protoplast fusion of lolium perenne and lotus corniculatus for gene
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Acknowledgments
My sincerest gratitude to my supervisors, Dr Mary Christey and Professor Tony Conner
whose guidance, constant encouragement, and support on all frontiers has made this project
a learning curve in my career. I am greatly priviledged to have worked under your guidance.
I thank my associate-supervisor Dr Hayley Ridgway for supporting and encouraging me at
crucial junctures during this project.
I would like to thank Pastoral Genomics, Auckland, New Zealand, and New Zealand
Institute for Crop & Food Research Ltd., New Zealand for financially supporting me. I am
thankful to Foundation for Research Science and Technology, New Zealand for awarding
me the Enterprise Scholarship. Special thanks to Pastoral Genomics, for extending the
financial support for an extra year, without which it would have been very difficult to
complete this project.
I would like to thank Robert H. Braun for his immense support in the laboratory, for having
long discussions and giving me tips which have contributed greatly to the completion of this
work. THANK YOU Robert.
Thanks to Ross Bicknell, Sylvia Erasmuson, and Andrew Catanach for helping me with the
AFLPs and flow cytometry. My grateful appreciation to Ruth Butler for helping me with the
statistical softwares. Thank you Ruth.
My sincere thanks to the Graphics team at Crop & Food, Robert Lamberts for taking
attractive pictures of my specimens, Astrid Erasmuson & Carolyn Barry for helping design
posters at various stages of this project. Thanks also to Sam Wakelin for designing the
graphic pictures and posters.
I would like to thank all members of the Plant Genetic Technologies Team at Crop & Food,
past and present. A big thanks to Annemarie Lokerse who has constantly supported me.
Thanks to Dianne Hall for watering my plants in the glass house.
My thanks to all my past teachers whose teachings have made me qualify for this project.
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Many thanks to my wife, Deepa, who has stood alongside me in the ups and downs of this
project. I would also like to thank my family members who have encouraged me all
throughout my career.
Lastly but most importantly, I would like to thank my parents who have constantly
supported me and guided me in my endeavours.
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Appendix 1
Species Description
a. Lolium perenne L. (Perennial ryegrass)
Lolium perenne (see inset) is a grass belonging to the graminae family Poaceae. It has a diploid
chromosome number of 2n=2x=14.
Leaves of perennial ryegrass are folded in the bud (in contrast to annual ryegrass (Lolium
multiflorum L.) in which the leaves are rolled). Leaf blades are 2 to 6 mm wide, 5 to 15 cm
long, sharply tapered, and keeled. Blades are bright green, prominently ridged on the adaxial
(upper) surface, and smooth, glossy, and glabrous (hairless) on the abaxial (lower) surface.
Leaf margins are slightly scabrous (rough to the touch, covered with minute serrations).
Blades increase in size from the first to the seventh leaf on a tiller, although tillers rarely have
more than three live leaves at one time (Balasko et al., 1995). Leaf sheaths usually are not
keeled, compressed but sometimes almost cylindrical, glabrous, pale green, and reddish at
the base. Sheaths may be closed or split. The collar is narrow, distinct, glabrous, and
yellowish to whitish-green. Auricles are small, soft, and
claw-like. The ligule is a thin membrane, from 0.5 to 2.5
mm, obtuse (rounded at the apex), and may be toothed
near the apex.
The shallow root system is highly branched and
produces adventitious roots from the basal nodes of
tillers. Perennial ryegrass has no rhizomes or stolons. It
will, however, produce a dense and closely knit sward or
turf with high plant densities and proper management
(Spedding & Diekmahns, 1972).
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b. Lotus corniculatus L. (Bird’s foot trefoil).
Lotus corniculatus (see inset) which belongs to the family Fabaceae, is a tetraploid with
2n=4x=24. It is a glabrous to sparsely pubescent perennial plant, though not a long-lived
perennial with a life span of 2-4 years. Growth form ranges from prostrate to erect with
numerous stems arising from a basal, well developed crown and branches arising from leaf
axils. Primary growth comes from the crown, but regrowth comes from buds formed at
nodes on the stubble left after defoliation. Leaves are pentafoliate, alternately on short stalks
with the two leaflets at the petiole base resembling stipules. The asymmetrical pointed
leaflets are mainly glabrous and are more slender and pale green.
Inflorescences with up to eight flowers are umbel-
like cymes at the end of long axillary branches.
The calyx is a dentate tube and the yellow five-
petalled corolla is often tinged red. The flowering
period is indeterminate and so seed is set over an
extended period in summer. The self-sterile plants
are cross-pollinated, mainly by honey bees. Seed
pods, 2-5 cm long, contain 15-20 seeds attached
to the ventral suture (Refer Fig 4.1); the seeds are
released by a sudden split of the pod along both
sutures after one to two weeks of ripening during
which the pods change from green to brown. The seeds vary from round to oval in
shape and from greenish yellow to dark brown in colour. The plant root system consists
of a deep taproot with numerous secondary roots that have a good lateral spread. Some
Moroccan genotypes produce rhizomes that arise from axillary buds on stem bases
(Skerman et al., 1988).
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Appendix 2: Flow cytometric analysis
Profiles generated from the flow cytometric analysis of the fusion parents (L. perenne and L.
corniculatus) and the 14 putative fusion colonies obtained. The fusion colony profiles all
resembled the L. corniculatus profile.
Lotus corniculatus
Lolium perenne cv Bronsyn
cell suspensions
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FC2 callus
L. corniculatus callus
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FC4 callus
FC3 callus
160
FC5 callus
FC6 callus
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FC7 callus
FC8 callus
162
FC9 callus
FC10 callus
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FC11 callus
FC12 callus
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FC13 callus
FC14 callus
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Appendix 3 Polyacrylamlide Gel Preparation
1.1. Thoroughly clean the small and the large glass plates with Jiff, followed by generous
acetone, then 95% ethanol and facial tissue. 1.2. In a chemical fume hood, prepare fresh binding solution by adding 6ul of Bind
Silane (methacryloxypropyl-trimethoxy-silane, Sigma M-6514, store at 4oC) to 2 ml of 0.5% acetic acid in 95% ethanol in a 15 ml Falcon tube. Wipe large glass plate using facial tissue saturated with the freshly prepared binding solution. Be certain to wipe the entire plate surface with the saturated tissue.
1.3. Wait 5 minutes for the binding solution to dry. Wipe the glass plate with 95%
ethanol and facial tissue to remove the excess binding solution. Note: Failure to wipe excess binding solution from the glass plate will cause the gel to stick to both plates, and the gel will be destroyed upon separation of the glass plates after electrophoresis. 1.4. Apply 3 cm splodge of Sigmacote® (Chlorinated organopolysiloxane SIGMA) onto
one side of the other glass plate. With a dry facial tissue tissue, spread the Sigmacote over the entire surface of the plate.
1.5. Wait 5 minutes for the Sigmacote to dry. Remove the excess Sigmacote with ethanol
and facial tissue. 1.6. Place 0.4mm spacers on the large glass plate. Put the small glass plate on the large
one. Assemble the glass plates by using clamps to hold them in place (2 clamps on each side). Place the gasket, bottom corner first, removing clamps when required.
Note: Take special care to prevent the treated surfaces from touching each other. 1.7. Pour 6% acrylamide solution (60ml for one gel) into a beaker. For each 60 ml
solution, add, 60ul of TEMED (N,N,N’,N’-Tetraethyl-ethylenediamine) and 600ul of 10% APS (ammonium persulfate) to the acrylamide solution, and mix gently.
1.8. Suck the acrylamide into a syringe. Take care to remove bubbles. Carefully inject the
acrylamide solution into the space in the gasket. 1.9. Insert a multi-well sharks-tooth comb, straight side into the gel, between the glass
plates (approximately 6-8 mm of the comb should be between the two glass plates). Secure the comb with four clamps.
1. 10. Allow polymerisation to proceed for at least 1 hour to overnight. Note: The gel may be stored overnight at room temperature if a paper towel saturated with dH20 and plastic wrap are placed around the well end of the gel to prevent the gel from
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drying out. As an alternative, the gel can be stored overnight by keeping in the electrophoresis apparatus with 1xTBE buffer in the bottom and top chamber of the apparatus (see steps 2.1 and 2.5). 2. Polyacrylamide Gel Electrophoresis For more efficient use of gel space, one may load one gel multiple times using samples from either the same or different loci. When loading samples from different loci, load the samples in order of smallest to largest size ranges, or load the mixed solution of two samples with equal quantity. 2.1. Remove the clamps from the polymerised acrylamide gel assembly and clean the
outside of the glass plates with paper towel saturated with dH20. 2.2. Shave any excess polyacrylamide away from the comb. Remove the comb, and shave
and wash any excess polyacrylamide from the top of the glass plates. Wash surfaces of the gel setup.
2.3. Gently lower the gel (glass plates) into the lower slot with the large plate facing out
and the well-side on top. Secure the glass plates, increasing the pressure a little at a time.
2.4. Ensure the tap between chambers of the electrophoresis apparatus is closed. Add
1xTBE (1 litre) to the top of the electrophoresis apparatus until the small plate is well covered. Pour the rest of the TBE into the bottom tank.
2.5. Using a disposable Pasteur pipette, remove the air bubbles and unpolymerized
acrylamide on the top of the gel. Be certain the well area is devoid of air bubbles, small pieces of polyacrylamide and urea.
2.6. Pre-run the gel to achieve a gel surface temperature of approximately 45-50°C. Set
the power constant at 70W and the voltage should be between 1400 and 1800V. Any higher and the plates may be getting too hot. Pre-run for about half an hour to an hour.
2.7. Prepare PCR samples by mixing 8ul of each sample with 5ul of STR 3x-loading
solution, or add 10ul STR to the whole solution.
2.8. Prepare 500ng molecular weight marker (Marker DNA + 3x STR) for each marker
lane. 2.9. Denature the samples and markers by heating at 95°C for 1.5-2 minutes and
immediately chill on ice.
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2.10. After the pre-run, use a disposable Pasteur pipette to flush the urea from the well
area and carefully insert the sharks-tooth comb teeth into the gel approximately 1-2mm.
2.11. Load 6ul of each sample and marker into the respective wells. To prevent the gel
from cooling, gel loading should not exceed 20 minutes. 2.12. Run the gel using the same conditions as the pre-run step (2.6), until Xylene cyanol is
about half way (1.5 to 2 hours). *In a 4% gel, Bromophenol blue migrates at approximately 40 bases, and Xylene Cyanol migrates at approximately 170 bases. 3. Silver Staining 3.1. After electrophoresis, empty the buffer chamber and carefully loosen the gel clamps.
Remove the glass plates from the apparatus. 3.2. Place the gel (glass plates) on a flat surface. Remove the comb and the side spacers.
Use a plastic wedge to carefully separate the two glass plates. The gel should be strongly affixed to the large glass plate.
3.3. Place the gel (attached to the small plate) in a shallow plastic tray. 3.4. For silver staining, follow the steps listed below:
Step Solution Time a. Fix/stop solution 30 minutes b. dH20 2 minutes c. Repeat step b. twice 2x2 minutes d. Staining solution 30 minutes e. dH20 10 seconds f. Developer solution (4-10°C) 2-5 minutes g. Fix/stop solution 5-6 minutes h. dH20 2-3 minutes
Note: All steps should be conducted on a shaker and the plates should be well covered by the solutions. 3.5. Position the gel upright and allow it to dry overnight.
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Composition of solutions 4% acrylamide solution (1000 ml) 420 g urea, 450 dH2O 100 ml 10x TBE 100 ml 40 % acrylamide/bis-acrylamide (19:1) 6% acrylamide solution (500mls) 75mls 40% acrylamide:bis 230g urea (=>7.67M) 50mls 10X TBE
Add acrylamide with some water and add half the urea, stir. Add rest of urea. Incubate at 37 dgrees to aid disolving in a shaking water-bath. Add the TBE and make up to volume.
0.5% acetic-acid in 95% ethanol
Add l ml of glacial acetic acid to 199 of 95% ethanol 10% APS (ammonium persulfate)
Add 1.0g of ammonium persulfate to 10 ml of dH2O Developer solution
sodium carbonate 90g 120gm 37% formaldehyde (H2CO) 4.5ml 6ml 10mg/ml sodium thiosulfate (Na2S20.5HO) 600 µl 800µl dH2O To 3L To 4l
Note: Prepare the solution and chill to 10'C before use. Prepare fresh before each use. The sodium carbonate can be dissolved and chilled overnight in the cold room. Add the formaldehyde and sodium thiosulfate before use. Staining solution:
2 g silver nitrate (AgNO3) 3g 4g 3 ml 37% formaldehyde (H2CO) 4.5 6ml 2000 ml dH2O To 3L To 4 L
Fix/stop solution (10% acetic acid) 200 ml glacial acetic acid 300ml 400ml 1800 ml dH20 2.7L 3.6 L
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Appendix 4 Publications and presentations
Publications
Raikar S. V., R. H. Braun, C. Bryant, T. Conner & M. C. Christey (2007). Efficient isolation,
culture and regeneration of Lotus corniculatus protoplasts. Plant Biotechnology Reports
(under review).
Raikar S.V., Bryant C., Braun R., Conner A.J. & Christey M.C. (2007). Whole genome
amplification from plant cell colonies of somatic hybrids using strand displacement
amplification. Plant Biotechnology Reports,1, 175-177.
Raikar S.V., M.C. Christey, A.J. Conner, R. Braun, & C. Bryant. (2006). Protoplast isolation,
colony formation and shoot regeneration from Lolium perenne, p. 41-44. In C.F. Mercer
(ed.), Advances in pasture plant breeding: Papers from the 13th Australasian Plant Breeding
Conference. Grassland Research and Practice Series No. 12. New Zealand Grassland
Association, Dunedin, New Zealand.
Oral Presentations
Raikar S. V., R. H. Braun, C. Bryant, T. Conner & M. C. Christey (2007). Asymmetric
somatic hybridisation between Lolium perenne (Monocot) and Lotus corniculatus (Dicot).
New Zealand Institute for Agricultural and Horticultural Science Convention, Lincoln,
New Zealand. 13-15th August 2007.
Raikar S. V., R. H. Braun, C. Bryant, T. Conner & M. C. Christey (2007). Asymmetric
somatic hybridization between Lolium perenne (Monocot) and Lotus corniculatus (Dicot)
17th Biennial meeting The New Zealand Branch IAPTC&B Rotorua, New
Zealand. February, 2007.
Raikar S. V., M. C Christey, T. Conner, & R. Braun (2005). Protoplast isolation and colony
formation from Lolium perenne 16th Biennial meeting The New Zealand
Branch IAPTC&B Methven, New Zealand. February, 2005.
170
Poster Presentations
Raikar S. V., R. H. Braun, C. Bryant, T. Conner & M. C. Christey (2007). Whole genome
amplification of small protoplast fusion colonies using strand displacement
amplification. 17th Biennial meeting The New Zealand Branch IAPTC&B
Rotorua, New Zealand. February, 2007.
Raikar S. V., M. C Christey, C. Bryant, T. Conner, & R. Braun (2006). Genetic Variation in
Lolium perenne for callus induction and protoplast isolation. 11th International Congress
for Plant Tissue Culture and Biotechnology Beijing, China.
Raikar S. V., M. C Christey, C. Bryant, T. Conner, & R. Braun (2006). Protoplast isolation,
colony formation and shoot regeneration from Lolium perenne L. 13th Australasian Plant
Breeding Conference, Christchurch New Zealand.