References
Akada S. & Hirai A. (1986). Studies on the mode of separation of chloroplast genomes in
parasexual hybrid calli. III. Random separation of two types of chloroplast genomes
in a hybrid callus. Japanese Journal of Genetics, 61, 437-445.
Akashi R., Hashimoto A. & Adachi T. (1993). Plant regeneration from seed-derived
embryogenic callus and cell suspension cultures of bahiagrass (Paspalum notatum).
Plant Science, 90, 30-80.
Akashi R., Harris S., Tsay-Hoffmann S.S. & Hoffmann F. (2000). Plants from protoplasts
isolated from long-term root culture (Super Root) of Lotus corniculatus. Journal of Plant
Physiology, 157, 215-221.
Ahuja P.S., Hadiuzzaman S., Davey M.R. & Cocking E.C. (1983). Prolific plant
regeneration from protoplast-derived
tissues of Lotus corniculatus L. (birdsfoot
trefoil). Plant Cell Reports, 2, 101-104.
Aljanabi S.M., Forget L. & Dookun A. (1999). An improved and rapid protocol for the
isolation of polysaccharide and polyphenol-free sugarcane DNA. Plant Molecular
Biology Reporter 17, 281.
Altpeter F. & Posselt U.K. (2000). Improved plant regeneration from cell suspensions of
commercial cultivars, breeding and inbred lines of perennial ryegrass (Lolium perenne
L.). Journal of Plant Physiology, 156, 790-796.
Ankele E., Heberle-Bors E., Pfosser M.F. & Hofinger B.J. (2005). Searching for
mechanisms leading to albino plant formation in cereals. Acta Physiologiae Plantarum,
27, 651-664.
Arcioni S., Bellucci M., Damiani F., Pupilli F. ((2000). Gene transfer for alfalfa
improvement.
Retrieved
on
August
22
2007
from
http://www.naaic.org/TAG/TAGpapers/arcioni/arcioni.html
Arumuganathan K, Earle ED. (1991). Estimation of Nuclear DNA content of plants by
flow cytometry. Plant Molecular & Biology Reports, 9, 229-233.
126
Asano Y. & Sugiura K. (1990). Plant regeneration from suspension culture-derived
protoplasts of Agrostis alba L. (redtop). Plant Science, 72, 267-273.
Asano Y., Ito Y., Sugiura K. & Fujiie A. (1994). Improved protoplast culture of bentgrass
(Agrostis L.) using a medium with increased agarose concentration. Journal of Plant
Physiology, 143, 122-124.
Assani A., Haicour R., Wenzel G., Foroughi-Wehr B., Bakry F. & Cote F.X. (2002).
Influence of donor material and genotype on protoplast regeneration in banana and
plantain cultivars (Musa spp.) Plant Science, 162, 355-362.
Aziz M.A., Chand P.K., Power J.B. & Davey M.R. (1990). Somatic hybrids between the
forage legumes Lotus corniculatus L. and L. tenuis Waldst et kit. Journal of Experimental
Botany, 41, 471-479.
Balasko J.A., Evers G.W., Duell R.W. (1995). Bluegrasses, ryegrasses, and bentgrasses. p.
357-372. In: R.F. Barnes, DA Miller, and CJ Nelson. (eds.), Forages. Vol. 1. An
Introduction to Grassland Agriculture, 5th ed. Iowa State Univ. Press. Ames, IA.
Barone A., Li J., Sebastiano A., Cardi T. & Frusciante L. (2002). Evidence for tetrasomic
inheritance in a tetraploid Solanum commersonii (+) S. tuberosum somatic hybrid
through the use of molecular markers. Theoretical andApplied Genetics, 104, 539-546.
Barry T.N. (1989). Condensed tannins: their role in ruminant protein and carbohydrate
digestion and possible effects upon the rumen ecosystem. In: Nolan JV, Leng RA
and Demeyer DI (eds.), The roles of protozoa and fungi in ruminant digestion, Perumbal
Books Armidale Australia. Pp. 153-169.
Bates G.W. (1985). Electrical fusion for optimal formation of protoplast heterokaryons in
Nicotiana. Planta, 165, 217-224.
Bates G.W., Hasenkampf C.A., Contolini C.L., & Piastuch W.C. (1987). Asymmetric
hybridization in Nicotiana by fusion of irradiated protoplasts. Theoretical and Applied
Genetics, 74, 718–726.
127
Bauer-Weston B., Keller W., Webb J. & Gleddie S. (1993). Production and
characterisation of asymmetric somatic hybrids between Arabidopsis thaliana and
Brassica napus. Theoretical and Applied Genetics, 86, 150-158.
Begum F., Paul S., Bag N., Sikdar S.R. & Sen S.K. (1995). Somatic hybrids between
Brassica juncea (L.) Czern. and Diplotaxis harra (Forsk.). Boiss and the generation of
backcross progenies. Theoretical and Applied Genetics, 91, 1167-1172.
Belgrave B.R., Watt P.C., Brock J.L., Wewala S., & Sedcole J.R. (1990). A survey of
farmer knowledge and use of pasture cultivars in New Zealand. New Zealand Journal
of Agricultural Research, 33, 199-211.
Bhaskaran S. & Smith R.H. (1990). Regeneration in cereal tissue culture: A Review. Crop
Science, 30, 1328-1336.
Bidlack J. E. (1990). Cell wall components and lignin Biosynthesis in Forages. Ph.D.
Dissertation, Iowa State Univ., Ames, IA
Blanco L., Bernad A., Lazaro J.M., Martin G., Garmendia C., Salas M. (1989). Highly
efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode
of DNA replication. Journal of Biological Chemistry, 264, 8935-8940.
Boss W.F. & Mott R.L. (1980). Effects of divalent cations and polyethylene glycol on the
membrane fluidity of protoplasts. Plant Physiology, 66, 835-837.
Bottley A., Xia G.M. & Koebner R.M.D. (2006). Homoeologous gene silencing in
hexaploid wheat. The Plant Journal, 47, 897-906.
Bradley D.E., Bruneau A.H. & Qu R. (2001). Effects of cultivar, explant treatment, and
medium supplements on callus induction and plantlet regeneration in perennial
ryegrass. International Turfgrass Society Research Journal, 9, 152-156.
Brière C., Harthou H., Jauneau A., Alibert G. &
Petitprez M. (2004). The actin
cytoskeleton is involved in signalling protoplast embryogenesis induced by agarose
embedding. Physiologia Plantarum, 122, 115-122.
128
Broadhurst R.B., & Jones T.J. (1978). Analysis of condensed tannins using acidified
vanillin. Journal of Science Food & Agriculture, 29, 788-794.
Brüdern A. & Thiel G. (1999). Effect of cell-wall digesting enzymes on physiological state
and competence of maize coleoptile cells. Protoplasma, 209, 24-255.
Buiteveld J., van de Valk J., Creemers-Molenaar J. & Colijn-Hooymans C.M. (1993).
Callus induction and plant regeneration from explants of commercial cultivars of
leek (Allium ampeloprasum L.). Plant Cell Reports, 12, 431-434.
Burgon A., Bondesen O.B., Verburgt W.H., Hall A.G., Bark N.S., Robinson M., Tim G.
(1997). The forage seed trade. In: D T Farey & G J Hampton (Eds.), Forage seed
production. 1. Temperate species, pp. 271-286 Wallingford, UK: CAB International.
Cai Y., Xiang F., Zhi D., Liu H. & Xia G. (2007). Genotyping of somatic hybrids between
Festuca arundinacea Schreb. And Triticum aestivum L. Plant Cell Reports, DOI
10.1007/s00299-007-0397-5
Carlson P.S., Smith H.H. & Dearing R.D. (1972). Parasexual plant hybridisation.
Proceedings of National Academy of Sciences USA, 69, 2292-2294.
Chen W.P., Wu Q.S., Liu D.J. & Jones M.G.K. (1992). Callus formation from somatic
hybridisation of Wheat (Triticum Aestivum L.) and Ryegrass (Lolium perenne L.) by
electrofusion. Acta Botanica Sinica, 34(4), 284-290.
Cheng A.X. & Xia G.M. (2004). Somatic hybridisation between common wheat and
Italian ryegrass. Plant Science, 166, 1219-1226.
Chevrier N., Qureshi J.A. Huel P. & Kartha K.K. (1990). Heritability of in vitro
regeneration in wheat. Canadian Journal of Plant Science, 70, 547-550.
Christey M. (2004). Protoplast culture and Somatic Hybridization.. In E.C. Pua and C.J.
Douglas (Eds.), Biotechnology in Agriculture and Forestry, 54, (pp. 119-148) SpringerVerlag, Berlin, Heidelburg.
Christey M.C. Makaroff C.A. & Earle E.D. (1991). Atrazine-resistant cytoplasmic malesterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile
129
Brassica oleracea and atrazine-resistant Brassica campestris. Theoretical and Applied Genetics,
83, 201-208.
Cocking E.C. (1960). A method for isolation of plant protoplasts and vacuoles. Nature,
187, 962-963.
Cocking E.C. (2000). Turning point article plant protoplasts. In Vitro Cell Developmental
Biology-Plant, 36, 77-82.
Collard B.C.Y. & Mackill D.J. (2008). Marker-assisted selection: An approach for
precision plant breeding in the twenty-first century. Philosophical Transactions of The
Royal Society B, 363, 557-572.
Colomba E.L., Grunberg K., Griffa S., Ribotta A., Mroginski L. & Biderbost E. (2006).
Effect of genotype and culture medium on somatic embryogenesis and plant
regeneration from mature embryos of fourteen apomictic cultivars of buffel grass
(Cenchrus ciliaris L.). Grass and Forage Science, 61, 2-8.
Commun K., Mauro M.C, Chupeau Y., Boulay M., Burrus M., & Jeandet P. (2003).
Phytoalexin production in grapevine protoplasts during isolation and culture. Plant
Physiology and Biochemistry, 41, 317-323.
Conner, A.J., Glare, T.R., & Nap, J.P. (2003). The release of genetically modified crops
into the environment: II. Overview of ecological risk assessment. The Plant Journal,
33, 19-46.
Crea F., Calderini O., Nenz E., Cluster P.D., Damiani F. & Arcioni S. (1997).
Chromosomal and molecular rearrangements in somatic hybrids between tetraploid
Medicago sativa and diploid Medicago falcata. Theoretical and Applied Genetics, 95, 11121118.
Creemers-Molenaar J., Loeffen J.P.M. & Van der Valk P. (1988). The effect of 2,4Dichlorophenoxyacetic acid and donor plant environment on plant regeneration
from immature inflorescence-derived callus of Lolium perenne L. and Lolium
multiflorum L. Plant Science, 57, 165-172.
130
Creemers-Molenaar J., Van der Valk P., Loeffen P.M. & Zaal M.A.C.M. (1989). Plant
regeneration from suspension cultures and protoplasts of Lolium perenne L. Plant
Science, 63, 167-176.
Creemers-Molenaar J., Hall R.D. & Krens F.A. (1992a). Asymmetric protoplast fusion
aimed at intraspecific transfer of cytoplasmic male sterility (CMS) in Lolium perenne
L. Theoretical and Applied Genetics, 84, 763-770.
Creemers-Molenaar J., van Eeuwijk F.A. & Krens F.A. (1992b). Culture optimization for
perennial ryegrass protoplasts. Journal of Plant Physiology, 139, 303-308.
Cutler A.J., Saleem M., Coffey M.A. & Loewen M.K. (1989). Role of oxidative stress in
cereal protoplast recalcitrance. Plant Cell Tissue and Organ Culture, 18, 113-127.
Dalton S.J. (1988a). Plant regeneration from cell suspension protoplasts of Festuca
arundinacea Schreb., Lolium perenne L. and L. multiflorum Lam. Plant Cell Tissue and
Organ Culture, 12, 137-140.
Dalton S.J. (1988b). Plant regeneration from cell suspension protoplasts of Festuca
arundinacea Schreb. (tall fescue) and Lolium perenne L. (perennial ryegrass). Journal of
Plant Physiology, 132, 170-175.
Dalton S.J., Bettany A.J.E., Timms E. & Morris P. (1998). Transgenic plants of Lolium
multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide
fibre mediated transformation of cell suspension cultures. Plant Science, 312, 31-43.
Dalton S.J., Bettany A.J.E., Timms E. & Morris P. (1999). Co-transformed diploid Lolium
perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) plants produced by
microprojectile bombardment. Plant Cell Reports, 18, 721-726.
Davey M.R., Blackhall N.W., Lowe K.C. & Power B.J. (1996). Stimulation of plant cell
division and organogenesis by short-term, high-voltage electrical pulses. In:
Electrical Manipulation of Cells, Lynch P.T., Davey M.R. (Eds.) Chapman and Hall,
New York pp 273-286.
Davey M.R., Anthony P., Power B.J. & Lowe K.C. (2005). Plant protoplasts: Status and
biotechnological perspectives. Biotechnological Advances, 23, 131-171.
131
Day A., & Ellis T.H.N. (1984). Chloroplast DNA deletions associated with wheat plants
regenerated from pollen: Possible basis for maternal inheritance of chloroplasts.
Cell, 39, 359-368.
Day A., & Ellis T.H.N. (1985). Deleted forms of plastid DNA in albino plants from
cereal anther culture. Current Genetics, 9, 671-678.
Dean F.B., Nelson J.R., Giesler T.L., Lasken R.S. (2001) Rapid Amplification of Plasmid
and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle
Amplification. Genome Research, 11, 1095-1099.
Derks F.H.M., Hakkert J.C. & Derbeek W.H.J. (1992). Genome composition of
asymmetric hybrids in relation to the phylogenetic distance between the parents
nucleus-chloroplast interaction. Theoretical and Applied Genetics, 84, 930-940.
Dijak M., Smith D.L., Wilson T.J. & Brown D.C.W. (1986). Stimulation of direct
embryogenesis from mesophyll protoplasts of Medicago sativa. Plant Cell Reports, 5,
468-470.
Donaldson P.A., Bevis E.E., Pandeya R.S. (1994). Random chloroplast segregation and
frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana
tabacum L. and N. glutinosa L. Theoretical and Applied Genetics, 87, 900-908.
Dudits D., Fejér O., Hadlaczky G., Koncz C., Lázár G.B. & Horváth G. (1980).
Intergeneric gene transfer mediated by plant protoplast fusion. Molecular & General
Genetics, 179, 283–288.
Duke J.A. (1981). Handbook of legumes of world economic importance, London: Plenum Press.
Dunford R. & Walden R.M. (1991). Plastid genome structure and plastid related transcript
levels in albino barley plants derived from anther culture. Current Genetics, 20, 339347.
Durieu P. & Ochatt S.J. (2000). Efficient intergeneric fusion of pea (Pisum sativum L.) and
grass pea (Lathyrus sativus L.) protoplasts. Journal of Experimental Botany, 51(348),
1237-1242.
132
Duzgunes N., Wilschut J., Fraley R. & Papahadjopoulos D. (1981). Studies on the
mechanism of membrane fusion. Role of head-group composition in calcium and
magnesium-induced fusion of mixed phospholipids vesicles. Biochimica et Biophysica
Acta, 642, 182-195.
Esau K. (1977). Cell Wall. In: Plant Anatomy, John Wiley & Sons, New York 43-60.
Fahleson J., Eriksson I., Landgren M., Stymne S. & Glimelius K. (1994). Intertribal
somatic hybrids between Brassica napus and Thlaspi perfoliatum with high content of
the T. perfoliatum-specific nervonic acid. Theoretical and Applied Genetics, 87, 795–804.
Forsberg J., Lagercrantz U. & Glimelius K. (1998a). Comparison of UV light, X-ray and
restriction enzyme treatment as tools in production of asymmetric somatic hybrids
between Brassica napus and Arabidopsis thaliana. Theoretical & Applied Genetics, 96,
1178–1185.
Forsberg J., Dixelius C., Lagercrantz U. & Glimelius K. (1998b). UV dose-dependent
DNA elimination in asymmetric somatic hybrids between Brassica napus and
Arabidopsis thaliana. Plant Science, 131, 65-76.
Gadkar V., Rillig M.C. (2005). Application of Phi29 DNA polymerase mediated whole
genome amplification on single spores of arbuscular mycorrhizal (AM) fungi.
FEMS Microbiology Letters, 242, 65-7.
Gamborg O.L. (2002). Plant tissue culture biotechnology, milestones. In Vitro Cellular and
Developmental Biology-Plant, 38, 81-92.
Ge T.M., Yu Y.J., Xie Y.F., Zhang D.P., Zhang R.D. & Qin F.L. (1997). Asymmetric
somatic hybridisation and plant regeneration between wheat and Italian ryegrass (in
Chinese). Journal of Huazhong Agricultural University, 16, 105-111.
Ge T.M., Lin X.H. Qin F.L., Yu S.W. & Yu Y.J. (2006) Protoplast electrofusion between
Common Wheat (Triticum Aestivum L.) and Italian ryegrass (Lolium multiflorum Lam.)
and regeneration of mature cybrids. In Vitro Cellular and Developmental Biology-Plant,
42, 179-187.
133
Gelvin S.B. (2003). Agrobacterium-mediated plant transformation: the biology behind the
“gene-jockeying” tool. Microbiology and Molecular Biology Reviews, 67, 16-37.
Gilliland T.J., Camlin M.S. & Wright C.E. (1982). Evaluation of phosphoglucoisomerase
allozyme electrophoresis for the identification and registration of cultivars of
perennial ryegrass (Lolium perenne). Seed Science Technology, 10, 415-430.
Glen A.E., Bacon C.W., Price R. & Hanlin R.T. (1996). Molecular phylogeny of
Acremonium and its taxonomic implications. Mycologia, 88, 369-383.
Goodacre R., Roberts L., Ellis D., Thorogood D., Reader S.M., Ougham H., et al. (2007).
From phenotype to genotype: Whole tissue profiling for plant breeding.
Metabolomics, DOI: 10.1007/s11306-007-0062-6
Gordon A.H., Lomax J.A., Dalgarno K. & Chesson A. (1985).
Preparation and
composition of mesophyll, epidermis, and fibre cell walls from leaves of perennial
ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum). Journal of Food Science
and Agriculture, 36, 509-519.
Gram T., Mattsson O. & Joersbo M. (1996). Division frequency of pea protoplasts in
relation to starch accumulation. Plant Cell Tissue and Organ Culture, 45, 179-183.
Grimes H.D. & Boss W.F. (1985). Intracellular calcium and calmodulin involvement in
protoplast fusion. Plant Physiology, 79, 253-258.
Gülly C. (1996). Mutationen im chloroplastengenom als auslöser für den Albinismus in
Pollenpflanzen von Weizen? Diploma Thesis, University of Vienna, Vienna.
Guo W.W., Prasad D., Serrano P., Gmitter Jr. F.G. & Grossier J.W. (2004). Citrus
somatic hybridisation with potential for direct tetraploid scion cultivar
development. Journal of Horticultural Science and Biotechnology, 79, 400-405.
Gupta H.S. & Pattanayak A. (1993). Plant regeneration from mesophyll protoplasts of
rice (Oryza sativa L.) Bio/Technology, 11, 90-94.
134
Guthridge K.M., Dupal M.P., Jones E.S., Kolliker R., Smith K.F. & Forster J.W. (2001).
AFLP analysis of genetic diversity within and between populations of perennial
ryegrass (Lolium perenne L.). Euphytica, 122, 191-201.
Hadwiger M.A. & Heberle-Bors E. (1986). Pollen plant production in Triticum turgidum
ssp. durum. In: Proceedings of International Symposium of Nuclear Techniques and in-vitro
culture for plant improvement, FAO/IAEA, Vienna, pp 213-220.
Hall R.D., Rouwendal G.J.A. & Krens F.A. (1992). Asymmetric somatic cell hybridisation
in plants. I. The early effects of (sub)lethal doses of UV and gamma irradiation on
the cell physiology and DNA integrity of cultured sugarbeet (Beta vulgaris L.)
protoplasts. Molecular General and Genetics, 234, 306-314.
Handberg K. & Stougaard J. (1992). Lotus japonicus an autogamous, diploid legume species
for classical and molecular genetics. Plant Journal, 2, 487-496.
Hansen L.N. & Earle E.D. (1994). Novel flowering and fatty acid characters in rapid
cycling Brassica napus L. resynthesised by protoplast fusion. Plant Cell Reports, 14,
151-156.
Hansen L.N. & Earle E.D. (1997). Somatic hybrids between Brassica oleracea L. and Sinapis
alba L. with resistance to Alternaria brassicae (Berk) Sacc. Theoretical and Applied
Genetics, 94, 1078-1085.
Hanzel J.J., Miller J.P., Brinkman M.A. & Fendos E. (1985). Genotype and media effects
on callus formation and regeneration in barley. Crop Science, 25, 27-31.
Harada T., Sato T., Asaka T. & Matsukawa I. (1991). Large-scale deletions of plastid
DNA in rice anther culture. Theoretical and Applied Genetics, 81, 157-161.
Harada T., Ishikawa R., Niizeki M. & Saito K (1992). Pollen-derived rice calli that have
large deletions in the plastid DNA do not require protein synthesis in plastid for
growth Molecular and General Genetics, 233, 145-150.
Hatfield R.D., Wilson J.R. & Mertens D.R. (1999). Composition of cell walls isolated
from cell types of grain sorghum stems. Journal of the Science of Food and Agriculture, 79,
891-899.
135
Hawken R.J., Cavanagh J.A.L., Meadows J.R.S., Khatkar M.S., Husaini Y., Zenger K.R., et
al. (2006). Whole-Genome Amplification of DNA Extracted from Cattle Semen
Samples. Journal of Dairy Science, 89, 2217-2221.
Hayward M.D. & McAdam N.J. (1977). Isozyme as a measure of distinctiveness and
stability in cultivars of Lolium perenne. Z. Pflanzenz, chtg, 79, 59-68.
Hensgens L.A.M., de Bakker E.P.H.M., van Os-Ruygrok E.P., Rueb S., van de Mark F.,
van der Maas H.M., et al. (1993). Transient and stable expression of gusA fusions
with rice genes in rice, barley and perennial ryegrass. Plant Molecular Biology, 22, 11011127.
Heseman C. U. & Schröder G. (1982). Loss of nuclear DNA in leaves of rye. Theoretical
and Applied Genetics, 62, 128-131.
Hofinger B. (1999). Molecular genetic analysis of plastid deficiencies in anther culturederived albino plants of wheat (Triticum aestivum). Diploma Thesis, University of
Vienna, Vienna.
Holme, I.B., Torp A.M., Hansen L.N. & Andersen S.B. (2004). Quantitative trait loci
affecting plant regeneration from protoplasts of Brassica oleracea. Theoretical and
Applied Genetics, 108, 1513-1520.
Horn M.E., Shillito R.D., Conger B.V. & Harms C.T. (1988). Transgenic plants of
orchardgrass (Dactylis glomerata L.) from protoplasts. Plant Cell Reports, 7, 469-472.
Hu Q., Hansen L, Laursen J., Dixelius C. & Andersen S. (2002). Intergeneric hybrids
between Brassica napus and Orychophragmus violaceus containing traits of agronomic
importance for oilseed rape breeding. Theoretical and Applied Genetics, 105, 834-840.
Ishibashi T., Kimura S., Furukawa T. & Sakaguchi K. (2006). DNA repair mechanisms in
UV-B tolerant plants. Japan Agricultural Research Quaterly, 40(2), 107-113.
Jain S.M. (2001). Tissue culture-derived variation in crop improvement. Euphytica, 118,
153-166.
136
Jain S.M., Shahin E. A. & Sun S. (1988). Interspecific protoplast fusion for the transfer of
atrazine resistance from Solanum nigrum to tomato (Lycoperscicon esculentum L.) Plant
Cell Tissue and Organ Culture, 12, 189-192.
Jain R.K., Khehra G.S., Lee S.H., Blackhall N.W., Marchant R., Davey M.R., et al. (1995).
An Improved procedure for plant regeneration from Indica and japonica rice
protoplasts. Plant Cell Reports, 14, 515-519.
Kaimori N., Senda M., Ishikawa R., Akada S., Harada T., & Niizeki M. (1998).
Asymmetric somatic cell hybrids between Alfaalfa and Birdsfoot Trefoil. Breeding
Science, 48, 29-34.
Kanno A., Kanzaki H. & Kameya T. (1997). Detailed analyses of chloroplast and
mitochondrial DNAs from the hybrid plant generated by asymmetric protoplast
fusion between radish and cabbage. Plant Cell Reports, 16, 479-484.
Kao K.N. (1977). Chromosomal behaviour in somatic hybrids of soybean and Nicotiana
glauca. Molecular & General Genetics, 150, 225-230.
Kao, K. N. & Michayluk M. R.. (1974). A method for high-frequency intergeneric fusion
of plant protoplasts. Planta, 115, 355-367.
Kao, K. N., Constabel F., Michayluk M.R. & Gamborg O.L. (1974). Plant protoplast
fusion and growth of intergeneric hybrid cells. Planta, 120, 215-227.
Kaul K. (1990). Potential biotechnological approaches. In: Kasperbauer MJ (ed)
Biotechnology in tall fescue improvement, CRC Press, Boca/ Raton, pp 59-77.
Kihara M., Cai K-N., Ishikawa R., Harada T., Niizeki M. & Saito K. (1992). Asymmetric
somatic hybrid calli between leguminous species of Lotus corniculatus and Glycine max
and regenerated plants from the calli. Japanese Journal of Breeding, 42, 55-64.
Kisaka H., Lee H., Kisaka M., Kanno A., Kang K. & Kameya T. (1994). Production and
analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and
dicotyledon (Daucus carota L.). Theoretical Applied Genetics, 89, 365-371.
137
Kisaka H., Kisaka M., Kanno A. & Kameya T. (1997). Production and analysis of plants
that are somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.).
Theoretical Applied Genetics, 94, 221-226.
Kisaka H., Kisaka M., Kanno A. & Kameya T. (1998). Intergeneric somatic hybridisation
of rice (Oryza sativa L.) and Barley (Hordeum vulgare. L) by protoplast fusion. Plant
Cell Reports, 17, 362-367.
Klercker J.A.F. (1892). Eine methode zur isolierung lebender protoplasten. Ofvers VetenskAkad Förh Stokh, 9, 463-475.
Knox M.R. & Ellis T.H.N. (2001). Stability and inheritance methylation states at PstI sites
in Pisum. Molecular Genetics and Genomics, 265, 497-507.
Kushnir S. G, Shlumukov L. R., Pogrebnyak N.J., Berger S. & Gleba Y. (1987).
Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome.
Molecular and General Genetics, 209, 159-163.
Kyozuka J., Hayashi Y. & Shimamoto K. (1987). High frequency plant regeneration from
rice protoplasts by novel nurse culture methods. Molecular and General Genetics, 206,
408-413.
Lallemand J., Michaud O. & Greneche M. (1991). Electrophoretical description of rye
grass varieties: a catalogue. Plant Variety Seeds, 4, 11-16.
Lasken R.S., Egholm M. (2003). Whole genome amplification: abundant supplies of DNA
from precious samples or clinical specimens. Trends in Biotechnology, 21, 531-535.
Lee L, Schroll R.E., Grimes H.D. & Hodges T.K. (1989). Plant regeneration from indica
rice (Oryza sativa L.) protoplasts. Planta, 178, 325-333.
Leonards K.S. & Haug A. (1979). Regulation of zoospore development: electron spin
resonance studies of the isolated lipid components from Blastocladiella emersonii
zoospores. Plant Physiology, 63, S-145.
138
Li Z.Y., Tanner G.J. & Larkin P.J. (1990). Callus regeneration from Trifolium subterraneum
protoplasts and enhanced protoplast division by low-voltage treatment and nurse
cells. Plant Cell Tissue and Organ Culture, 21, 67-73.
Linsmaier E.M. & Skoog F. (1965). Organic growth factor requirements of tobacco tissue
cultures. Physiology Plantarum, 8, 100-127.
Liu J.H. & Deng X.X. (1999). Regeneration of hybrid calluses via donor-recipient fusion
between Microcitrus papuana and Citrus sinensis. Plant Cell Tissue and Organ culture, 59,
81-87.
Liu J., Xu X. & Deng X. (2005). Intergeneric somatic hybridization and its application to
crop genetic improvement. Plant Cell Tissue and Organ Culture, 82, 19-44.
Lotfy S., Luro F., Carreel F., Froeloicher Y., Rist D. & Ollitrault P. (2003). Application of
cleaved amplified polymorphic sequence method for analysis of cytoplasmic
genome among Aurantioideae intergeneric somatic hybrids. Journal of American
Society of Horticultural Science, 128, 225-230.
Löschen-berger F. & Heberle-Bors E. (1992). Anther culture responsiveness of Austrian
winter wheat (Triticum aestivum L.) cultivars. Die Bodenkultur, 43, 115-122.
Lowe K.C., Anthony P., Power J.B. & Davey M.R. (2003). Novel approaches for
regulating gas supply to plant systems in vitro: application and benefits of artificial
gas carriers. In Vitro Cellular and Developmental Biology-Plant, 39, 557-566.
Lührs, R. & Lörz H. (1987). Plant regeneration in vitro from embryogenic cultures of
spring and winter type barley (Hordeum vulgare L.) varieties. Theoretical and Applied
Genetics, 75, 16-25.
Ma R., Guo Y.D. & Pulli S. (2003). Somatic embryogenesis and fertile green plant
regeneration from suspension cell –derived protoplast of rye (Secale cereale L.). Plant
Cell Reports, 22, 320-327.
Mathieu Y., Lapous D., Thomine S., Lauriére C., & Guern J. (1996). Cytoplasmic
acidification as an early phosphorylation-dependent response of tobacco cells to
elicitors. Planta, 199, 416-424.
139
McMillin D.E. (1983). Plant isozymes: a historical perspective. In: Tanksley SD and
Orton TJ (Eds.), Isozymes in Plant Genetics and Breeding, part A. (pp. 3-13). Elsevier,
Amsterdam.
Mian M.A.R., Zwonitzer J.C. & Hopkins A.A. (2000). Genetic diversity assessment in
Festuca arundinacea Schreb. Using AFLP markers. Abstr. In: Molecular Breeding of Forage
Crops, Second International Symposium (p. 146). Nov. 19-24, 2000 Victoria Australia.
Min B.R., McNabb W.C., Barry T.N. & Kemp P.D. (1998). The effect of condensed
tannins on the production of wool and on its processing characteristics in sheep
grazing Lotus corniculatus. Australian Journal of Agricultural Research, 49, 597-605.
Min B.R., Fernandez J.M., Barry T.N., McNabb W.C. & Kemp P.D. (2001). The effect of
condensed tannins in Lotus corniculatus upon reproductive efficiency and wool
production in ewes during autumn. Animal Feed Science and Technology, 92, 185-202.
Michalczuk L., Cooke T.J. & Cohen J.D. (1992a). Auxin levels at different stages of carrot
somatic embryogenesis. Phytochemistry, 31, 1097-1103.
Michalczuk L., Ribnicky D.M., Cooke T.J. & Cohen J.D. (1992b). Regulation of indole-3acetic acid biosynthetic pathways in carrot cell cultures. Plant Physiology, 100, 13461353.
Mohmand A.S. & Nabors M.W. (1990). Somaclonal variant plants of wheat derived from
mature embryo explants of three genotypes. Plant Cell Reports, 8, 558-560.
Moieni A. & Sarrafi A. (1995). Genetic analysis for haploid-regeneration responses of
hexaploid-wheat anther cultures. Plant Breeding, 114, 247-249.
Moshelion M., Moran N. & Chaumont F. (2004). Dynamic changes in the osmotic water
permeability of protoplast plasma membrane. Plant Physiology 135, 2301-2317.
Mouritzen P. & Bach-Holm P. (1994) Chloroplast genome breakdown in microspore
culture of barley (Hordeum vulgare L.) occurs primarily during regeneration. Journal of
Plant Physiology, 144, 586-593.
140
Mueller U.G. & Wolfenbarger L.L. (1999). AFLP genotyping and fingerprinting. Tree, 10,
389-394.
Murashige T. & Skoog F. (1962). A revised medium for rapid growth and bioassays with
tobacco tissue culture. Physiology Plantarum, 15, 473-497.
Nagata T.G. & Melchers G. (1978). Surface charge of protoplasts and their significance in
cell-cell interaction. Planta, 142, 235-238.
Nakajo S., Niizeki M., Harada T., Ishikawa R. & Saito K. (1994). Somatic cell
hybridisation in rice (Oryza sativa L.) and birsfoot trefoil (Lotus corniculatus L.).
Breeding Science, 44, 79-81.
Nap J.P., Metz P.L.J., Escaler M. & Conner A.J. (2003). The release of genetically
modified crops into the environment: I. Overview of current status and regulations.
The Plant Journal, 33, 1-18.
Nenz E., Pupilli F., Paolocci F., Damiani F., Cenci C.A., & Arcioni S. (1996) Plant
regeneration and genetic transformation of Lotus angustissimus. Plant Cell Tissue and
Organ Culture, 45, 145-152.
Nielsen G. (1980). Identification of all genotypes in tetraploid ryegrass (Lolium spp.)
segregating for four alleles in a Pgi-enzyme locus. Hereditas, 92, 49-52.
Nielsen K.A., Larsen E. & Knudsen E. (1993). Regeneration of protoplast-derived green
plants of Kentucky blue grass (Poa pratensis L.). Plant Cell Reports, 12, 537-540.
Niizeki M. & Grant W.F. (1971). Callus, plantlet formation, and polyploidy from cultured
anthers of Lotus and Nicotiana. Canadian Journal of Botany, 49, 2041-2051.
Niizeki M., & Saito K. (1986). Plant regeneration from protoplasts of bridsfoot trefoil,
Lotus corniculatus L. Japanese Journal of Breeding, 36, 177-180.
Niizeki M., Nakajo S. & Harada T. (1992). Somatic cell hybridisation in rice and birdsfoot
trefoil. Lotus Newsletter, 23, 18-22.
Niizeki M., Kihara M. & Cai K-N. (1994). Somatic hybridisation between Birdsfoot
Trefoil (Lotus corniculatus L.) and Soybean (Glycine max L.). In: Bajaj Y.P.S. (Ed.),
141
Biotechnology in Agriculture and Forestry, 27, (pp. 132-144) Springer-Verlag, Berlin
Germany.
Niizeki M. (2001). Somatic hybridisation between Medicago sativa L. (Alfalfa) and Lotus
corniculatus L. (Birdsfoot trefoil). In: Nagata and Bajaj (Eds.), Biotechnology in
Agriculture and Forestry, 49, (pp. 341-355) Springer-Verlag, Berlin Germany.
Oberwalder B., Rouß B., Schilde-Rentschler L., Hemleben V. & Ninnemann H. (1997).
Asymmetric fusion between wild and cultivated species of potato (Solanum spp.)
detection of asymmetric hybrids and genome elimination. Theoretical and Applied
Genetics, 94, 1104-1112.
Olesen A., Storgaard M. Madsen S. & Andersen S.B. (1995). Somatic in vitro culture
response of Lolium perenne L.: genetic effects and correlations with anther culture
Euphytica, 86, 199-209.
Olesen A., Storgaard M. & Madsen S. (1996). Suspension culture performance in
commercial varieties of perennial ryegrass (Lolium perenne L.). Euphytica, 88, 151-157.
Ozais-Akins P., Ferl R.J. & Vasil I.K. (1986). Somatic hybridization in the gramineae:
Pennisetum americanum (L.) K. Schum. (pearl millet)+ Panicum maximum Jacq. (Guinea
grass). Molecular & General Genetics, 203, 365-370.
Papadakis A.K. & Roubelakis-Angelakis K.A. (2002). Oxidative stress could be
responsible for the recalcitrance of plant protoplasts. Plant Physiology and Biochemistry,
40, 549-559.
Papadakis A.K., Paschalidis K.A. & Roubelakis-Angelakis K.A. (2005). Biosynthesis
profile and endogenous titers of polyamines differ in totipotent and recalcitrant
plant protoplasts. Physiologia Plantarum 125, 10-20.
Papahadjopoulos D. (1978). Calcium-induced phase changes and fusion in natural and
model membranes. In: Poste G., Nicolson G.L. (Eds.), Membrane Fusion, (Pp 765790). Elsevier, New York .
142
Pasternak T.P., Prinsen E., Ayaydin F., Miskolczi P., Potters G., Asard H., et al. (2002).
The role of auxin, pH, and stress in the activation of embryogenic cell division in
leaf protoplast-derived cells of alfalfa. Plant Physiology, 129, 1807-1819.
Pati P.K. Sharma M. & Ahuja P.S. (2005). Extra thin alginate film: An efficient technique
for protoplast culture. Protoplasma, 226, 217-221.
Pelletier G., Primard C., Vedel F., Chetrit P., Remy R., Rouselle P. & Renard M. (1983).
Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion. Molecular
and General Genetics, 191, 244-250.
Petolino J. (2002). Direct DNA delivery into intact cells and tissues. In: Khachatourians
G.G., McHughen A., Scorza R., Nip W.K. & Hui Y.H. (Eds), Transgenic Plants and
Crops, pp. 137-143. Marcel Dekker, Inc, New York.
Piccirilli M., Pupilli P. & Arcioni S. (1988). Lotus tenuis Wald. & Kit.: in vitro conditions for
plant regeneration from protoplasts and callus of various explants. Plant Science, 55,
77-82.
Potrykus I. (1990). Gene transfer to cereals. An assessment. Biotechnology, 8, 535-542.
Potrykus I. & Shillito R.D. (1986). Protoplasts: isolation, culture, plant regeneration.
Methods in Enzymology, 118, 549-578.
Power J.B., Cummins S.E. & Cocking E.C. (1970). Fusion of isolated plant protoplasts.
Nature, 225, 1016-1018.
Puolimatka M. & Karp A. (1993). Effect of genotype on chromosome variation in tissue
culture of inbred and outbred rye. Heredity, 71, 138-144.
Pupilli F., Arcioni S., Damiani F. & Pezzotti M. (1990). Plant regeneration from callus and
protoplast cutures of Lotus pedunculatus. Plant Cell Tissue and Organ Culture, 23, 193199.
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.
143
Ramulu K.S., Dijkhuis P., Famelaer I., Cardi T. & Verhoeven H.A. (1994). Cremart: A
new chemical for efficient induction of micronuclei in cells and protoplasts for
partial genome transfer. Plant Cell Reports, 13, 687-691.
Rasheed J.H., Al-Mallah M.K., Cocking E.C. & Davey M.R. (1990). Root hair protoplasts
of Lotus corniculatus L. (birdsfoot trefoil) express their totipotency. Plant Cell Reports,
8, 265-269.
Roldan Ruiz I., Van Laecke K., De Riek J., Dendauw J., Depicker A., Van Bockstaele E.,
et al. (1997). The use of AFLP markers for the identification of ryegrass (Lolium
spp.). In: Van Lacke K, De Riek J, Van Bockstaele E, De Loose M, Krajewski P,
Kaczmarek Z. (Eds.), Advances in Biometrical genetics. Proc. Tenth meeting of the
EUCARPIA Section Biometrics in Plant Breeding, (Pp 231-237.). Institute of Plant
Genetics Poznan Poland. 14-16 May.
Roldan Ruiz I., Dendauw J., Van Bockstaele E., Depicker A. & De Loose M. (2000).
AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.) Molecular
Breeding, 6, 125-134.
Rose R.J., Thomas M.R. & Fitter J.T. (1990). The transfer of cytoplasmic and nuclear
genomes by somatic hybridisation. Australian Journal of Plant Physiology, 17, 303-321.
Sakamoto K. & Takeguchi T. (1991). Regeneration of intergeneric somatic hybrid plants
between Lycopersicon esculentum and Solanum muricatum. Theoretical and Applied Genetics,
81, 509-513.
Salisbury F. B. & Ross, C. W (1992). Plant Physiology and Plant Cells. In: Plant Physiology,
Wadsworth, Inc., Belmont, CA 3-26.
Scott D., Maunsell L.A., Keoghan J.M., Allan B.E., Lowther W.L. & Cossens G.G.
(1995). A Guide to Pastures and Pasture Species for the New Zealand High
Country: In: Round-Turner Nick & David Ryde (Eds.) Grassland Research & Practice
Series No. 4, New Zealand Grassland Association Incorporated, Palmerston North,
New Zealand.
144
Sharma H.C., Crouch J.H., Sharma K.K., Seetharama N. & Hash C.T. (2002).
Applications of biotechnology for crop improvement: prospects and constraints.
Plant Science, 163, 381-395.
Sherraf I., Tizroutine S., Chaput M.H., Allot M., Mussio I., Sihachakr D., Rossignol L. &
Ducreux G. (1994). Production and characterisation of intergeneric somatic hybrids
through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon
pennellii. Plant Cell Tissue and Organ Culture, 37, 137-144.
Shiba T. & Sugawara K. (2005). Resistance to the rice leaf bug, Trigonotylus caelestialium, is
conferred by Neotyphodium endophyte infection of perennial ryegrass, Lolium perenne.
Entomologia Experimentalis et Applicata, 115, 387–392.
Shillito R.D., Paszkowski J. & Potrykus I. (1983). Agarose plating and a bead type culture
technique enable and stimulate development of protoplast derived colonies in a
number of plant species. Plant Cell Reports, 2, 244-247.
Sidorov V.A., Menczel L., Nagy F. & Maliga P. (1981). Chloroplast transfer in Nicotiana
based on metabolic complementation between irradiated and iodoacetate treated
protoplasts. Planta, 152, 342-345.
Sidorov V. A., . Yevtushenko D. P, Shakhovskyand A. M., & Gleba Y. Y (1994). Cybrid
production based on mutagenic inactivation of protoplasts and rescuing of mutant
plastids in fusion products: Potato with a plastome from S. bulbocastanum and S.
Pinnatisectum. Theoretical and Applied Genetics, 88, 525-529.
Siegel M.R., Latch G.C.M. & Johnson M.C. (1985). Acremonium fungal endophytes of
tall fescue and perennial ryegrass: significance and control. Plant Disease, 69, 179183.
Sinha A., Wetten A.C. & Caligari P.D.S. (2003). Effect of biotic factors on the isolation of
Lupinus albus protoplasts. Australian Journal of Botany, 51,103-109.
Skarzhinskaya M., Landgren M. & Glimelius K. (1996). Production of intertribal somatic
hybrids between Brassica napus L. and Lesquerella fendleri (Gray) Wats. Theoretical and
Applied Genetics, 93, 1242–1250.
145
Skerman P.J., Cameron D.G. & Riveros F. (1988). Tropical Forage Legumes FAO
publication.
Skoog F. & Miller C.O. (1957). Chemical regulation of growth and organ formation in
plant tissues cultured in vitro. Symposium of Society of Experimental Biology, 11, 118-131.
Spangenberg G., Vallés M.P., Wang Z.Y., Montavon P., Nagel J. & Potrykus I. (1994).
Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.)
and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts. Theoretical and
Applied Genetics, 88, 509-519.
Spangenberg G., Wang Z.Y., Wu X.L., Nagel J., Iglesias V. & Potrykus I. (1995a).
Transgenic tall fescue (Festuca arundinacea ) and red fescue (F. rubra) plants from
microprojectile bombardment of embryogenic suspension cells. Journal of Plant
Physiology, 145, 693-701.
Spangenberg G. Wang Z.Y., Wu X.L., Nagel J. & Potrykus I. (1995b). Transgenic
perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of
embryogenic suspension cells. Plant Science, 108, 209-217.
Spangenberg G., Wang Z.Y., Legris G., Montavon P., Takamizo T., Pérez-Vicente R., et
al. (1995c). Intergeneric symmetric and asymmetric somatic hybridisation in Festuca
and Lolium. Euphytica, 85, 235-245.
Spedding C.R.W. & Diekmahns E.D. (1972). Grasses and Legumes in British Agriculture. Bull.
49, Commonwealth Bureau of Pastures and Field Crops. Farnham Royal, Bucks,
England: Commonwealth Agriculture Bureaux.
Sundberg E. & Glimelius K. (1991). Effects of parental ploidy level and genetic
divergence on chromosome elimination and chloroplast segregation in somatic
hybrids within Brassicaceae. Theoretical and Applied Genetics, 83, 81-88.
Sundberg E., Landgren M. & Glimelius K. (1987). Fertility and chromosome stability in
Brassica napus resynthesised by protoplast fusion. Theoretical and Applied Genetics, 75,
96-104.
146
Tabaeizadeh Z., Ferl R.J. & Vasil I.K. (1986). Somatic hybridization in the Gramineae:
Saccharum officinarum L. (Sugarcane)+ Pennisetum americanum (L.) K. Schumm. (Pearl
millet). Proceedings of National Academy of Sciences, USA, 83, 5616-5619.
Takami K., Matsumara A., Yahata M., Imayama T., Kunitake H. & Komatsu H. (2004).
Production of intergeneric somatic hybrids between round Kumquat (Fortunella
japonica Swingle) and ‘Morita navel’ orange (Citrus sinensis Osbeck). Plant Cell Reports,
23, 39-45.
Takamizo T., Spangenberg G., Suginobu K. & Potrykus I. (1991). Intergeneric somatic
hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca
arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.). Molecular &
General Genetics, 231, 1-6.
Taylor P.W.J., Ko H.L. & Adkins W.S. (1992). Factors affecting protoplast isolation and
the regeneration of shoot-like structures from protoplast-derived callus of
Sugarcane (Saccharum spp. Hybrids). Australian Journal of Botany, 40, 863-876.
Terakawa T., Sato T. & Koike M. (1992). Plant regeneration from protoplasts isolated
from embryogenic suspension cultures of creeping bentgrass (Agrostis palustris
Huds). Plant Cell Reports, 11, 457-461.
Thom E.R., Burggraaf V.T., Watts R.J. & Hooper R.J. (2003). Relationship of tillering and
morphological characteristics of two perennial ryegrass lines to pulling when grazed
by diary cows. New Zealand Journal of Agricultural Research, 46, 15-25.
Tian D. & Rose R.J. (1999). Asymmetric somatic hybridisation between the annual
legumes Medicago truncatula and Medicago scutellata. Plant Cell Reports, 18, 989-996.
Torello W.A. & Symington A.G. (1984). Regeneration from perennial ryegrass callus
tissue. HortScience, 19(1), 56-57.
van der Maas H.M., de Jong E.R., Rueb S., Hensgens L.A.M. & Krens F.A. (1994). Stable
transformation and long term expression of the gusA reporter gene I callus lines of
perennial ryegrass (Lolium perenne L.) Plant Molecular Biology, 24, 401-405.
147
van Overbeck, J. (1935). Auxin production in the seedlings of dwarf
maize. Plant
Physiology, 13, 587-598.
Vardi A., Breiman A. & Galun E. (1987). Citrus cybrids: production by donor-recipient
protoplast fusion and verification by mitochondrial-DNA restriction profiles.
Theoretical and Applied Genetics, 75, 51-58.
Vasil I.K. (1988). Progress in the regeneration and genetic manipulation of cereal crops
Biotechnology, 6, 397-402.
Vedel F., Chetrit P., Mathieu C., Pelletier G. & Primard C. (1986). Several different
mitochondrial DNA regions are involved in intergenomic recombination in Brassica
napus cybrid plants. Current Genetics, 11, 17-24.
Vessabutr S. & Grant W.F. (1995). Isolation culture and regeneration of protoplasts from
birdsfoot trefoil (Lotus corniculatus). Plant Cell Tissue and Organ Culture, 41: 9-15.
Vos P., Hogers R., Bleeker M., Reijans M., van de Lee T., Hornes M., Frijters A., et al.
(1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research, 23,
4407-4414.
Waghorn G.C., Ulyatt M.J., John A. & Fisher M.T. (1987). The effect of condensed
tannins on the site of digestion of amino acids and other nutrients in sheep fed on
Lotus corniuculatus L. British Journal of Nutrition, 57, 115-126.
Wang Z.Y., Nagel J., Potrykus I. & Spangenberg G. (1993). Plants from cell suspensionderived protoplasts in Lolium species. Plant Science, 94, 179-193.
Wang Y., Waghorn G.C., Barry T.N. & Shelton I.D. (1994). The effect of condensed
tannins in Lotus corniculatus upon plasma metabolism of methionine, cysteine and
inorganic sulphate by sheep. British Journal of Nutrition, 72, 923-935.
Wang Z.Y., Legris G., Vallés M.P., Potrykus I. & Spangenberg G. (1995). Plant
regeneration from suspension and protoplast cultures in the temperate grasses
Festuca and Lolium. In: Terzi M. et al. (eds.), Current Issues in Plant Molecular and Cellular
Biology, (pp. 81-86). Netherlands: Kluwer Academic.
148
Wang Y., Douglas G.B., Waghorn G.C., Barry T.N. & Foote A.G. (1996a). Effect of
condensed tannins in Lotus corniculatus upon lactation performance in ewes. Journal of
Agricultural Science Cambridge, 126, 353-362.
Wang Y., Douglas G.B., Waghorn G.C., Barry T.N., Foote A.G. & Purchas R.W. (1996b).
Effect of condensed tannins upon the performance of lambs grazing Lotus
corniculatus and lucerne (Medicago sativa). Journal of Agricultural Science Cambridge, 126,
87-98.
Wang G.R., Binding H. & Posselt U.K. (1997). Fertile transgenic plants from direct gene
transfer to protoplasts of Lolium perenne L. and Lolium multiflorum Lam. Plant
Physiology, 151, 83-90.
Wang Z.Y., Scott M. & Hopkins A. (2002a). Plant regeneration from embryogenic cell
suspension cultures of Lolium temulentum. In Vitro Cellular & Developmental Biology, 38,
446-451.
Wang Z., Lehmann D., Bell J. & Hopkins A. (2002b). Development of an efficient plant
regeneration system for Russian wildrye (Psathyrostachys juncea). Plant Cell Reports, 20,
797-801.
Wang Y.P., Sonntag K. & Rudloff E. (2003). Development of rapeseed with high erucic
acid content by asymmetric somatic hybridisation between Brassica napus and Cranbe
abyssinica. Theoretical and Applied Genetics, 106, 1147-1155.
Wang Y.P., Sonntag K., Rudloff E. & Chen J.M. (2005). Intergeneric somatic
hybridisation between Brassica napus L. and Sinapis alba L. Journal of Integrative Plant
Biology, 47, 84-91.
Watanabe M., Suzuki K., Kawasaki H. & Watanabe Y. (2002). Differential responses of
Brassica napus and Petunia hybrida to leaf protoplast isolation stress. Physiologia
Plantarum, 114, 645-651.
Webb JK., & Watson EJ. (1991). Lotus corniculatus L.: Morphological and cytological
analysis of regenerants from three sources of tissue and selected progeny. Plant Cell
Tissue and Organ Culture, 25, 27-33.
149
Webb J.K., Woodcock S. & Chamberlain D.A. (1987). Plant regeneration from
protoplasts of Trifolium repens and Lotus corniculatus. Plant Breeding, 98, 111-118.
Widholm J.M. (1972). The use of fluorescein diacetate and phenosafranine for
determining viability of cultured plant cells. Stain Technology, 47, 189-194.
Wilkins P.W. & Humphreys M.O. (2003). Progress in breeding perennial forage grasses
for temperate agriculture. Journal of Agricultural Science, 140 (2), 129-150.
Wright R.L., Somers D.A. & McGraw R.L. (1987). Somatic hybridization between
birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd. Theoretical and
Applied Genetics, 75, 151-156.
Wu L., Ye X.D., Wang Z.Y., Potrykus I. & Spangenberg G. (1997). Gene transfer to
ryegrasses: down-regulation of major pollen allergens in transgenic plants. In,
Proceedings of XVIII International Grassland Congress, 1, 35-36. Winnipeg and
Saskatoon.
Xia G.M., Wang H. & Chen H.M. (1996). Plant regeneration from intergeneric
asymmetric hybridisation between wheat (Triticum aestivum L.) and Russian wildrye
(Psathyrostachys juncea (Fisch) nevski) and Couch grass (Agropyron elongatum (Host)
nevski). Chinese Science Bulletin, 41, 1382-1386.
Xia G.M., Li Z.Y., Wang S.L., Xiang F.N., Liu J.Y., Chen P.D. & Liu D.J. (1998).
Asymmetric somatic hybridisation between haploid common wheat and UVirradiated Haynaldia villosa. Plant Science, 137, 217-223.
Xia G.M., Xiang F.N., Zhou A.F., Wang H., He S.X. & Chen H.M. (1999). Fertile hybrid
plant regeneration from somatic hybridisation between Triticum aestivum and
Agropyron elongatum. Acta Botanica Sinica, 41, 349-352.
Xia G.M., Xiang F.N., Zhou A.F., Wang H. & Chen H.M. (2003). Asymmetric somatic
hybridisation between wheat (Triticum aestivum L.) and Agropyron elongatum (Host )
Nevishi. Theoretical and Applied Genetics, 107, 299-305.
150
Xiang F.N., Xia G.M., & Chen H.M. (2003) Effect of UV dosage on somatic
hybridisation between common wheat (Triticum aestivum L.) and Avena sativa L. Plant
Science, 164, 697-707.
Xiao Z., Wan L. & Han B. (2004). An interspecific somatic hybrid between Actinidia
chinensis and Actinidia kolomikta and its chilling tolereance. Plant Cell Tissue and Organ
Culture, 79, 299-306.
Xu X.Y., Liu J.H. & Deng X.X. (2004). Production and characterisation of intergeneric
diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle
and sour orange (Citrus aurantium). Euphytica, 136, 115-123.
Xu X.Y., Hu Z.Y., Li J.F., Liu J.H. & Deng X.X. (2007) Asymmetric somatic
hybridization between UV-irradiated Citrus unshiu and C. sinensis: regeneration and
characterization of hybrid shoots. Plant Cell Reports, DOI 10/1007/s00299-0070350-7.
Yamada T. & Kishida T. (2003). Genetic analysis of forage grasses based on heterologous
RFLP markers detected by rice cDNAs. Plant Breeding, 122, 57-60.
Yamada Y., Yang Y.Q. & Tang D.T. (1986). Plant regeneration from protoplast derived
callus of rice (Oryza sativa L.). Plant cell reports, 5, 85-88.
Yan C.-Q., Qian K.-X., Yan Q.-S., Zhang X.-Q., Xue G.-P., Huangfu W.-G., et al. (2004)
Use of asymmetric somatic hybridization for transfer of the bacterial blight
resistance trait from Oryza meyeriana L.to O. sativa L. ssp. japonica. Plant cell Reports, 22,
569-575.
Zaghmout O.M.F. & Torello W.A. (1990). Isolation and culture of protoplasts from
embryogenic suspension cultures of red fescue (Festuca rubra L.). Plant Cell Reports, 9,
340-343.
Zaghmout O.M.F. & Torello W.A. (1992). Plant regeneration from callus and protoplast
of perennial ryegrass (Lolium perenne L.). Journal of Plant Physiology, 140, 101-105.
151
Zhou A., Xia G., Zhang X., Chen H. & Hu H. (2001). Analysis of chromosomal and
organellar DNA of somatic hybrids between Triticum aestivum and Haynaldia villosa
Schur. Molecular Genetics and Genomics, 265, 387-393.
Zhou A.F., Chen X.L., Xia G.M. & Chen H.M. (2002). UV-fusion between protoplasts of
common wheat and Haynaldia villosa. Journal of Plant Physiology and Molecular Biology,
28, 305-310.
Zubko M.K., Zubko E.I. & Gleba Y.Y. (2002). Self-fertile cybrids Nicotiana tabacum (+
Hyoscyamus aureus) with a nucleo-plastome incompatibility. Theoretical and Applied
Genetics, 105, 822-828.
152
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.
153
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.
154
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).
155
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 umbellike cymes at the end of long axillary branches.
The calyx is a dentate tube and the yellow fivepetalled 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).
156
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
157
L. corniculatus callus
FC2 callus
158
FC3 callus
FC4 callus
159
FC5 callus
FC6 callus
160
FC7 callus
FC8 callus
161
FC9 callus
FC10 callus
162
FC11 callus
FC12 callus
163
FC13 callus
FC14 callus
164
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
165
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.
166
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
a.
b.
c.
d.
e.
f.
g.
h.
Solution
Fix/stop solution
dH20
Repeat step b. twice
Staining solution
dH20
Developer solution (4-10°C)
Fix/stop solution
dH20
Time
30 minutes
2 minutes
2x2 minutes
30 minutes
10 seconds
2-5 minutes
5-6 minutes
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.
167
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
37% formaldehyde (H2CO)
10mg/ml sodium thiosulfate (Na2S20.5HO)
90g
4.5ml
600 µl
120gm
6ml
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)
3 ml 37% formaldehyde (H2CO)
2000 ml dH2O
Fix/stop solution (10% acetic acid)
200 ml glacial acetic acid
1800 ml dH20
168
3g
4.5
To 3L
300ml
2.7L
4g
6ml
To 4 L
400ml
3.6 L
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.
169
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.
170