old seedlings were transferred to one pint Mason jars
with respective media types; MS mineral salts with MS vitamins, MS mineral salts with
B5 vitamins, octoploid strawberry formulation media, with 1 % and 3% sucrose
concentrations, respectively (Table 2.1). The jars were sealed with Parafilm M Barrier
Film (West Chester, PA). After two months, three different media types with the 1% and
38
3% sucrose concentrations were evaluated qualitatively by observing the plant vigor.
The rating scale consisted of healthiest (5), healthy (4), moderate (3), unhealthy (2), and
the unhealthiest (1), with respective score as shown in Figure 2.1. The healthiest is
defined when the plant showed long petioles, robust leaf, better vigor and green
chlorophyll color. The unhealthiest is define as a brown, truncated, small, less leaves
and low chlorophyll color plants. The highest scores were used to select the optimal
sucrose concentration.
The experiment was repeated on MS mineral salts with MS vitamins, MS mineral
salts with Gamborg vitamins, and Gamborg mineral salts with B5 vitamins, with one
selected sucrose concentration. The plantlets growth (number of leaves) and fresh
weight were measured after two months (Table 2.2). The experiment was set up in a
Complete Randomized Design (CRD) and was repeated three times. The data were
analyzed by proc glm procedure, using SAS 9.2 software, with α= 0.05.
2.3 Results
Qualitative observations on sucrose level effect on YW5AF7 in vitro plant
demonstrated a distinct pattern of 1% and 3% sucrose. All media types with 3% sucrose
showed a reduction in plant vigor compared to plants grown on 1% sucrose (Figure 2-1
and Figure 2-2). Plants grown on various media types were scored by qualitative
observations ranged from the healthiest to the unhealthiest. The healthiest is defined
when the plant showed long petioles, robust leaf proliferation, better vigor and green
chlorophyll color. The unhealthiest is defined as brown, truncated, small, less leaves
and low chlorophyll content. MS mineral salts with B5 vitamins scored the highest,
followed by MS mineral salts with MS vitamins, and finally Gamborg mineral salts with
B5 vitamins (Figure 2-1 and Figure 2-2). All plants cultured on the octoploid media
39
formulation (Table 2-1) had the unhealthiest condition (Figure 2-2) when observed after
two months (Figure 2-1). Based on the results from these experiments, 1% sucrose was
chosen as the optimal concentration.
There was no significant difference among fresh weight (g) for in vitro plants after
being grown on the three different media types; MS mineral salts with MS vitamins, MS
mineral salts with B5 vitamins and Gamborg mineral salts with B5 vitamins (Table 2-3).
However, there were significantly differences on the leaf numbers between the
treatments. The highest leaf production was observed on MS mineral salts with B5
vitamins (16 leaves per plant), followed by MS mineral salts with MS vitamins(14 leaves
per plants) and finally Gamborg mineral salts with B5 vitamins (12 leaves per plants)
(Table 2-3). From this simple experiment it was concluded that MS mineral salts with B5
vitamins and 1% sucrose yielded the highest leaf number compared with other media
types.
2.4 Discussion
The experiment did not focus necessarily to define a media for YW5AF7
strawberry, instead, three media types commonly used in strawberry tissue culture were
investigated; MS mineral salts with MS vitamins, MS mineral salts with B5 vitamins and
Gamborg mineral salts with B5 vitamins on the growth of YW5AF7 seedlings. The
choice of medium is very important for plant tissue culture to ensure the plants are able
to receive the nutrients required for optimal growth and development in an otherwise
minimal in vitro environment (Murashige and Skoog, 1962; Gamborg et al., 1968;
Gamborg et al., 1976).
The result for MS mineral salts with MS vitamins, MS mineral salts with B5
vitamins and the octoploid strawberry formulation media demonstrated that the
40
YW5AF7 genotype responded differently based on a number of easily scored
observations (Figure 2-1). Only two sucrose concentrations were tested, 1% and 3%.
The most widely used sucrose concentration in octoploid strawberry media is 3%
(Passey et al., 2003; Kaur et al., 2005; Yonghua et al., 2005; Landi and Mezzetti, 2006;
Mohamed, 2007; Mohamed et al., 2007; Sakila et al., 2007; Biswas et al., 2009) while
1% is less prevalent. For YW5AF7 the 1% sucrose concentration is favored compared
to 3 % sucrose concentration based on seedling vigor (Figure 2-1). Decreasing the
concentration of sucrose in the medium increased seedling fresh weight and general
plant vigor in those plantlets (Langford and Wainwright, 1988). The in vitro grown
seedlings grown in the jars depend on exogenous sucrose in the culture medium, as
their photosynthesis ability is impaired by decreasing of the carbon-fixing enzyme
RubPase. The exogenous sucrose depresses the activity of enzyme RubPase and
PEPcase, both are part of photosynthetic machinery (Grout, 1988). It also was observed
that YW5AF7 seedlings performed better on basal medium, sodium phosphate, and
adenine sulfate (Figure 2-2). Hyperhydricity was also observed with seedlings grown on
octoploid strawberry formulation media by 100% (data not shown). The octoploid media
formulation contains auxin and cytokinin which may lead to hyperhydricity on the
seedlings (Debnath, 2009). Plus, medium supplemented with IAA and BA did not
support prolific growth and lead to 100 % tissue browning (see chapter 3: regeneration
and transformation).
Sucrose has a significant roles in accumulation of anthocyanin, as apparently did
other factors, such as light intensity, culture conditions, plant growth regulators and
media components (Miyanaga et al., 2000). Sucrose synthase is a prominent
41
component of the catalytic unit and functions by catalyzing the formation of UDP-
glucose from sucrose which will help to promote robust cellulose synthesis (Nakai et al.,
1999; Fujii et al., 2009). The different responses on sucrose concentrations are possibly
related to the polyploid genome—possibly higher expression of sucrose synthase as the
polyploid number increases (Comai, 2005). Other factors include Parafilm sealing
around the jar increasing the anaerobic stress response through time to strawberry
seedlings or plantlets, thus potentially inducin the transcription and translation of
sucrose synthase (Richard et al., 1991) .
Truncated growth and brown seedlings were observed on all media formulations
containing 3% sucrose. One possible explanation is that the high sucrose level creates
an osmotic stress resulting the inhibition of cell growth (Figure 2-1, Figure 2-2) (Ibrahim,
1987). The octoploid strawberry media tested with YW5AF7 seedlings demonstrated
100% seedlings mortality when using 3% sucrose, indicating that what works well for
the octoploid does not translate directly to the diploids. In preparations with 1% sucrose
and approximately 80% of seedlings died. The remaining 20% were weak and brown in
color (Figure 2-1). One explanation for this result is that the PGRs used in the octoploid
media (IAA and BA) negatively affected diploid strawberry growth and development
(Table 2-1). These observations demonstrated that the 1% sucrose concentration is
superior for the in vitro cultivation of the inbred YW5AF7 genotype.
MS mineral salts with MS vitamins and MS mineral salts with B5 vitamins
demonstrated significant qualitative differences in plant vigor (Figure 2-2). These two
media used for seed germination, regeneration or transformation in strawberry
(Haymes, 1998; Folta et al., 2006; Landi and Mezzetti, 2006; Slovin et al., 2009). MS
42
mineral salts with B5 vitamins produced healthier plants, with 20% of the seedlings
being in the ‘healthiest’ class and 100% survival. The MS mineral salts with MS vitamins
yielded no plants in the 'healthiest' class and about 90% seedlings survival on 1 %
sucrose concentration (Figure 2-1). MS mineral salts with MS vitamins is a very
common for regeneration and transformation media in the literatures. However use of
MS mineral salts with B5 vitamins can only be found in a small subset of report (Husaini
and Abdin, 2007), and Gamborg mineral salts with B5 vitamins is used for cell
suspension media for the most part (Gamborg et al., 1968).
Replication of the media formulation experiments with 1 % sucrose demonstrated
significantly differences in the average shoot number between MS mineral salts with B5
vitamins and Gamborg mineral salts with B5 vitamins (Table 2-3). MS mineral salts with
B5 vitamins produced more shoots, with approximately 1.4 more leaves compared to
MS mineral salts with MS vitamins, and 3.4 more leaves compared to Gamborg mineral
salts with B5 vitamins (Table 2-3). However, there are no significant differences in fresh
weight between MS mineral salts with MS vitamins and MS mineral salts with B5
vitamins (Table 2-3). MS mineral salts with B5 vitamins resulted the highest leaf
number, and fresh weight. MS mineral salts with MS vitamins gave 0.04 g more weight
compared to MS mineral salts with B5 vitamins. MS media gave 0.071 g and o.o316g
when treated with MS vitamins and B5 vitamins, respectively, compared to Gamborg
media with B5 vitamins. The fresh weight possibly reflected the differences in root
nutrient uptake on respective media. Different vitamin formulations have an effect on
leaf number between MS mineral salts with MS vitamins and MS mineral salts with B5
vitamins. The B5 vitamins and MS vitamins have identical inositol concentration, but B5
43
vitamins are two times higher in Nicotic Acid and Pyroxidine-HCl and ten times higher in
Thiamine-HCl. The B5 vitamins contained the 2,4-D hormone while MS vitamins
contained IAA hormones and also glycine (Gamborg et al., 1976). The PGRs; AA and
2,4-D are the key to obtain higher shoot number, as auxin type and concentration effect
shoot differentiation (Barcelo et al., 1998).
Table 2-1. Media types tested, A; MS mineral salts with MS vitamins, B; MS mineral salts with B5 vitamins and C; an octoploid strawberry formulation with 1 % and 3 % of sucrose concentration on Yellow Wonder 5AF7 seedlings. The volume for each media is 1 liter with pH 5.8.
A B C Sucrose (1% or 3%) 10 g/L 30 g/L 10 g/L 30 g/L 10 g/L 30 g/L MS mineral salts with MS vitamins
4.4 g/L 4.4 g/L - - 4.4 g/L 4.4 g/L
MS mineral salts with B5 vitamins
- - 4.4 g/L 4.4 g/L - -
Na2H2PO4 - - - - 0.17 g/L 0.17 g/L Adenine sulphate - - - - 0.08 g/L 0.08 g/L 6- BA (2mg/L) - - - - 0.5 ml/L 0.5 ml/L IAA (2mg/L) - - - - 0.5 ml/L 0.5 ml/L MES (3.5 mM) 0.5 g/L 0.5 g/L 0.5 g/L 0.5 g/L - - 0.7% Phytoagar 7.0 g/L 7.0 g/L 7.0 g/L 7.0 g/L 7.0 g/L 7.0 g/L
44
Table 2-2. Three different media types treated with 1% sucrose concentration on Yellow wonder 5AF7 seedlings. Media A; MS mineral salts with MS vitamins, B; MS mineral salts with B5 vitamins, D; Gamborg mineral salts with B5 vitamins. The volume for each media types is 1 liter, with pH 5.8.
A B C MS mineral salts with MS vitamins
4.4 g/L - -
MS mineral salts with B5 vitamins
- 4.4 g/L -
Gamborg mineral salts with B5 vitamins
- - 3.16 g/L
MES (3.5 mM) 0.5 g/L 0.5 g/L 0.5 g/L 0.7% Phytoagar 7.0 g/L 7.0 g/L 7.0 g/L Sucrose (1% or 3 %) 10 g/L 10 g/L 10 g/L
Table 2-3. Average of leaf number and average of fresh weight of YW5AF7 seedlings on 3 different media types; MS mineral salts with MS vitamins, MS mineral salts with B5 vitamins and Gamborg mineral salts with B5 vitamins. The average leaf number and average fresh weight of seedlings with ± standard error (SE). The data were analyzed by using proc glm SAS 9.2 software with α = 0.05, n=8 with 3 independent replicates on each media type.
Media types Leaf numberx Seedlings fresh weight (g)y MS mineral salts with MS vitamins
14.9 ± 0.708ab 0.1732 ± 0.0388a
MS mineral salts with B5 vitamins
16.3 ± 0.695a 0.1338 ± 0.0163a
Gamborg mineral salts with B5 vitamins
12.9 ± 1.397b 0.1022 ± 0.0182a
x is when the p-value is <.0001 y is when the p-value is 0.015
45
Figure 2-1. Plant health score experiment on (A) MS mineral salts with MS vitamins, (B) MS mineral salts with B5 vitamins, and an (C) octoploid strawberry formulation media (score; the healthiest (5), healthy (4), moderate (3), unhealthy(2) and unhealthiest (1) (each treatment replicates; n=8 and error bar with percentage). The healthiest is defined when the plant showed long petioles, robust leaf, better vigor and green chlorophyll color. The unhealthiest is define as a brown, truncated, small, less leaves and low chlorophyll color plants.
46
Figure 2-2. The morphology of 5AF7 seedlings on different media type after 6 weeks. (A, B; MS mineral salts with B5 vitamins (1% sucrose), C, D; MS mineral salts with B5 vitamins (3% sucrose), E, F; MS mineral salts with MS vitamins (1% sucrose), G, H; MS mineral salts with MS vitamins (3% sucrose), I, J; octoploid strawberry media formulation (1% sucrose), K, L; octoploid media formulation (3% sucrose). Hyperhydricity was observed on MS mineral salts with MS vitamins treated with 3 % sucrose; H and octoploid media formulation media; I, J, K and L.
10 mm
47
CHAPTER 3 REGENERATION AND TRANSFORMATION OF YW5AF7
3.1 Introduction
The diploid strawberry line YW5AF7 has been proposed as a functional genomics
tool because of its homozigosity and ability to be transformed and regenerated (Slovin
et al. 2009). The goal of this work is to determine if YW5AF7's regeneration efficiency
could be increased beyond what was observed in the previous reports. As noted in
previous chapters, each strawberry genotype has specific optimal culture conditions
with regard to sucrose concentrations and plant growth regulators (Landi and Mezzetti,
2006; Mohamed et al., 2007; Slovin et al., 2009). TDZ has been successfully used to
regenerate YW5AF7, as well as other diploid strawberries, but this growth regulator
leads to short regenerated shoots. This causes a problem especially during the rooting
stage, because the shoots are short and decrease the frequency of rooting. The
YW5AF7 line initiates shoots on all media tested by Slovin et al. (2009), but tissue
regeneration and vigor were best supported by combinations of 1.50 µM IBA and 10.00
µM TDZ; a media formulation chosen for high efficiency with some genotype (Zhao et
al., 2004).
In other studies the highest shoot regeneration frequency was observed in petiole
segments. Zhao and colleagues (2004) defined a specific medium that develop shoots
after 9 weeks in culture. The number of transformation events is closely correlated to
the highest number of explants exhibiting shoots. This would be highly desired if the
protocol were to be generated in a potential functional genomics model. The choice of
explant for regeneration and transformation is also very important as greenhouse tissue
and in vitro tissue have been shown to give different results for regeneration and
48
transformation efficiency. Even though ex vitro explants are favored for robust
regeneration, the period for adjustment to in vitro environment and fungus
contamination lowers the attractiveness of using ex vitro explants.
There are two goals in this chapter. The first is to identify the best combination and
type of PGRs to maximize YW5AF7 regeneration by using leaves as explant. The
second is to produce transgenic shoots by using A. tumifaciens mediated transformation
by using leaves and petioles as explants.
3.2 Materials and Methods
3.2.1 Plant Material
This experiment was carried out using leaf pieces and petioles from in vitro plant
grown in jars with MS medium with B5 vitamins, 1% sucrose, 0.7% phytoagar (Table 2-
2). In vitro derived leaves cut were 0.5 cm x 0.5 cm with few horizontal cuts at the
middle of leaf, while the petiole segments cut were 0.5 cm.
3.2.2 Preliminary Auxin and Cytokinin Experiment
The plant materials were transferred to different combinations of auxin and
cytokinin treatments (Table 3-1) on 4 Petri plates (replicate) on 3 explants (sub
replicate) for each plate. The plates were sealed with one layer of Parafilm. The plate
then incubated under 16/8 h light cool-white/ dark cycle at 25± 2 ºC for 8 weeks. After
that period, the number of regenerated shoots per explant was collected and the data
were treated as factorial design. Statistical analyses were conducted within each of
auxin groups. Data were analyzed by the proc glm procedure using SAS 9.2 software
with alpha (α) =0.05.
49
3.2.3 Plant Growth Regulators (PGRs) Optimization
The highest average number of shoots per explant was determined from each
auxin and cytokinin group combinations. From the data collected at the end of
preliminary experiment (Table 3-2, Table 3-3 and Table 3-4), five combinations of
PGRs were identified as the best candidates for producing high numbers of shoots
within the eight week experimental period.
New sets of experiments were conducted with following PGR combinations (in
micromolar (µM); 1.5 IBA & 15 BA, 15 IAA & 10 TDZ, 0.2 2,4-D & 4.0 TDZ, 3.0 IBA &
4.0 TDZ, 0.45 2,4-D & 10 BA) with 4 each on four plates (replicates) with three explants
(sub replicates) for each plate. The experiments were repeated three times, and the
plates then incubated under 16/8 h light/ dark cycle at 25± 2 ºC for 6-8 weeks.
After that period, the number of regenerated shoots per explant was collected and
the data were treated as Randomized Completed Block Design (RCBD). Statistical
analyses were conducted between PGR treatment groups. Data were analyzed with
Pairwise Multiple Comparisons by the proc glm procedure using SAS 9.2 software with
alpha (α) =0.05.
3.2.4 Comparison Between 5AF7 Medium & Hawaii-4 medium
Comparison of medium defined for YW5AF7, named "5AF7 medium" (MS medium
+ 2% sucrose + 1.5 µM IBA + 15 µM BA; pH 5.8 + 0.7% phytoagar) with Hawaii-4
medium were conducted for YW5AF7 leaves. The Hawaii-4 medium is defined by MS
mineral salts with MS vitamins, 2% sucrose -supplemented with 0.83 µM IBA and 13.32
µM BA (Oosumi et al., 2006). Four media plates were made for both 5AF7 media and
Hawaii-4 medium, and five in vitro leaves were transferred on each of them. The reason
five leaves were used instead of three leaves is to obtain larger sub replicate for
50
statistical analysis as 5AF7 medium and Hawaii-4 medium were used to compare their
efficiency in shoot production.
The experiments were repeated three times, and the plates were then incubated
under 16/8 h light/ dark cycle at 25± 2 ºC for 8 weeks. The numbers of shoots per
explant after 6- 8 weeks of observation on both media types were taken. The data were
treated as RCBD and student's t-test was conducted between 5AF7 medium and
Hawaii-4 medium, counting the shoots per explant by the proc glm procedure using
SAS 9.2 software. The percentage number of explants with shoots also were calculated
for each media.
3.2.5 Regeneration of Six Cultivars of Diploid F. vesca on 5AF7 Medium
Six cultivars of diploid strawberries were tested with this medium to determine how
it may be applied to ther genotypes. The diploids tested were 'Rodluvan', 'Reugen',
'Baron Solemacher', 'Fragole di Bosco', 'Mignonette' and 'Alexandria'. The cultivars were
tested on 5AF7 medium (MS medium + 2% sucrose + 0.7% phytoagar + 1.5 µM IBA +
15 µM BA, pH5.8) to determine the degree by which the YW5AF7 medium may be
applied to these other potentially useful accessions. Young leaves were used as
explants on four plates (replicates) with three explants (sub replicate) and the
experiment were repeated three times.
3.2.6 Transformation
The night before co-cultivation, a single fresh colony of Agrobacterium tumifeciens
GV3101 containing the construct of interest was inoculated into LB broth with 50 µg/ml
gentamycin, 10µg/ml rifampicilin, and 100 µg/ml kanamycin. The culture was incubated
on shaker at 200 rpm overnight at 28◦C (16-18 hrs).
51
After overnight incubation, 500 µl of overnight culture was spun down in a sterile
1.5 ml tube. The supernatant was discarded, and the pellet was resuspended in 20 ml
of liquid co-cultivation medium (1 x MS medium, 2% sucrose; pH 5.8) in a 50 ml tube.
Acetosyringone (ACS) was prepared by mixing 20 mg ACS in 400uL of 70% ethanol,
then 8 µl of the mixture was added to the 20 ml co-cultivation medium and was mixed
completely. The mixture was measured by spectrophotometry to an OD600 of 0.1. The
leaves and petioles were added into 50 ml tube containing the 20 ml of co-cultivation
medium with ACS and incubated for 1 hour at room temperature.
After co-cultivation, the explants were dry on sterile paper and inoculated on 5AF7
medium (Table 3-2). The plates then place under darkness at 4ºC. After 2 days, the
tissues were washed five times with sterile water with vigorous shaking. The tissues
then were incubated in 10 ml water with 500 µg/L timentin for 1 hour. Then, the tissues
were dried on sterile paper and transferred to pre-selection medium (Table 3-2) to
inhibit the growth of A. tumifaciens. The plates then incubated under 16/8 h light/ dark
cycle at 25± 2 ºC and sub-cultured every 4 weeks. On every subculture, necrotic tissue
was not transferred to new media. This was done to limit phytotoxicity caused by
phenolic exudation from dead tissue.
After four weeks, the explants with green callus were transferred on Selection
Medium I (Table 3-2) for one week. Then the explants were transferred on Selection
Medium II (Table 3-2) for another two weeks. Finally they were moved to Selection
Medium III (Table 3-2). About 5-6 weeks on the Selection Medium III, the regenerated
shoots were transferred to Recovery Medium (Table 3-2) for 4 weeks to allow recovery
before they were transferred to Rooting Medium (Table 3-2). After 2 months on rooting
52
medium, plantlets were transferred to Magenta box containing MS medium with MS
vitamins + 1% sucrose + 0.7% agar).
3.3 Results
3.3.1 Preliminary Shoot Regeneration Experiment
Preliminary experiments demonstrated that MS medium with MS vitamins, 2%
sucrose, 1.5 µM IBA and 15 µM BA gave better shoots regeneration shoot growth and
faster regeneration from leaf explants. Combinations of different types of auxin and
cytokinin in varying concentrations demonstrated a significantly varied response with
regard to regeneration from leaf disk explants. Any combinations of auxin with kinetin
did not yield any regenerated shoots on any explant; leaf disk or petioles. Instead the
leaf disk and petioles turned brown after 4 weeks. The incompatibility of kinetin with
YW5AF7 genotype was verified by repeating the same auxin combinations with kinetin
three times and still there are no shoots regenerated (data not shown). Observations
after eight weeks of shoot regeneration demonstrated that shoots also grew to different
lengths among the PGR combination groups. Plant length ranged between 4-6 mm on
optimum combinations of IBA with BA and IAA with BA tested and vary with any auxins
in combinations with TDZ tested, between 3 -6 mm.
Leaf explants were found to be most efficient for regenerating shoots. A
combination of 3.0 µM IBA with 4.0 µM TDZ resulted in five shoots per explant
compared to combinations of 1.5 µM IBA with 15 µM BA, 0.45 µM 2,4-D with 10 µM BA,
0.2 µM 2,4-D with 4 µM TDZ, and 15 µM IAA with 10 µM TDZ that yielded about three
shoots per explant (Table 3-3 and Table 3-4). Green callus was observed to grow
vigorously on YW5AF7 leaf explants compared to petioles after 4 weeks (Figure 3-1).
Leaf explants performed better compared to petioles in YW5AF7 regeneration when
53
treated with any combinations of auxin and cytokinin. Shoot regeneration on leaf
explants was generally via indirect shoot morphogenesis, with a green callus forming by
four weeks, followed by indirect shoot initiation from the callus after the subsequent four
weeks. The shoot regeneration rates on petiole explants was only one or two shoots per
explant. The percentage of shoots per explant was also very low as only about 10% of
petioles in every treatment group produced shoot (data not shown).
3.3.2 Optimization of 5AF7 Medium Candidates
Five potential candidate combinations of PGRs on shoot regeneration from leaf
disks demonstrated that more than 50% of leaf explants would produce shoots (Table 3-
5). The highest percentage of explants with shoots observed was approximately 70%,
on medium supplemented with 1.5 µM IBA and 15 µM BA followed about 64 % explants
with shoots on medium supplemented with 0.2 µM 2,4-D and 4 µM TDZ. The
combinations of 1.5 µM IBA with 15 µM BA and 3.0 µM IBA with 4 µM TDZ produced
the highest number of shoots per explant among the five treatments, with a mean of
approximately four shoots per explant (Table 3-2). However, combinations of 3.0 µM
IBA and 4 µM TDZ resulted in fewer shoots regenerated, as only 22% of shoots per
explant were observed compared to combinations of 1.5 µM IBA with 15 µM BA. The
highest number of shoots per explant and percentage of explants with shoots favors the
medium supplemented with 1.5 µM IBA and 15 µM BA as the best candidate for
YW5AF7 shoot regeneration. This combination was adopted as the new 5AF7 medium.
Comparison of 5AF7 medium and Hawaii-4 medium on YW5AF7 leaf disk
demonstrated a robust regeneration of shoots with average of five and four shoots per
explants, respectively (Table 3-6). There are no significant differences between the two
media, as both have the same auxin and cytokinin types. The main differences are the
54
concentrations, with the YW5AF7 medium having 0.67 µM of IBA and 1.68 µM of BA.
The Hawaii-4 medium with the combination 0.83 µM IBA and 13.32 µM BA (Oosumi et
al., 2006) are expected to be in the range of 0.5 - 1.5 µM IBA and 10 - 15 µM BA and
the Hawaii-4 medium concentrations are relatively close with 5AF7 medium. The
relative ratio and absolute quantity of IBA and BA is very crucial in promoting
acceptable levels of shoots regeneration, with high efficiency in YW5AF7.
The six genotypes of F. vesca showed different responses to shoot regeneration
after treated with 5AF7 medium for 8 weeks. Cultivars 'Alexandria', 'Mignonette' and
'Fragole di Bosco' demonstrated prolific shoot regeneration with average of five, four
and two shoots per explant (Table 3-7). Other cultivars of 'Rodluvan', 'Reugen', and
'Baron Solemacher' produced shoots an average less than one shoot per explant.
Regeneration was also observed with cultivar 'Alexandria'(Table 3-7) and YW5AF7
(Table 3-5 and Table 3-6), regenerated about five shoot per explant in average.
3.3.3 Agrobacterium-Mediated Transformation
Overall, it took 12 weeks for green callus to initiate after co-cultivation. After 8
weeks on Pre-selection medium (Table 3-2), green callus was clearly observed.
Treatment with 250 mg/L cabernicilin + 500 mg/L timentin showed a delay green callus
formation compare straight regeneration in the absence of antibiotics. After 8 weeks on
pre-selection media the kanamycin concentrations used were 5 mg/L and increased
after one week to 10 mg/L, and finally to 20 mg/L after 2 weeks (Table 3-2). This is one
of a strategies used to limit escapes, non-transgenic shoots that regenerated despite
selection. Control leaf disks also generated callus after 6 weeks and shoots after 10
weeks on 5 mg/L. It was also observed that 10 mg/L kanamycin selection gave less
than 50% of callus formation on in vitro leaf disk (data not shown).
55
Shoots resistant to kanamycin were observed. Only four out of twenty shoots were
truly resistant to kanamycin after being subcultured two times, once every 4 weeks on
Selection III media (Table 3-2). On every subculture, necrotic tissue was not transferred
to new media for two times. This was done to ensure no phytotoxicity cause by
phenolic compound exudation from dead tissue. After 8 weeks on the Selection media
III, apparently kanamycin resistant green shoots were observed on 10 mg/L and 5 mg/L
of kanamycin selection (Figure 3-2). The Recovery media (Table 3-2) was used to
initiate rooting. Recovery media contains glucose instead of sucrose, and causes
development of adventitious roots faster than sucrose-containing media (K. Folta,
unpublished).In general, the process took approximately six months to progress from
explant to rooted plant (Table 3-1).
3.4 Discussion
To adopt YW5AF7 as a useful alternative to the H4 genotype it was important to
have an efficient regeneration and transformation protocol. Regeneration of cultivar F.
vesca was optimal on MS medium with MS vitamins without any addition of
supplemental nitrogen, unlike several cultivars of F. × ananassa (El-Mansouri et al.,
1996). In accordance with previous results obtained for cultivated strawberry (James et
al., 1990; Mathews et al., 1995; Barcelo et al., 1998; Borkowska, 2001) and diploid
strawberry (El-Mansouri et al., 1996; Alsheikh et al., 2002; Oosumi et al., 2006), the
combinations of IBA with BA yielded excellent results for F. vesca regeneration. Callus
formation is a must for YW5AF7 before it can initiate shoots via indirect shoot
organogenesis (Slovin et al., 2009).
The regeneration capacity of leaf disk and petioles was tested during the
preliminary experiments. The results indicate that explant source vary in the number of
56
shoots produced and the time required to produce them. Best shoot regeneration was
observed on young leaf disks. The several horizontal cuts in the middle of leaf disk also
contributed the high shoot regeneration from injured tissues. The choice of using in vitro
grown plants helped to save time and resources. There is no need for surface
sterilization, which means the explants are free from stress and damage occurring from
bleach and ethanol treatments. Each genotype has specific requirements for plant
growth regulators and media (Passey et al., 2003; Folta and Dhingra, 2006). However,
a “universal” media type would be of great value, so a number of different genotypes
were tested on the optimized YW5AF7 medium. The identical number of shoots per
explant with genotypes YW5AF7 and three cultivars of "Alexandria', 'Mignonette' and
'Fragole di Bosco' showed a common response to the optimized combination of IBA
with BA. Comparison of 5AF7 medium with Hawaii-4 medium gave an idea of how the
defined combination of PGRs can improve the number of shoots. Even though there is
average about one shoot per explant different, the combinations of BA with BA provided
a general optimum range for Hawaii-4 and YW5AF7 regeneration that may apply more
broadly to F. vesca accessions, or perhaps Fragaria accessions overall.
In transgenic studies it is necessary to transform plant materials as well as
regenerate them, so it was important to test how transgenic plant materials could be
generated on this media formulation. We know that YW5AF7 can be transformed, as it
has been demonstrated previously (Slovin et al., 2009). It is possible that there was a
problem with the vector used in transformation, either in the raw material used or
perhaps how the Tnt1 retrotransposon could be affecting regeneration of transformed
tissues. The experiments in this chapter test a broad range of PGRs concentrations to
57
empirically determine the optimal medium for use of 5AF7. The optimum concentration
was identified and tested on a series of plant genotypes. The medium for YW5F7
regeneration from leaves explant was defined on medium supplemented with 1.5 µM
IBA and 15 µM BA. While transgenic experiments were not successful, the used a
single plasmid and construct that could be detrimental to transformation or regeneration
of transformed tissue. Further experimentation can resolve this problem.
Table 3-1. Auxin and cytokinin group PGRs combinations were presented in groups. List of sections contained in the template. Each group were observed within same timeline; 8 weeks. The experiments were conducted with 3 groups in one time; e.g. group 1 until group3, group 4 until group 6 and group 7 until group 9. The statistical analyses were conducted within the 3 horizontal groups.
Cytokinin (µM) BA TDZ Kinetin 2 10 15 2 4 10 1.21 2.21 3.21
Auxin (µM) IBA
0.1 1 1 1 2 2 2 3 3 3 0.5 1 1 1 2 2 2 3 3 3 1.5 1 1 1 2 2 2 3 3 3 2.5 1 1 1 2 2 2 3 3 3 3.0 1 1 1 2 2 2 3 3 3
IAA 1.0 4 4 4 5 5 5 6 6 6 5.0 4 4 4 5 5 5 6 6 6 10 4 4 4 5 5 5 6 6 6 15 4 4 4 5 5 5 6 6 6 20 4 4 4 5 5 5 6 6 6
2,4-D 0.2 7 7 7 8 8 8 9 9 9
0.45 7 7 7 8 8 8 9 9 9 0.68 7 7 7 8 8 8 9 9 9 0.9 7 7 7 8 8 8 9 9 9 1.2 7 7 7 8 8 8 9 9 9
58
Table 3-2. Media compositions used in transformation procedure of Tnt1 retrotransposon YW5AF7.
Media Compositions 5AF7 medium MS mineral salts with MS vitamins + 1.5 µM IBA + 15 µM IBA +
2% sucrose + 0.7% TC agar , pH 5.8 Pre-selection medium 5AF7 medium + 250 mg/L carbenicillin + 500 mg/L timentin Selection Medium I Pre-selection medium + 20 mg/L kanamycin Selection Medium II Pre-selection medium + 10 mg/L kanamycin Selection Medium III Pre-selection medium + 5 mg/L kanamycin Recovery Medium MS medium with MS vitamins + 1% glucose + 0.7% TC agar;
pH 5.8 Rooting Medium 0.5 x MS medium + 0.01 mg l-1 + 1% sucrose + 0.7% TC agar;
pH 5.8
Table 3-3. Adventitious shoot regeneration from leaves explant following treated with combinations of two different auxins types with BA on respective concentrations. A combination of IAA with BA was not shown because there was no shoots regeneration observed. The statistical analysis factorial design were conducted within each auxin groups by proc glm procedure using SAS 9.2 software (Alpha (α) = 0.05). The experiments were conducted with 3 groups in one time. Each treatment has 4 replicates with 3 sub replicate. The shoot regeneration per explant were observed after 8 weeks
x when p-value is <.0001 z when p-value is 0.0053 % is percentage of explants with shoots for each PGRs combination.
BA (µM) 2 10 15
Auxin (µM)
IBAx % % % 0.1 0 a 0 0 a 0 0.23 ± 0.17 a 16.7 0.5 0 a 0 0.17 ± 0.167 a 8.3 0 a 0 1.5 0 a 0 - - 3.0 ± 1.17 b 41.6 2.5 0 a 0 0 a 0 0.08 ± 0.083 a 16.7 3.0 0 a 0 0.41 ± 0.23 a 25 - -
2,4-Dz 0.2 0.58 ± 0.43 de 8.3 0.92 ± 0.38 cd 41.7 0.3 ± 0.26 de 8.3
0.45 0.83 ± 0.51 cd 41.6 2.8 ± 0.81 a 66.7 1.2 ± 0.68 bce 44.4 0.68 1.0 ± 0.28 cd 66.7 1.5 ± 0.72 ade 66.7 1.0 ± 0.28 cdf 66.7 0.9 0.83 ± 0.3 cd 66.7 2.6 ± 0.69 ab 75 0.83 ± 0.34 cd 41.7 1.2 0 de 0 2.2 ± 0.6 acf 66.7 0.83 ± 0.37 cd 41.7
59
Table 3-4. Adventitious shoot regeneration from leaves explant following treated with combinations of three different auxins types with TDZ on respective concentrations. The statistical analysis Pairwise Multiple Comparison by proc glm procedure using SAS 9.2 software (Alpha (α) = 0.05) were conducted within each auxin groups. The experiments were conducted with 3 groups in one time. Each treatment has 4 replicates with 3 sub replicate. Number of shoots per explant was observed after 8 weeks.
TDZ (µM) 2 4 10
Auxin (µM)
IBAx % % % 0.1 - - 0.4 ± 0.24 a 33.3 0 a 0 0.5 - - 0.3 ± 0.19 a 25 0 a 0 1.5 0.83 ± 0.67 a 16.7 0.7 ± 0.67 a 16.7 0 a 0 2.5 1.3 ± 0.98 a 33.3 0 a 0 0.3 ± 0.3 a 8.3 3.0 - - 4.7 ± 2.9 b 33.3 0.92 ± 0.83 a 16.7
IAAy 1.0 0 a 0 0 a 0 0 a 0 5.0 0 a 0 0 a 0 0 a 0 10 0 a 0 0 a 0 2.1 ± 0.82 b 66.7 15 0 a 0 0 a 0 3.3 ± 1.23 c 75 20 0 a 0 0.17 ± 0.11 a 25 0.4 ± 0.38 a 22.2
2,4-Dz 0.2 0.92 ± 0.43 cde 33.3 2.5 ± 0.71 abc 77.8 0.75 ± 0.41 de 25
0.45 0 e 0 2.6 ± 0.53 ac 91.6 1.2 ± 0.44 bcde 41.7 0.68 1.7 ± 0.76 ad 44.4 0.92 ± 0.45 cde 16.7 1.2 ± 0.72 bcde 25 0.9 2.3 ± 0.73 ac 66.7 1.0 ± 0.37 cde 41.7 1.2 ± 0.51 bcde 50 1.2 0.85 ± 0.42 de 25 0.92 ± 0.36 cde 41.7 0.7 ± 0.47 de 22.2
x when p-value is 0.009 y when p-value <.0001 z when p-value is 0.006 % is the percentage of explants with shoots for each PGRs combination.
60
A B
C D
Figure 3-1. Regeneration of YW5AF7 leaf disks and petiole on 5AF7 medium and Hawaii-4 medium. A, B) 8 weeks old media with shoot regenerated on 5AF7 medium, C,D) 8 weeks old medium with shoot regenerated on Hawaii-4 medium. The letter P represent petiole explant and letter L represent leaves explant.
P
L L
L
L
L
L
P
L
L
L
L L
L P
61
Table 3-5. Adventitious shoot regeneration from YW5AF7 leaves explant following treated with 5 different combinations and concentrations of PGRs. Data were treated as RCBD and analyzed with Pairwise Multiple Comparison using t-test by proc glm procedure SAS 9.2 software (Alpha (α) = 0.05).
Auxin (µM) Cytokinin (µM) Number of shoots per explant x
Percentage of explants with shoots (%)
1.5 IBA 15 BA 4.59 ± 0.692a 69.44 15 IAA 10 TDZ 1.38 ± 0.309 c 48.15 0.2 2,4-D 4.0 TDZ 2.77 ± 0.546 bc 63.33 3.0 IBA 4.0 TDZ 4.14 ± 0.907 ab 48.15 0.45 2,4-D 10 BA 2.0 ± 0.381 c 56.48
x is when p-value is <.0001
A B
C D
Figure 3-2. Callus formation was observed about 5 days after transformation. A -B) Callus formation on leaf disk explant after 7 days of treatment, arrow pointed at the callus, C) Shoots formation after 8 weeks on Selection Medium II with10 µg/ml Kan, D) Shoots formation after 8 weeks on 5 µg/ml Kan with arrows pointed at the shoots. Arrows pointed at the shoots.
1 mm 1 mm
10 mm 10 mm
62
Table 3-6. Average number of shoots per explant on YW5AF7 leaf disks on 5AF7 medium and Hawaii-4 medium after 8 weeks of treatments. Data were treated as RCBD and analyzed with Pairwise Multiple Comparison by proc glm procedure using SAS 9.2 software (Alpha (α) = 0.05).
Media Number of shoots per explantx
Percentage of explants with shoots (%)
5AF7 medium 5.07 ± 1.272 a 39.5 Hawaii-4 medium 4.10 ± 0.696 a 54
x is when the Least Significant Difference= 2.2035
Table 3-7. Average shoot number per explant for six F. vesca accessions on 5AF7 medium. Data were treated as RCBD and analyzed with Pairwise Multiple Comparison by proc glm procedure using SAS 9.2 software (Alpha (α) = 0.05).
Cultivars Number of shoots per explant x Percentage of explants with shoots (%)
Rodluvan 0.31 ± 0.221 b 5.5 Mignonette 4.4 ± 1.77 a 16.7 Baron Solemacher 0.8 ± 0.226 b 27.8 Alexandria 5.0 ± 1.97 a 22.2 Reugen 0.5 ± 0.31 b 8.3 Fragole di Bosco 2.2 ± 0.723 ab 36.1
x is when p-value is 0.0002
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CHAPTER 4 MUTAGENESIS WITH Tnt1 RETROTRANSPOSON
4.1 Introduction
Science witnessed the great discovery of transposon element, (TEs) in early 50's
in Zea mays (McCllintock, 1951). This stimulated many interests around the world in the
plant and animal fields, including how TEs could drive evolution and affect genome
expansion. One type of TEs, called retrotransposons have unique features that make
them unique compared to other insertion elements (Mazier et al., 2007). These TEs can
mobilize throughout the genome and replicate to a high copy number. These TEs
possess active long terminal repeat (LTR) regions. Retrotransposons such as Tnt1
(Melayah et al., 2004; Le et al., 2007) could be possibly teamed with a high efficiency
transgenic system like 5AF7 and be used as a valuable mutagenesis tool. The
experimental trails in this chapter test the possibility of using a specific class of
retrotransposon for mutagenesis in strawberry.
The Tnt1 retrotransposon is an active 5.334-kb-long copia-like LTR retroelement
that was isolated from tobacco (Nicotina tobacum) (Figure 4-1) (Grandbastien et al.,
1989). The Tnt1 retrotransposon has been successfully introduced to Arabidopsis
thaliana (Lucas et al., 1995), Medicago truncatula (Ratet et al., 2006) and lettuce
(Lactuca sativa), (Mazier et al., 2007). The mutation induced by Tnt1 retrotransposon
are stable because the element transposes via a replicative mode (Grandbastien et al.,
1989; Vernhettes et al., 1998; Mazier et al., 2007), leaving potentially disruptive inserts
in the genome while mobilizing to new locations. Mazier et al. (2007) demonstrated the
Tnt1 insertions in lettuce are genetically independent and transcriptionally active even in
64
the early stages of plant regeneration. Like any other retrotransposons, Tnt1 elements
are able to transpose into gene-rich regions (Mazier et al., 2007).
There are three different subfamilies of Tnt1 elements; Tnt1A, Tnt1B, and Tnt1C.
All share a conserved ability to transpose in different species (Vernhettes et al., 1998).
Moreover, U3 regions of the Tnt1 subfamilies are completely different among them and
have independently evolved in the different genomes, leading to differential regulation of
retrotransposon (Vernhettes et al., 1998). Other reports demonstrated that the Tnt1
retrotransposon is linked to a plant’s response to external stress (Grandbastien et al.,
2005), and is also involved in plant defense mechanisms during pathogen attack
(Grandbastien et al., 1997).
Extensive studies of gene function in the Rosaceae family are limited due to the
fact that most rosaceous plants are physically large and are long generation tree crops.
Their large size and long juvenility imposes substantial space and time commitments,
consistent with most woody plants (Slovin et al., 2009). Diploid strawberry could be an
excellent system to perform tests of gene function. Like Arabidopsis, F. vesca is small,
has as small genome, is fully sequenced (Shulaev et al., 2010) and can be transformed.
The plant has great potential as a model to test forward and reverse genetics for the
Rosaceae family (Slovin et al., 2009).
The objective of this chapter is to test if the Tnt1 retrotransposon may be useful in
strawberry. The pilot experiments attempt to install the Tnt1retrotransposon into the F.
vesca YW5AF7 genome. Accomplishing this end provides a means to later test
transposition and use as a mutagenesis tool.
65
4.2 Materials and Methods
4.2.1 Plant Material
Transformation and regeneration was accomplished as indicated in Chapter 3.
The Tnt1 retrotransposon was introduced to plant material using Agrobacterium-
mediated transformation on leaves and petioles. Four plants showed strong resistance
to kanamycin and were plant grown in MS mineral salts in sterile Magenta boxes with
2% sucrose. The plants were labeled as plant A, B, C and D.
4.2.2 Polymerase Chain Reaction (PCR) Analysis
Plant DNA extraction from leaf tissue was performed using DNAeasyR Plant Mini
Kit (Qiagen, 2006) following the manufacturer's instructions on four putative transgenic
plant. The presence of the Tnt1 retrotransposon in YW5AF7 regenerating lines was
determined using PCR analysis. Primers for Tnt1 retrotransposon were ordered through
Integrated DNA Technologies (IDT; Coralville, IA).
The Tnt1 primer sequences used were termed forward; 5'-ACC ATC CTG CAC
GGT AAG AC-3' (Tm= 57.1 ºC) and reverse; 5'-TAC ATG CCG CAC ATC CTT TA-3'
(Tm= 54.3 ºC). A single copy gene encoding F-box protein was used as a positive
control for PCR analysis. The F-box primers are 5'-AGA AGT GGC ACC ATC CGA
CGA TTT C-3' and 5'-GGC GTG ACC GCA TGA AGT AAA GTG-3'. Approximately 50
ng of template DNA was mixed with the PCR mixture (Table 4-1). The PCR cycling
conditions consisted of an initial denaturation step at 94 ºC for 1 min, followed by 40
cycles at 92ºC for 30 s, 52ºC for 30 s and 72ºC for 30 s, and the final elongation step at
72ºC for 5 min.
66
4.2.3 Electrophoresis
PCR products were resolved using agarose electrophoresis. Tris-acetate-EDTA
(TAE) buffer is used as both a running buffer and in agarose gel. 1% agarose mixture
(0.5 g of DNA agarose + 0.5 x TAE buffer) was heated in microwave until the agarose
fully dissolved in the TAE buffer solution. After that, the agarose mixture was allowed to
cool, decanted into a gel forming tray and allowed to solidify. Samples were loaded onto
the gel using a glycerol-based loading dye (Sambrook et al., 1993).
4.3 Results
At first, it was thought PCR result with Tnt1 primers gave us a promising result on
Tnt1 insertion is successfully transformed in plant A, B and D, but not plant C.
Amplification of a fragment about 550 bp which is close with expected result is observed
(Figure 4.1). This result was the first try on Tnt1 primers with the plant DNA extractions.
However, results for strawberry (kanamycin) primers on the DNA extraction gave
negative result without any fragment amplified. A single-copy gene encoding an F-box
protein was used as positive control. The experiment lacked a negative control (Figure
4-2), leaving a question whether amplified fragments by Tnt1 primers are correct. Two
new sets of PCR reactions were conducted with Tnt1 plasmid as positive control and
one negative (no template) control. Another set of PCR analyses with Tnt1 primers
using the same DNA plant extraction showed negative results for all DNA extracted
(Figure 4-3). Another set of PCR reactions was conducted and generated the same
negative result (data not shown). There was no fragment amplified by Tnt1 primers on
DNA extractions from plant; Lane 2, 3, 4 and 5. The negative control; Lane 7 also
showed no band which indicates no contamination of other DNA occurred during
preparation of PCR mixture (Figure 4-2).
67
The positive control at lane 6 showed a band size about 550 bp, similar size with
Tnt1 retrotransposon (Figure 4-1). The length of the fragment approximately 550 bp that
was similar with expected size. This demonstrated that the primers and PCR reaction
are correct and working, but no target was detected in the putatively transgenic tissues.
An exclusive positive control PCR using F-box primers was conducted to
determine whether the templates extracted from four kanamycin resistant plants and the
YW5AF7 control were reliable. The F-box gene-specific primers amplified a product
successfully in all DNA preparations, including the kanamycin resistant plants and
YW5AF7 by amplifying a fragment length approximately 420 bp (Figure 4-4). This
meant that the DNA extractions were successful and the template concentrations were
sufficient for PCR amplification.
4.4 Discussion
4.4.1 Understanding Tnt1 Retrotransposons
Retrotransposons are the largest class of transposable elements (TEs). Long
terminal repeat (LTR) Tnt1 retroransposons are reported to produce stable insertions
when transformed to Arabidopsis thaliana, lettuce and Mediago truncatula (Lucas et al.,
1995; Ratet et al., 2006; Mazier et al., 2007). So far, there are no reports of Tnt1
transformation in the genus Fragaria. Grandbastien et al. (1989) isolated a Tnt1
retrotransposon from subfamily Tnt1A, from root in low copy numbers that are not found
in healthy parts of the tobacco plant. Tnt1 subfamily members, Tnt1A, Tnt1B and Tnt1C
have exclusive U3 regions that regulate Tnt1 expression in host genomes, and can
evolve independently within respective plant genomes (Vernhettes et al., 1998). Plus,
the expression of Tnt1 retrotransposons in tobacco plants showed that the RNA is not a
68
unique sequence but a population of different but closely related sequences
(Casacuberta et al., 1995).
4.4.2 Strawberry Retrotransposons
Large genomes of an octoploid strawberry, F. × ananassa show exclusive
retrotransposons, Ty1-copia and Ty3-gypsy groups are present in high copy numbers
(Ma et al., 2008; Pontaroli et al., 2009). The LTR retrotransposons are normally silenced
until a certain threshold number is reached or it is released due to environmental or
physiological circumstances (Perez-Hormaeche et al., 2008). In strawberries, FaRE1,
with size about 5.1kb was recently isolated is an active-copy of Ty1-copia in cultivated
strawberry (He et al., 2009). This is possible, as there are chances of a silenced
retrotransposon to be active due to external factors such as biotic or abiotic stress (Mhiri
et al., 1997; Grandbastien et al., 2005; Ma et al., 2008). In future experiments the use of
a strawberry retrotransposon may be preferred over the use of Tnt1, especially if Tnt1
has some unknown deleterious effect in strawberry tissues that would limit regeneration
of transgenic tissues.
4.4.3 Experiment Observations
Positive results in generating the Tnt1 fragment of approximately 550 bp are
shown on three plants but only one time. However, there is no amplification of the
control kanamycin marker observed (Figure 4-2). The Tnt1 fragments were absent
when another two independent sets of PCR reaction conducted with the same DNA
extractions, under same PCR program and PCR thermalcycler (data not shown). The
lack of reproducibility suggests that this was contamination or possibly residual A.
tumifaciens on the transgenic plant.
69
These conclusions were supported by the amplification of the strawberry F-box
gene fragment of about 420 bp (Figure 4-4). This result meant the strawberry DNA
extractions were of sufficient quality and quality for PCR amplification.
4.4.4 Future Suggestions
The experiments should be repeated after culture conditions have been more
thoroughly tested, mostly with kan selection relative to YW5AF7. This is important
because Fragaria is very sensitive to kanamycin, as reported in LF9 lines (Folta et al.,
2006). The optimum kanamycin concentration for YW5AF7 need to be determined in
future experiments, as the recommended concentrations for F. vesca regeneration vary
greatly (Folta and Dhingra, 2006), but generally are around 25 mg/L (El-Mansouri et al.,
1996; Oosumi et al., 2006). It is possible that selection could be optimized for this
accession and would lead to better results.
Carbenicillin and timentin with respective concentrations in the selection media
must be included in every selection media formulation as it is necessary to suppress the
growth of A. tumifaciens in co-cultivated tissues. It is also possible that there is
sensitivity to these compounds unique to YW5AF7. While not likely, it is easily tested,
and could also be a reason why no transfomants were generated. Transformation of
Tnt1 insertions should also be tested with F. vesca genotype Hawaii-4. Even tough
retrotransposons are abundant in plants, the sequence of strawberry and tobacco
retrotransposons can be differentiated within specific primers, constructed based on
each species.
The ability of Tnt1 retrotransposons to transpose upon tissue injury and generate
many potential insertion events is one of the reason it is an excellent mutagenesis tool.
The recently isolated active strawberry LRT retrotransposon, FaRE1, is another
70
potential candidate as a mutagenesis tool within the genus Fragaria. However, limited
information is known about this newly isolated retrotransposon, its mobility, copy
number and sequence variation among strawberry species and cultivars.
More molecular analyses should be considered instead of PCR. Southern blot
hybridization using the entire T-DNA region could be used to test the possibility that
DNA insertions were present, but then were lost. At this time the limited transgenic plant
material postpones this option.
Mutagenesis using the Tnt1 retrotransposon is an excellent potential way to
generate forward and reverse-genetic tools in strawberry. The results of this trail
indicate that there is no evidence of Tnt1 insertion in strawberry via Agrobacterium
mediated transformation. It is possible that this was due to small amounts of starting
material or some unknown effect of the Tnt1 retrotransposon in strawberry that limits
regeneration and/or transformation of strawberry tissues.
71
Figure 4-1. A schematic representation of the tnk23 T-DNA region showing Hinc II restriction enzyme sites, RB, Right border; LB, Left border redrawn from Mazier et al. (2007).
Table 4-1. PCR mixture for 1X reaction. PCR mixture component 1X experimental
tube (µl) 1X Positive
control tube (µl) 1X negative control (µl)
10 x buffer MgSO4 5 5 5 2.5 mM dNTP 1 1 1 Taq DNA polymerase 0.5 0.5 0.5 1 nM/ml Forward primer 1 1 1 1 nM/ml Reverse primer 1 1 1 Nanopure filtered water 39.5 41 40.5 Template (50-100ng) Plant DNA extract 2 - - Tnt1 plasmid - 0.5 - Total volume 50 50 50
RB LB LTR Tnt1 LTR nptII
Hinc II Hinc II Hinc II
Hinc II Hinc II Hinc II
72
Figure 4-2. Agarose gel size fractionation of PCR amplification products from DNA extracts isolated from kanamycin resistant plants. Lanes 1 to 9 represent the 2-Log DNA ladder, PCR products produced by Tnt1 primers for plants A, B, C, and D and the PCR products produced with the kanamycin primers for plants A, B, C, and D, respectively.
1 2 3 4 5 6 7 8 9
~550 bp
73
Figure 4-3. Four DNA extracts of kanamycin resistant plant with Tnt1 primers. Lanes; 1) 2-Log DNA ladder, 2) plant A, 3) plant B, 4) plant C, 5) plant D, 6) Tnt1 plasmid (positive control), 7) negative control (no DNA template).
1 2 3 4 5 6 7
~550 bp
74
Figure 4-4. Four DNA extracts of kanamycin resistant plant with F-box protein primers. Lanes; 1) 2-Log DNA ladder, 2) plant A, 3) plant B, 4) plant C, 5) plant D, 6) YW5AF7 plant (positive control), 7) negative control (no template).
1 2 3 4 5 6 7
~430 bp
75
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BIOGRAPHICAL SKETCH
Mohamad Fadhli Mad' Atari was born in Perak, Malaysia. He graduated from
Universiti Sains Malaysia with a Bachelor of Science degree, majoring in applied
science (biotechnology). He then joined Synergy group, and after two months, he
became one of the pioneer for the new subsidiary company, World Lab Tissue Culture
and Research Centre. This company's focus is on production of Cavendish banana
tissue culture propagation to meet local and South Asia demand on the seedlings and
plantlets. About one year after that, he was offered by Universiti Sains Malaysia for
Academic Staff Training Scheme (ASTS), and then pursuing his master's degree in
strawberry tissue culture and transformation in University of Florida, Gainesville.
