Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant

Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.

[1]  M. Moniruzzaman,et al.  Comparative analysis of bacterial and fungal endophytes responses to Candidatus Liberibacter asiaticus infection in leaf midribs of Citrus reticulata cv. Shatangju , 2020 .

[2]  S. He,et al.  Citrus CsACD2 Is a Target of Candidatus Liberibacter Asiaticus in Huanglongbing Disease. , 2020, Plant physiology.

[3]  M. Moniruzzaman,et al.  Exploration of Susceptible Genes with Clustered Regularly Interspaced Short Palindromic Repeats–Tissue-Specific Knockout (CRISPR-TSKO) to Enhance Host Resistance , 2020 .

[4]  A. Ciacciulli,et al.  New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review , 2020, Frontiers in Plant Science.

[5]  Chongde Sun,et al.  Physiochemical changes in Citrus reticulata cv. Shatangju fruit during vesicle collapse , 2020, Postharvest Biology and Technology.

[6]  L. Palou,et al.  Control of major citrus postharvest diseases by sulfur-containing food additives. , 2020, International journal of food microbiology.

[7]  G. Zhong,et al.  Citrus Origin, Diffusion, and Economic Importance , 2020 .

[8]  Rafael de Felício,et al.  Structure-function relationship of a citrus salicylate methylesterase and role of salicylic acid in citrus canker resistance , 2019, Scientific Reports.

[9]  E. Baldwin,et al.  Effect of Huanglongbing or Greening Disease on Orange Juice Quality, a Review , 2019, Front. Plant Sci..

[10]  J. Grosser,et al.  Genetic transformation of the ‘W Murcott’ tangor: comparison between different techniques , 2018, Scientia Horticulturae.

[11]  M. Sohani,et al.  Efficient genetic transformation of sour orange, Citrus aurantium L. using Agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus , 2018, Plant Gene.

[12]  J. Graham,et al.  Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice , 2018, Transgenic Research.

[13]  Dominica Rohozinski,et al.  Accurate measurement of transgene copy number in crop plants using droplet digital PCR , 2017, The Plant journal : for cell and molecular biology.

[14]  Xiaojun Chang,et al.  A simple and efficient in planta transformation method for pommelo (Citrus maxima) using Agrobacterium tumefaciens , 2017 .

[15]  B. Staskawicz,et al.  CRISPR-Cas9 mediated mutagenesis of a DMR6 ortholog in tomato confers broad-spectrum disease resistance , 2016 .

[16]  Aili Li,et al.  CRISPR/Cas9: A powerful tool for crop genome editing , 2016 .

[17]  P. Roussos.  Orange (Citrus sinensis (L.) Osbeck) , 2016 .

[18]  A. Omar,et al.  Somatic Embryogenesis: Still a Relevant Technique in Citrus Improvement. , 2016, Methods in molecular biology.

[19]  B. Snel,et al.  DOWNY MILDEW RESISTANT 6 and DMR6-LIKE OXYGENASE 1 are partially redundant but distinct suppressors of immunity in Arabidopsis. , 2015, The Plant journal : for cell and molecular biology.

[20]  S. Gardiner,et al.  Breeding better cultivars, faster: applications of new technologies for the rapid deployment of superior horticultural tree crops , 2014, Horticulture Research.

[21]  Tolga Izgu,et al.  Genetic Transformation in Citrus , 2013, TheScientificWorldJournal.

[22]  M. Qaim,et al.  Genetically Modified Crops and Food Security , 2013, PloS one.

[23]  L. Navazio,et al.  Plant cell suspension cultures. , 2013, Methods in molecular biology.

[24]  I. Gribaudo,et al.  Genetic transformation of fruit trees: current status and remaining challenges , 2012, Transgenic Research.

[25]  G. Moore,et al.  Citrus Transformation: Challenges and Prospects , 2011 .

[26]  C. Srinivasan,et al.  In Vitro Systems for Propagation and Improvement of Tropical Fruits and Palms , 2011 .

[27]  J. Grosser,et al.  Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus , 2011, Plant Cell, Tissue and Organ Culture (PCTOC).

[28]  J. Grosser,et al.  An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus , 2010, Plant Cell Reports.

[29]  J. Grosser,et al.  Protoplast Fusion Technology – Somatic Hybridization and Cybridization , 2010 .

[30]  Y. Mu,et al.  Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs , 2009, PloS one.

[31]  J. Grosser,et al.  Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus , 2009, Plant Cell, Tissue and Organ Culture (PCTOC).

[32]  Y. Duan,et al.  Citrus Transgenics : Current Status and Prospects , 2009 .

[33]  R. Huibers,et al.  Arabidopsis DMR6 encodes a putative 2OG-Fe(II) oxygenase that is defense-associated but required for susceptibility to downy mildew. , 2008, The Plant journal : for cell and molecular biology.

[34]  M. Talón,et al.  Citrus Genomics , 2008, International journal of plant genomics.

[35]  L. Peña,et al.  Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. , 2008, Tree physiology.

[36]  N. K. Koç,et al.  The effects of some carbohydrates on growth and somatic embryogenesis in citrus callus culture , 2006 .

[37]  L. Peña,et al.  Early events in Agrobacterium-mediated genetic transformation of citrus explants. , 2004, Annals of botany.

[38]  L. Vieira,et al.  Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo (Citrus paradisi Macf.×Poncirus trifoliata L. Raf.) using thin epicotyl sections , 2004 .

[39]  L. Vieira,et al.  Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments , 2003, Plant Cell, Tissue and Organ Culture.

[40]  N. Maclean,et al.  Copy Number Related Transgene Expression and Mosaic Somatic Expression in Hemizygous and Homozygous Transgenic Tilapia (Oreochromis Niloticus) , 2000, Transgenic Research.

[41]  F. Carimi,et al.  Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.] , 1998, Plant Cell, Tissue and Organ Culture.

[42]  K. S. Lee,et al.  Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp. , 1997, Plant Cell, Tissue and Organ Culture.

[43]  M. Toonen,et al.  Description of somatic-embryo-forming single cells in carrot suspension cultures employing video cell tracking , 1994, Planta.

[44]  F. Carimi,et al.  Somatic embryogenesis from styles of lemon (Citrus limon) , 1994, Plant Cell, Tissue and Organ Culture.

[45]  S. Lawrence,et al.  Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants , 1992, Plant Cell Reports.

[46]  N. Nito,et al.  In vitro plantlet formation from juice vesicle callus of satsuma (Citrus unshiu Marc.) , 1990, Plant Cell, Tissue and Organ Culture.

[47]  F. Gmitter,et al.  Plant regeneration from undeveloped ovules and embryogenic calli of Citrus: Embryo production, germination, and plant survival , 2004, Plant Cell, Tissue and Organ Culture.

[48]  T. Warkentin,et al.  Transgene copy number can be positively or negatively associated with transgene expression , 2004, Plant Molecular Biology.

[49]  B. J. Mendes,et al.  Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments , 2003 .

[50]  P. von Aderkas,et al.  Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. , 2000, Tree physiology.

[51]  J. Mariath,et al.  Histological analysis of somatic embryogenesis in Brazilian cultivars of barley, Hordeum vulgare vulgare, Poaceae , 1999, Plant Cell Reports.

[52]  J. Chan,et al.  Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis , 1998, Plant Cell Reports.

[53]  Michael G. K. Jones,et al.  Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum , 1998 .

[54]  L. Peña,et al.  Virulence and supervirulence ofAgrobacterium tumefaciensin woody fruit plants , 1998 .

[55]  L. Navarro,et al.  Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C. aurantium) and alemow (C. macrophylla) , 1998 .

[56]  R. Henry.  Molecular and Biochemical Characterization of Somaclonal Variation , 1998 .

[57]  J. Mathur,et al.  Establishment and maintenance of cell suspension cultures. , 1998, Methods in molecular biology.

[58]  S. Jain,et al.  Somaclonal Variation and Induced Mutations in Crop Improvement , 1998, Current Plant Science and Biotechnology in Agriculture.

[59]  Z. Singh,et al.  Somatic embryogenesis and plantlet regeneration in mandarin (Citrus reticulata Blanco) , 1995 .

[60]  C. H. T. Cate,et al.  Differential methylation and expression of the β-glucuronidase and neomycin phosphotransferase genes in transgenic plants of potato cv. Bintje , 1993 .

[61]  P. Meyer,et al.  The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. , 1992, The Plant journal : for cell and molecular biology.

[62]  C. Gasser,et al.  Genetically Engineering Plants for Crop Improvement , 1989, Science.

[63]  J. Juárez,et al.  Aberrant Citrus Plants Obtained by Somatic Embryogenesis of Nucelli Cultured in Vitro , 1985, HortScience.

[64]  日高 哲志,et al.  Plantlet Formation by Anther Culture of Citrus aurantium L. , 1982 .

[65]  B. Haccius.  Question of unicellular origin of nonzygotic embryos in callus culture , 1978 .

[66]  J. Cameron,et al.  Tree and fruit characters of Citrus triploids from tetraploid by diploid crosses , 1969 .

[67]  T. Murashige,et al.  Growth factor requirements of Citrus tissue culture , 1969 .

[68]  T. Murashige,et al.  In Vitro Initiation of Nucellar Embryos in Monoembryonic Citrus , 1968, Journal of the American Society for Horticultural Science.