You're using an outdated browser. Please upgrade to a modern browser for the best experience.
Submitted Successfully!
Thank you for your contribution! You can also upload a video entry or images related to this topic. For video creation, please contact our Academic Video Service.
Version Summary Created by Modification Content Size Created at Operation
1 Md Moniruzzaman + 1881 word(s) 1881 2021-04-06 06:18:20 |
2 format correct Vivi Li Meta information modification 1881 2021-04-15 06:00:14 | |
3 format correct Vivi Li Meta information modification 1881 2021-04-15 06:01:52 | |
4 format correct Vivi Li Meta information modification 1881 2021-04-15 06:02:28 |

Video Upload Options

We provide professional Academic Video Service to translate complex research into visually appealing presentations. Would you like to try it?
No, upload directly Yes
Cite
If you have any further questions, please contact Encyclopedia Editorial Office.
Moniruzzaman, M. Citrus Cell Suspension Culture. Encyclopedia. Available online: https://encyclopedia.pub/entry/8702 (accessed on 26 December 2025).
Moniruzzaman M. Citrus Cell Suspension Culture. Encyclopedia. Available at: https://encyclopedia.pub/entry/8702. Accessed December 26, 2025.
Moniruzzaman, Md. "Citrus Cell Suspension Culture" Encyclopedia, https://encyclopedia.pub/entry/8702 (accessed December 26, 2025).
Moniruzzaman, M. (2021, April 15). Citrus Cell Suspension Culture. In Encyclopedia. https://encyclopedia.pub/entry/8702
Moniruzzaman, Md. "Citrus Cell Suspension Culture." Encyclopedia. Web. 15 April, 2021.
Citrus Cell Suspension Culture
Edit
This entry is adapted from the peer-reviewed paper 10.3390/plants10040664

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.

cell suspension Citrus tissue culture de novo organogenesis genetic engineering

1. Introduction

The importance of Citrus spp. is linked to their enormous economic and nutritional values [1]. However, citrus cultivation has been confronting many challenges, including control of diseases [2][3][4][5]. Solutions to the problems will rely mostly on breakthroughs in breeding, which is also hindered by problems, such as sterility, self- and cross-incompatibility [6], widespread nucellar embryony, and long juvenile periods that are associated with traditional breeding practices [7].

Genetic engineering by transformation has been widely adopted for crop improvement [8][9][10], including citrus [11]. The main advantage of the technique is that it allows modification of interestingtrait(s) without altering the overall genetic makeup, which is useful in making desirable changes in elite cultivar(s) [12][13]. The common practice of citrus genetic transformation studies is Agrobacterium tumefaciens [14][15]-mediated transformation of epicotyl segments of in vitro-germinated seedlings [14][16][17]. However, seed availability is seasonal and genotype-dependent. For example, many citrus cultivars are seedless or few-seeded. In addition, genotypes showed a strong impact on citrus organogenesis and genetic transformation [18][19][20]. Notably, juvenile tissues from mandarins hybrids are more difficult to be transformed by A. tumefaciens [21][22], reducing seriously genetic transformation efficiency [15]. On the other hand, the use of mature materials for A. tumefaciens-mediated transformation could result in earlier fruit production, bypassing or reducing the juvenile phase [23][24][25]. However, mature tissues show recalcitrance for de novo organogenesis induction in tissue culture and have a high occurrence of chimeric transformation and losing transformed cell lines in transgenic plants [26].

Genetic transformation using embryogenic cell suspension cultures could be a better alternative for having higher organogenetic potential [27][28]. Regeneration of putatively transformed cells and subsequent grafting of transgenic micro-shoots on rootstocks may shorten the juvenile period for flowering and fruiting [29]. The classical conception of somatic embryogenesis (SE) is based on the unicellular origin of somatic embryos [30], and this mode of somatic embryo development was the most frequently noticed in embryogenic cell suspensions of D. carota [31]. However, both a multicellular and a unicellular origin of somatic embryos in the same regeneration system is quite a common phenomenon, as was observed in several species, including Musa spp. [32], Cocos nucifera [33], Santalum album and S. spicatum [34], and H. vulgare [35].

The “Sweet orange”-Egyptian cultivar, Citrussinensis (L.), is the most common and important species among Citrus [36].“Shatangju” (Citrus reticulata) [37][38] is a popular local mandarin and “W. Murcott”’ (C. reticulata Blanco x C. sinensis L. Osbeck) [39], a tangor of unknown parentage, is one of the main fresh cultivars in the world. By following the general procedure (establishment and maintenance of the cell suspension, transformation of the cells, and subsequent plant organogenesis from putative transgenic cells), we successfully established a cell suspension culture and an associated Agrobacterium tumefaciens-mediated transformation system for the three Citrus cultivars was also established. Finally, corresponding transgenic plants were recovered with high efficiency. The developed methods should be useful for Citrus genetic improvements through genome engineering experiments.

2. Embryogenic Callus Induction

The EME, DOG and H+H have commonly used media for somatic embryogenesis [28][40]. In this experiment, embryonic calli were successfully induced from ovules of all three cultivars (“Sweet orange”-Egyptian cultivar, “Shatangju” and “W. Murcott”) on all three different solid media (EME, DOG and H+H). As shown in Figure 1A, the highest callus induction occurred on EME medium, while the lowest was on H+H, although no statistically significant difference was found among the three media used in the study. However, in some other cases and other plant species, media have shown to have significant effects on callus induction [41].

Plants 10 00664 g001 550

Figure 1. Somatic embryo induction.Data were recorded after 6–8 weeks of incubation. The statistical difference (p ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using Statistical Package for the Social Sciences (SPSS-version 23), and results were expressed as mean ± standard error of three independent experiments. (A) Culture media effect, all explants on the individual medium were considered as one group irrespective of genotypes (B) Genotypes effect, all explants of the individual genotype on all three media were considered as one group.

Figure 1B is the callus induction rates of the 3 cultivars in the case of 8 to 10 weeks old ovules. The highest induction rate, around 74%, was from “Sweet orange”*, whereas the lowest, around 71%, was from “W. Murcott”. Previous studies used excised nucelli [42], abortive ovules [43], unfertilized ovules [44], undeveloped ovules [45][46], isolated nucellar embryos [47], juice vesicles [48], anthers [49], styles and stigmas [50], leaves, epicotyls, cotyledons and root segments of in vitro grown nucellar seedling [51] for somatic embryogenesis in Citrus. We chose undeveloped ovules as callus induction material because previous studies showed that undeveloped ovule is a preferable material for somatic embryogenesis not only for having higher regeneration capacity but also for being mostly virus-free [52]. Gmitter and Moore reported the explants regeneration percentage from undeveloped ovule was between 0% and 70%, depending on genotypes [45], but all the 3 genotypes used in the study showed a higher than 70% induction rate.

Embryonic callus induction is closely associated with the differentiation status of the material (ovule) used [40][53]. In our experiment, the age of ovules was indeed showed a significant influence on the embryonic callus induction. As shown in Figure 2, the callus induction percentage varied from around 41% to 74% across the whole age group used in the study. However, it was neither the younger nor, the older age groups, but the middle age group (8 to 10 weeks) was the best in terms of callus induction rate.

Plants 10 00664 g002 550

Figure 2. Effect of ovule age on embryonic callus induction. Data were recorded after 6–8 weeks of incubation. All explants of the individual age group (i.e., 4 to 6) from all three cultivars on all three media were considered as one group. The statistical difference (p ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using SPSS (version 23), and results were expressed as mean ± standard error of three independent experiments.

3. Suspension Cell Culture and Plant Regeneration

In this experiment, suspension cell culture for all three genotypes was established in liquid H+H medium, as previous studies demonstrated that the medium (H+H) was suitable for citrus cell suspension culture [28][54]. Maintenance of suspension culture involves regular subculture (every two to three weeks), which is laborious but important for subsequent experiments and plant regeneration [28][55]. In this experiment, we investigated factors affecting intervals of suspension cell subculture and subsequent embryo development. Our results showed that adding a smaller amount (1ml) of suspension cells to fresh media (~50 mL) could extend subculture intervals to 8 weeks without affecting the following embryo production rate (15~16 per plate) (Figure 3). However, there was a significant reduction in the number of embryos (~8) produced per plate when the same 1 mL of suspension cells was used from 2 weeks subculture intervals, perhaps from an insufficient founder population. When larger volumes of suspension cells were used in the subculture, the subculture intervals were proportionally shortened. For example, using 5 mL of inoculation volume reduced subculture intervals to 2 weeks since longer intervals significantly reduced embryogenic capacity, possibly a result of nutrient exhaustion. Three milliliters inocula in 50 mL fresh medium was good for regular experiments (Figure 4B). This allowed the cells to grow and ensured sufficient cells for the experiments. However, for the maintenance of suspension cells, 1 mL inocula in 50 mL fresh medium was suitable for its significantly extended subculture intervals.

Plants 10 00664 g003 550

Figure 3. Effects of inoculation volume of suspension cells and subculture intervals on embryo production. The suspension cells were subcultured in H+H medium, and then the embryos were produced on EME-malt medium. The embryos were counted after 10 to 12 weeks of incubation on an EME-malt medium. All embryos produced from all genotypes of an individual subculture interval (i.e., 2 W) of the same inoculation volume (i.e., 1 mL) were considered as one group. The statistical difference (p ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using SPSS (version 23), and results were expressed as mean ± standard error of three independent experiments.

Plants 10 00664 g004 550

Figure 4. Embryonic callus induction, somatic embryo production, suspension cell culture establishment and plant regeneration of the “Sweet orange” (Citrus sinensis) cultivar. (Aa) Embryonic callus induction from 8 weeks old ovule on EME medium (Ab) and somatic embryo development, (B) Suspension cell culture establishment in an H+H medium, (C,D) Callus formation and embryo germination from suspension cell culture on an EME malt medium (the bars represent 0.5 mm), (E) Axis elongation of germinated embryos on an B+ medium, (F) Plants on RMAN rooting medium for root induction, (G) In vitro shoot grafted rootstock plant and (H) In vitro rooted plant transplanted on the soil. The bars represent 1 cm, except C and D.

In this study, Agrobacterium-mediated transformation and regeneration of suspension cellsderived from “Sweet orange”, “Shatangju” and “W. Murcott” were successfully accomplished (Figure 4). BASTA (20 mg/L) was added to the media to suppress the growth of nontransgenic cells. The transformation percentage was 32 to 35 and not significantly different among the cultivars. Genetic transformation with desirable genes is an effective alternative for Citrus improvement [56][57][58][59]. Apparently, higher transformation efficiency is preferable since more transformants mean the chance of selecting an ideal transgenic line is high. In this regard, cell suspension culture is better than other materials, such as commonly used epicotyl segments prepared from in vitro germinated seedlings [15][21][22] and mature stem pieces [22] that normally showed a very low transformation rate (less than 10%).

4. Transgenic Plant Recovery and Molecular Analysis of Transgenic Plants

The BASTA-survived in vitro micro-shoots were propagated in two ways: grafted on rootstocks (Figure 4G) or rooted on RMAN medium and then planted on soil (Figure 4F,H). Both methods gave a survival rate of higher than 90%. However, the growth of the in vitro rooted shoots was poorer than the grafted (Figure 4H,G). All plants regenerated from selection pressure of 20 mg/L BASTA contained the transgenes, as shown by PCR analysis of leaf genomic DNA (Figure 5), indicating that a concentration of 20 mg/L BASTA was high enough to screen out the transformants in our case. Tissues from different organs, including the apical and the basal leaves and even roots from invitro rooted plants, were examined by PCR. It seemed that no chimeric plant was detected, demonstrating that the chances were very slim for single-cells and/or a very small group of cells to produce chimeras (Figure 4C,D). Additionally, no visual phenotypic changes were observed on all transgenic lines so far.

Plants 10 00664 g005 550
Figure 5. Gel electrophoresis of PCR amplified DNA from transgenic plants. 2000 kb DNA ladder (M), transgenic plants samples (1–5), positive control (+), negative control (−). (A) PCR amplicon (1139 bp) from CaMV35S forward and CsDMR6 reverse primers.(B) PCR amplicon (429 bp) from Bar (bacterial bialaphos resistance gene) forward and reverse primers. (C) T-DNA constructs showing corresponding primer sites.

RT–PCR results showed that the DMR6 in all three tested transgenic lines had a higher expression level than the control, and particularly, transgenic line 2 showed the highest expression level (10-fold) (Figure 6). This may be because of the insertion of different numbers of gene copies in different transgene lines. Transgene expression level depends on transgene copy number and/or site of gene integration [60][61][62][63]. Different copy numbers in different transgenic lines could lead to variable gene expression levels in independent transformants [64]. Gene silencing could be induced by transgene [65], but no silencing was observed in PCR-tested transgenic lines in this study.

Plants 10 00664 g006 550

Figure 6. RT–PCR-mediated expression level analysis of CsDMR6 in transgenic plants. Results were expressed as mean ± standard error of three independent experiments.

References

  1. Zhong, G.; Nicolosi, E. Citrus origin, diffusion, and economic importance. In The Citrus Genome. Compendium of Plant Genomes; Gentile, A., La Malfa, S., Deng, Z., Eds.; Springer: Cham, Switzerland, 2020; pp. 5–21.
  2. Martínez-Blay, V.; Taberner, V.; Pérez-Gago, M.B.; Palou, L. Control of major citrus postharvest diseases by sulfur-containing food additives. Int. J. Food Microbiol. 2020, 330, 108713.
  3. Moniruzzaman, M.; Zhong, Y.; Yan, H.; Yuanda, L.; Jiang, B.; Zhong, G. Exploration of Susceptible Genes with Clustered Regularly Interspaced Short Palindromic Repeats–Tissue-Specific Knockout (CRISPR-TSKO) to Enhance Host Resistance. Crit. Rev. Plant Sci. 2020, 39, 387–417.
  4. Pang, Z.; Zhang, L.; Coaker, G.L.; Ma, W.; He, S.-Y.; Wang, N. Citrus CsACD2 Is a Target of CandidatusLiberibacterAsiaticus in Huanglongbing Disease. Plant Physiol. 2020, 184, 792–805.
  5. Dala-Paula, B.M.; Plotto, A.; Bai, J.; Manthey, J.A.; Baldwin, E.A.; Ferrarezi, R.S.; Gloria, M.B.A. Effect of Huanglongbing or Greening Disease on Orange Juice Quality, a Review. Front. Plant Sci. 2019, 9, 1976.
  6. Soost, R.; Cameron, J. Tree and fruit characters of Citrus triploids from tetraploid by diploid crosses. Hilgardia 1969, 39, 569–579.
  7. Salonia, F.; Ciacciulli, A.; Poles, L.; Pappalardo, H.D.; La Malfa, S.; Licciardello, C. New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review. Front. Plant Sci. 2020, 11, 1234.
  8. Qaim, M.; Kouser, S. Genetically modified crops and food security. PLoS ONE 2013, 8, e64879.
  9. Gasser, C.S.; Fraley, R.T. Genetically engineering plants for crop improvement. Science 1989, 244, 1293–1299.
  10. Song, G.; Jia, M.; Chen, K.; Kong, X.; Khattak, B.; Xie, C.; Li, A.; Mao, L. Crispr/cas9: A powerful tool for crop genome editing. Crop. J. 2016, 4, 75–82.
  11. Talon, M.; Gmitter, F.G., Jr. Citrus genomics. Int. J. Plant Genom. 2008, 2008, 528361.
  12. Gambino, G.; Gribaudo, I. Genetic transformation of fruit trees: Current status and remaining challenges. Transgenic Res. 2012, 21, 1163–1181.
  13. Van Nocker, S.; Gardiner, S.E. Breeding better cultivars, faster: Applications of new technologies for the rapid deployment of superior horticultural tree crops. Hortic. Res. 2014, 1, 14022.
  14. Peña, L.; Pérez, R.M.; Cervera, M.; Juárez, J.A.; Navarro, L. Early events in agrobacterium-mediated genetic transformation of Citrus explants. Ann. Bot. 2004, 94, 67–74.
  15. Dutt, M.; Grosser, J.W. Evaluation of parameters affecting Agrobacterium-mediated transformation of Citrus. Plant Cell Tissue Organ Cult. 2009, 98, 331–340.
  16. De Almeida, W.A.B.; Filho, F.D.A.A.M.; Mendes, B.M.J.; Pavan, A.; Rodriguez, A.P.M. Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments. Sci. Agric. 2003, 60, 23–29.
  17. Molinari, H.B.C.; Bespalhok, J.C.; Kobayashi, A.K.; Pereira, L.F.P.; Vieira, L.G.E. Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo (Citrus paradisi macf.×Poncirus trifoliata l. Raf.) using thin epicotyl sections. Sci. Hortic. 2004, 99, 379–385.
  18. Ghorbel, R.; Navarro, L.; Duran-Vila, N. Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C. Aurantium) and alemow (C. Macrophylla). J. Hortic. Sci. Biotechnol. 1998, 73, 323–327.
  19. Moore, G.A.; Jacono, C.C.; Neidigh, J.L.; Lawrence, S.D.; Cline, K. Agrobacterium-mediated transformation of citrus stem segments and regeneration of transgenic plants. Plant Cell Rep. 1992, 11, 238–242.
  20. Febres, V.; Fisher, L.; Khalaf, A.; Moore, G.A. Citrus transformation: Challenges and prospects. In Genetic Transformation; Alvarez, M., Ed.; IntechOpen: London, UK, 2011.
  21. Cervera, M.; López, M.M.; Navarro, L.; Peña, L. Virulence and supervirulence of Agrobacterium tumefaciensin woody fruit plants. Physiol. Mol. Plant Pathol. 1998, 52, 67–78.
  22. Dutt, M.; Erpen, L.; Grosser, J.W. Genetic transformation of the ‘W Murcott’ tangor: Comparison between different techniques. Sci. Hortic. 2018, 242, 90–94.
  23. Almeida, W.A.B.; Mourão Filho, F.A.A.; Pino, L.E.; Boscariol, R.L.; Rodriguez, A.P.M.; Mendes, B.M.J. Genetic transformation and plant recovery from mature tissues of Citrus sinensis l. Osbeck. Plant Sci. 2003, 164, 203–211.
  24. Cervera, M.; Navarro, A.; Navarro, L.; Peña, L. Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. Tree Physiol. 2008, 28, 55–66.
  25. Kobayashi, A.K.; Bespalhok, J.C.; Pereira, L.F.P.; Vieira, L.G.E. Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments. Plant Cell Tissue Organ Cult. 2003, 74, 99–102.
  26. Aderkas, P.V.; Bonga, J. Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol. 2000, 20, 921–928.
  27. Henry, R. Molecular and biochemical characterization of somaclonal variation. In Somaclonal Variation and Induced Mutations in Crop Improvement. Current Plant Science and Biotechnology in Agriculture; Jain, S.M., Brar, D.S., Ahloowalia, B.S., Eds.; Springer: Dordrecht, The Netherlands, 1998; Volume 32.
  28. Omar, A.A.; Dutt, M.; Gmitter, F.G.; Grosser, J.W. Somatic embryogenesis: Still a relevant technique in citrus improvement. In In Vitro Embryogenesis in Higher Plants; Humana Press: New York, NY, USA, 2016; Volume 1359, pp. 289–327.
  29. Dutt, M.; Grosser, J.W. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus. Plant Cell Rep. 2010, 29, 1251–1260.
  30. Haccius, B. Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorphology 1978, 28, 74–81.
  31. Toonen, M.A.J.; Hendriks, T.; Schmidt, E.D.L.; Verhoeven, H.A.; van Kammen, A.; de Vries, S.C. Description of somatic-embryo-forming single cells in carrot suspension cultures employing video cell tracking. Planta 1994, 194, 565–572.
  32. Lee, K.S.; Zapata-Arias, F.J.; Brunner, H.; Afza, R. Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp. Plant Cell Tissue Organ Cult. 1997, 51, 1–8.
  33. Chan, J.L.; Saénz, L.; Talavera, C.; Hornung, R.; Robert, M.; Oropeza, C. Regeneration of Coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis. Plant Cell Rep. 1998, 17, 515–521.
  34. Rugkhla, A.; Jones, M.G.K. Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum. J. Exp. Bot. 1998, 49, 563–571.
  35. Nonohay, J.S.; Mariath, J.E.A.; Winge, H. Histological analysis of somatic embryogenesis in brazilian cultivars of barley, Hordeum vulgare vulgare, poaceae. Plant Cell Rep. 1999, 18, 929–934.
  36. Roussos, P. Orange (Citrus sinensis (l.) Osbeck). In Nutritional Composition of Fruit Cultivars; Academic Press: Cambridge, UK, 2016; pp. 469–496.
  37. Cao, J.; Kang, C.; Chen, Y.; Karim, N.; Wang, Y.; Sun, C. Physiochemical changes in Citrus reticulata cv. Shatangju fruit during vesicle collapse. Postharvest Biol. Technol. 2020, 165, 111180.
  38. Yan, H.; Zhou, B.; Jiang, B.; Lv, Y.; Moniruzzaman, M.; Zhong, G.; Zhong, Y. Comparative analysis of bacterial and fungal endophytes responses to Candidatus liberibacterasiaticus infection in leaf midribs of Citrus reticulata cv. Shatangju. Physiol. Mol. Plant. Pathol. 2021, 113, 101590.
  39. Omar, A.A.; Murata, M.M.; El-Shamy, H.A.; Graham, J.H.; Grosser, J.W. Enhanced resistance to citrus canker in transgenic mandarin expressing xa21 from rice. Transgenic Res. 2018, 27, 179–191.
  40. Grosser, J.W.; Calović, M.; Louzada, E.S. Protoplast fusion technology—Somatic hybridization and cybridization. In Plant Cell Culture; Davey, M.R., Anthony, P., Eds.; John Wiley & Sons, Ltd.: New York, NY, USA, 2010; pp. 175–198.
  41. Kayim, M.; Koc, N.K. The effects of some carbohydrates on growth and somatic embryogenesis in citrus callus culture. Sci. Hortic. 2006, 109, 29–34.
  42. Rangan, T.S.; Murashige, T.; Bitters, W.P. In vitro initiation of nucellar embryos in monoembryonic Citrus. HortScience 1968, 3, 226–227.
  43. Bitters, W.P.; Murashigi, T.; Rangan, T.S.; Nauer, E.M. Investigations on established virusfree plants through tissue culture. Calif. Citrus Nurs. Soc. 1970, 9, 27–30.
  44. Button, J.; Bornman, C.H. Development of nucellar plants from unpollinated and unfertilized ovules of the washington navel orange in vitro. J. S. Afr. Bot. 1971, 37, 127–134.
  45. Gmitter, F.G.; Moore, G.A. Plant regeneration from undeveloped ovules and embryogenic calli of Citrus: Embryo production, germination, and plant survival. Plant Cell Tissue Organ Cult. 1986, 6, 139–147.
  46. Carimi, F.; Tortorici, M.C.; De Pasquale, F.; Crescimanno, F.G. Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [Citrus sinensis (l.) osb.]. Plant Cell Tissue Organ Cult. 1998, 54, 183–189.
  47. Litz, R.E.; Moore, G.A.; Srinivasan, C. In vitro systems for propagation and improvement of tropical fruits and palms. Hortic. Rev. 1985, 7, 157–200.
  48. Nito, N.; Iwamasa, M. In vitro plantlet formation from juice vesicle callus of satsuma (Citrus unshiu Marc.). Plant Cell Tissue Organ Cult. 1990, 20, 137–140.
  49. Hidaka, T.; Yamada, Y.; Shichijo, T. Plantlet formation by anther culture of Citrus aurantium L. Ikushugaku Zasshi 1982, 32, 247–252.
  50. Carimi, F.; De Pasquale, F.; Crescimanno, F.G. Somatic embryogenesis from styles of lemon (Citrus limon). Plant Cell Tissue Organ Cult. 1994, 37, 209–211.
  51. Gill, M.I.S.; Singh, Z.; Dhillon, B.S.; Gosal, S.S. Somatic embryogenesis and plantlet regeneration in mandarin (Citrus reticulata blanco). Sci. Hortic. 1995, 63, 167–174.
  52. Navarro, L.; Ortíz, J.; Juárez, J. Aberrant citrus plants obtained by somatic embryogenesis of nucelli cultured in vitro. HortScience 1985, 20, 214–215.
  53. Grosser, J.W.; Gmitter, F.G. Protoplast fusion for production of tetraploids and triploids: Applications for scion and rootstock breeding in citrus. Plant Cell Tissue Organ Cult. 2011, 104, 343–357.
  54. Moscatiello, R.; Baldan, B.; Navazio, L. Plant cell suspension cultures. In Plant Mineral Nutrients; Maathuis, F., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, USA, 2013; Volume 953, pp. 77–93.
  55. Mathur, J.; Koncz, C. Establishment and maintenance of cell suspension cultures. In Arabidopsis Protocols; Martinez-Zapater, J.M., Salinas, J., Eds.; Methods in Molecular Biology; Humana Press: New York, NY, USA, 1988; Volume 82.
  56. Guo, W.; Li, D.; Duan, Y. Citrus transgenics: Current status and prospects. Transgenic Plant J. 2007, 1, 202–209.
  57. Ghaderi, I.; Sohani, M.M.; Mahmoudi, A. Efficient genetic transformation of sour orange, Citrus aurantium l. Using agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus. Plant Gene 2018, 14, 7–11.
  58. Zhang, D.-M.; Zhong, Y.; Chang, X.-J.; Hu, M.-L.; Cheng, C.-Z.; Zhang, Y.-Y. A simple and efficient in planta transformation method for pommelo (Citrus maxima) using Agrobacterium tumefaciens. Sci. Hortic. 2017, 214, 174–179.
  59. Donmez, D.; Simsek, O.; Izgu, T.; Kacar, Y.A.; Mendi, Y.Y. Genetic transformation in citrus. Sci. World J. 2013, 2013, 491207.
  60. Kong, Q.; Wu, M.; Huan, Y.; Zhang, L.; Liu, H.; Bou, G.; Luo, Y.; Mu, Y.; Liu, Z. Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs. PLoS ONE 2009, 4, e6679.
  61. Rahman, M.; Hwang, G.; Abdul Razak, S.; Sohm, F.; Maclean, N. Copy number related transgene expression and mosaic somatic expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus). Transgenic Res. 2001, 9, 417–427.
  62. Hobbs, S.L.A.; Warkentin, T.D.; DeLong, C.M.O. Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol. Biol. 1993, 21, 17–26.
  63. Collier, R.; Dasgupta, K.; Xing, Y.P.; Hernandez, B.T.; Shao, M.; Rohozinski, D.; Kovak, E.; Lin, J.; de Oliveira, M.L.P.; Stover, E.; et al. Accurate measurement of transgene copy number in crop plants using droplet digital PCR. Plant J. 2017, 90, 1014–1025.
  64. Prols, F.; Meyer, P. The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in petunia hybrida. Plant J. 1992, 2, 465–475.
  65. Ottaviani, M.-P.; Smits, T.; Cate, C.H.H.T. Differential methylation and expression of the β-glucuronidase and neomycin phosphotransferase genes in transgenic plants of potato cv. Bintje. Plant Sci. 1993, 88, 73–81.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
Upload a video for this entry
Information
Subjects: Plant Sciences
Contributor MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register : Md Moniruzzaman
View Times: 1.2K
Revisions: 4 times (View History)
Update Date: 15 Apr 2021
Table of Contents
    Notice
    You are not a member of the advisory board for this topic. If you want to update advisory board member profile, please contact office@encyclopedia.pub.
    OK
    Confirm
    Only members of the Encyclopedia advisory board for this topic are allowed to note entries. Would you like to become an advisory board member of the Encyclopedia?
    Yes
    No
    ${ textCharacter }/${ maxCharacter }
    Submit
    Cancel
    There is no comment~
    ${ textCharacter }/${ maxCharacter }
    Submit
    Cancel
    ${ selectedItem.replyTextCharacter }/${ selectedItem.replyMaxCharacter }
    Submit
    Cancel
    Confirm
    Are you sure to Delete?
    Yes No
    Academic Video Service
    Feedback