Callus (cell biology)

Plant callus (plural calluses or calli) is a growing mass of unorganized plant parenchyma cells. In living plants, callus cells are those cells that cover a plant wound. In biological research and biotechnology callus formation is induced from plant tissue samples (explants) after surface sterilization and plating onto tissue culture medium in vitro (in a closed culture vessel such as a Petri dish).^[1] The culture medium is supplemented with plant growth regulators, such as auxin, cytokinin, and gibberellin, to initiate callus formation or somatic embryogenesis. Callus initiation has been described for all major groups of land plants.

Nicotiana tabacum parenchyma cells in culture

Callus induction and tissue culture

 

Callus cells forming during a process called "induction" in Pteris vittata

Plant species representing all major land plant groups have been shown to be capable of producing callus in tissue culture.^{[2][3][4][5][6][7][8][9][10][11][12]} A callus cell culture is usually sustained on gel medium. Callus induction medium consists of agar and a mixture of macronutrients and micronutrients for the given cell type. There are several types of basal salt mixtures used in plant tissue culture, but most notably modified Murashige and Skoog medium,^[13] White's medium,^[14] and woody plant medium.^[15] Vitamins are also provided to enhance growth such as Gamborg B5 vitamins.^[16] For plant cells, enrichment with nitrogen, phosphorus, and potassium is especially important. Plant callus is usually derived from somatic tissues. The tissues used to initiate callus formation depends on plant species and which tissues are available for explant culture. The cells that give rise to callus and somatic embryos usually undergo rapid division or are partially undifferentiated such as meristematic tissue. In alfalfa, Medicago truncatula, however callus and somatic embryos are derived from mesophyll cells that undergo dedifferentiation.^[17] Plant hormones are used to initiate callus growth. After the callus has formed, the concentration of hormones in the medium may be altered to shift the development from callus to root formation, shoot growth or somatic embryogenesis. The callus tissues then undergo further cell growth and differentiation, forming the respective organ primordia. The fully developed organs can then be used for the regeneration of the new mature plants.

 

Callus induced from Pteris vittata gametophytes

Morphology

Specific auxin to cytokinin ratios in plant tissue culture medium give rise to an unorganized growing and dividing mass of callus cells. Callus cultures are often broadly classified as being either compact or friable. Compact calluses are typically green and sturdy, while friable calluses are white to creamy yellow in color, fall apart easily and can be used to generate cell suspension cultures and somatic embryos. In maize, these two callus types are designated as type I (compact) and type II (friable).^[18] Callus can directly undergo direct organogenesis and/or embryogenesis where the cells will form an entirely new plant.

Callus cell death

Callus can brown and die during culture, mainly due to oxidation of phenolic compounds. In Jatropha curcas callus cells, small organized callus cells became disorganized and varied in size after browning occurred.^[19] Browning has also been associated with oxidation and phenolic compounds in both explant tissues and explant secretions.^[20] In rice, presumably, a condition which is favorable for scutellar callus induction induces necrosis too.^[21]

Uses

Callus cells are not necessarily genetically homogeneous because a callus is often made from structural tissue, not individual cells.^{[clarification needed]} Nevertheless, callus cells are often considered similar enough for standard scientific analysis to be performed as if on a single subject. For example, an experiment may have half a callus undergo a treatment as the experimental group, while the other half undergoes a similar but non-active treatment as the control group.

Plant calluses derived from many different cell types can differentiate into a whole plant, a process called regeneration, through addition of plant hormones to the culture medium. This ability is known as totipotency. A classical experiment by Folke Skoog and Carlos O. Miller on tobacco pith used as the starting explant shows that the supplementation of culture media by different ratios of auxin to cytokinin concentration induces the formation of roots – with higher auxin to cytokinin ratio, the rooting (rhizogenesis) is induced, applying equal amounts of both hormones stimulates further callus growth and increasing the auxin to cytokinin ratio in favor of the cytokinin leads to the development of shoots.^[22] Regeneration of a whole plant from a single cell allows transgenics researchers to obtain whole plants which have a copy of the transgene in every cell. Regeneration of a whole plant that has some genetically transformed cells and some untransformed cells yields a chimera. In general, chimeras are not useful for genetic research or agricultural applications.

Genes can be inserted into callus cells using biolistic bombardment, also known as a gene gun, or Agrobacterium tumefaciens. Cells that receive the gene of interest can then be recovered into whole plants using a combination of plant hormones. The whole plants that are recovered can be used to experimentally determine gene function(s), or to enhance crop plant traits for modern agriculture.

Callus is of particular use in micropropagation where it can be used to grow genetically identical copies of plants with desirable characteristics. To increase the yield, efficiency and explant survivability of micropropagation, a thorough care is taken for the optimization of the micropropagation protocol. For example, using explants composed of low totipotency cells may prolong the time necessary to obtain callus of sufficient size, increasing the total length of the experiment. Similarly, various plant species and explant types require specific plant hormones for callus induction and subsequent organogenesis or embryogenesis – for the formation and growth of maize calluses, auxin 2,4-Dichlorophenoxyacetic acid (2,4-D) was superior to 1-Naphthaleneacetic acid (NAA) or Indole-3-acetic acid (IAA), while the development of callus was hindered in prune explants after applying auxin Indole-3-butyric acid (IBA) but not IAA.^[23][24]

History

Henri-Louis Duhamel du Monceau investigated wound-healing responses in elm trees, and was the first to report formation of callus on live plants.^[25]

In 1908, E. F. Simon was able to induce callus from poplar stems that also produced roots and buds.^[26] The first reports of callus induction in vitro came from three independent researchers in 1939.^[27] P. White induced callus derived from tumor-developing procambial tissues of hybrid Nicotiana glauca that did not require hormone supplementation.^[14] Gautheret and Nobecourt were able to maintain callus cultures of carrot using auxin hormone additions.^{[citation needed]}

See also

• Embryo rescue
• Somatic embryogenesis
• Chimera (genetics)
• Hyperhydricity

References

1. What is Plant Tissue Culture?
2. Takeda, Reiji; Katoh, Kenji (1981). "Growth and sesquiterpenoid production by Calypogeia granulata inoue cells in suspension culture". Planta. 151 (6): 525–530. doi:10.1007/BF00387429. PMID 24302203. S2CID 21074846.
3. Peterson, M (2003). "Cinnamic acid 4-hydroxylase from cell cultures of the hornwort Anthoceros agrestis". Planta. 217 (1): 96–101. doi:10.1007/s00425-002-0960-9. PMID 12721853. S2CID 751110.
4. Beutelmann, P.; Bauer, L. (1 January 1977). "Purification and identification of a cytokinin from moss callus cells". Planta. 133 (3): 215–217. doi:10.1007/BF00380679. PMID 24425252. S2CID 34814574.
5. Atmane, N (2000). "Histological analysis of indirect somatic embryogenesis in the Marsh clubmoss Lycopodiella inundata (L.) Holub (Pteridophytes)". Plant Science. 156 (2): 159–167. doi:10.1016/S0168-9452(00)00244-2. PMID 10936522.
6. Yang, Xuexi; Chen, Hui; Xu, Wenzhong; He, Zhenyan; Ma, Mi (2007). "Hyperaccumulation of arsenic by callus, sporophytes and gametophytes of Pteris vittata cultured in vitro". Plant Cell Reports. 26 (10): 1889–1897. doi:10.1007/s00299-007-0388-6. PMID 17589853. S2CID 20891091.
7. Chavez, V. M.; Litz, R. E.; Monroy, M.; Moon, P. A.; Vovides, A. M. (1998). "Regeneration of Ceratozamia euryphyllidia (Cycadales, Gymnospermae) plants from embryogenic leaf cultures derived from mature-phase trees". Plant Cell Reports. 17 (8): 612–616. doi:10.1007/s002990050452. PMID 30736513. S2CID 29050747.
8. Jeon, MeeHee; Sung, SangHyun; Huh, Hoon; Kim, YoungChoong (1995). "Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves". Plant Cell Reports. 14 (8): 501–504. doi:10.1007/BF00232783. PMID 24185520. S2CID 20826665.
9. Finer, John J.; Kriebel, Howard B.; Becwar, Michael R. (1 January 1989). "Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.)". Plant Cell Reports. 8 (4): 203–206. doi:10.1007/BF00778532. PMID 24233136. S2CID 2578876.
10. O'Dowd, Niamh A.; McCauley, Patrick G.; Richardson, David H. S.; Wilson, Graham (1993). "Callus production, suspension culture and in vitro alkaloid yields of Ephedra". Plant Cell, Tissue and Organ Culture. 34 (2): 149–155. doi:10.1007/BF00036095. S2CID 25019305.
11. Chen, Ying-Chun; Chang, Chen; Chang, Wei-chin (2000). "A reliable protocol for plant regeneration from callus culture of Phalaenopsis". In Vitro Cellular & Developmental Biology - Plant. 36 (5): 420–423. doi:10.1007/s11627-000-0076-5. S2CID 30272969.
12. Burris, Jason N.; Mann, David G. J.; Joyce, Blake L.; Stewart, C. Neal (10 October 2009). "An Improved Tissue Culture System for Embryogenic Callus Production and Plant Regeneration in Switchgrass (Panicum virgatum L.)". BioEnergy Research. 2 (4): 267–274. doi:10.1007/s12155-009-9048-8. S2CID 25527007.
13. Murashige, Toshio; F. Skoog (July 1962). "A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures". Physiologia Plantarum. 15 (3): 473–497. doi:10.1111/j.1399-3054.1962.tb08052.x. S2CID 84645704.
14. White, P. R. (Feb 1939). "Potentially unlimited growth of excised plant callus in an artificial nutrient". American Journal of Botany. 26 (2): 59–4. doi:10.2307/2436709. JSTOR 2436709.
15. Lloyd, G; B McCown (1981). "Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture". Combined Proceedings, International Plant Propagators' Society. 30: 421–427.
16. Gamborg, OL; RA Miller; K Ojima (April 1968). "Nutrient requirements of suspension cultures of soybean root cells". Experimental Cell Research. 50 (1): 151–158. doi:10.1016/0014-4827(68)90403-5. PMID 5650857.
17. Wang, X.-D.; Nolan, K. E.; Irwanto, R. R.; Sheahan, M. B.; Rose, R. J. (10 January 2011). "Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells". Annals of Botany. 107 (4): 599–609. doi:10.1093/aob/mcq269. PMC 3064535. PMID 21224270.
18. Sidorov, Vladimir; Gilbertson, Larry; Addae, Prince; Duncan, David (April 2006). "Agrobacterium-mediated transformation of seedling-derived maize callus". Plant Cell Reports. 25 (4): 320–328. doi:10.1007/s00299-005-0058-5. ISSN 0721-7714. PMID 16252091. S2CID 22588581.
19. He, Yang; Guo, Xiulian; Lu, Ran; Niu, Bei; Pasapula, Vijaya; Hou, Pei; Cai, Feng; Xu, Ying; Chen, Fang (2009). "Changes in morphology and biochemical indices in browning callus derived from Jatropha curcas hypocotyls". Plant Cell, Tissue and Organ Culture. 98 (1): 11–17. doi:10.1007/s11240-009-9533-y. S2CID 44470975.
20. Dan, Yinghui; Armstrong, Charles L.; Dong, Jimmy; Feng, Xiaorong; Fry, Joyce E.; Keithly, Greg E.; Martinell, Brian J.; Roberts, Gail A.; Smith, Lori A.; Tan, Lalaine J.; Duncan, David R. (2009). "Lipoic acid—an [sic] unique plant transformation enhancer". In Vitro Cellular & Developmental Biology - Plant. 45 (6): 630–638. doi:10.1007/s11627-009-9227-5. S2CID 19424435.
21. Pazuki, Arman & Sohani, Mehdi (2013). "Phenotypic evaluation of scutellum-derived calluses in 'Indica' rice cultivars" (PDF). Acta Agriculturae Slovenica. 101 (2): 239–247. doi:10.2478/acas-2013-0020. Retrieved February 2, 2014.
22. Skoog, F.; Miller, C. O. (1957). "Chemical regulation of growth and organ formation in plant tissues cultured in vitro". Symposia of the Society for Experimental Biology. 11: 118–130. ISSN 0081-1386. PMID 13486467.
23. Sheridan, William F. (1975). "Tissue Culture of Maize I. Callus Induction and Growth". Physiologia Plantarum. 33 (2): 151–156. doi:10.1111/j.1399-3054.1975.tb03783.x. ISSN 1399-3054.
24. Štefančič, Mateja; Štampar, Franci; Osterc, Gregor (2005-12-01). "Influence of IAA and IBA on root development and quality of Prunus 'GiSelA 5' leafy cuttings". HortScience. 40 (7): 2052–2055. doi:10.21273/HORTSCI.40.7.2052. ISSN 0018-5345.
25. Razdan, M. K. (2003). Introduction to plant tissue culture (2. ed.). Enfield, NH [u.a.]: oxford Publishers. ISBN 1-57808-237-4.
26. Gautheret, Roger J. (1 December 1983). "Plant tissue culture: A history". The Botanical Magazine Tokyo. 96 (4): 393–410. doi:10.1007/BF02488184. S2CID 26425105.
27. Chawla, H.S. (2002). Introduction to plant biotechnology (2nd ed.). Enfield, N.H.: Science Publishers. ISBN 1-57808-228-5.