Genetic Transformation for Functional Genomics of Sorghum

  • Monika DalalEmail author
Part of the Compendium of Plant Genomes book series (CPG)


Plant transformation is an essential requirement for fundamental research in functional biology and for crop improvement. Sorghum is primarily a recalcitrant crop for tissue culture and transformation. It has taken three decades of painstaking optimization efforts to reach a transformation efficiency of 20 and 30 % through particle bombardment and Agrobacterium-mediated genetic transformation in sorghum, respectively. This chapter describes the different variables that were analyzed for the success of tissue culture and transformation in sorghum. These factors include type of explants, culture media, hormone combinations, methods of gene transfer, vectors, selection marker genes, and so on. Furthermore, efforts for deployment of this technique for sorghum improvement in the area of biotic and abiotic stress tolerance, and improvement of nutritional quality are discussed.


Regeneration Transformation Callus Transgenic Quality Stress 


  1. Ahmad N, Sant R, Bokan M, Steadman KJ, Godwin ID (2012) Expression pattern of the alpha-kafi rin promoter coupled with a signal peptide from Sorghum bicolor L. Moench J Biomed Biotechnol 2012:1–8CrossRefGoogle Scholar
  2. Baskaran P, Rajeswari BR, Jayabalan N (2006) Development of an in vitro regeneration system in sorghum [Sorghum bicolor (l.) Moench] using root transverse thin cell layers (tTCLs). Turk J Bot 30:1–9Google Scholar
  3. Battraw M, Hall YC (1991) Stable transformation of Sorghum bicolor protoplasts with chimeric neomycin phosphotransferase II and β-glucuronidase genes. Theor Appl Genet 82:161–168CrossRefPubMedGoogle Scholar
  4. Bevan MW, Flavell RB, Chilton MD (1983) A chimeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184–187CrossRefGoogle Scholar
  5. Brandizzi F, Irons S, Kearns A, Hawes C (2003) BY-2 cells: culture and transformation for live cell imaging. Curr Protoc Cell Biol 19:1.7.1–1.7.16Google Scholar
  6. Cai T, Butler L (1990) Plant-regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums. Plant Cell Tiss Org Cult 20:101–110CrossRefGoogle Scholar
  7. Can ND, Nakamura S, Haryanto TAD, Yoshida T (1998) Effects of physiological status of parent plants and culture medium composition on the anther culture of sorghum. Plant Prod Sci 1:211–215CrossRefGoogle Scholar
  8. Carvalho CHS, Zehr UB, Gunaratna N, Anderson J, Kononowicz HH, Hodges TK, Axtell JD (2004) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet Mol Biol 27:259–269CrossRefGoogle Scholar
  9. Casas AM, Kononowicz AK, Haan TG, Zhang LY, Tomes DT, Bressan RA, Hasegawa PM (1997) Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences. In Vitro Cell Dev Biol Plant 33:92–100CrossRefGoogle Scholar
  10. Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell John D, Butlers Larry G, Bressan Ray A, Hasegawa Paul M (1993) Transgenic sorghum plants via microprojectile bombardment. Agric Sci 90:11212–11216Google Scholar
  11. Cho MJ, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138:229–244CrossRefGoogle Scholar
  12. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  13. Da Silva LS, Taylor J, Taylor JRN (2011) Transgenic sorghum with altered kafi rin synthesis: Kafi rin solubility, polymerization, and protein digestion. J Agric Food Chem 59:9265–9270CrossRefPubMedGoogle Scholar
  14. Elkonin LA, Pakhomova NV (2000) Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. Plant Cell Tiss Org Cult 61:115–123CrossRefGoogle Scholar
  15. Elkonin IA, Lopushanskaya RF, Pakhomova NV (1995) Initiation and maintenance of friable, embryogenic callus of sorghum (Sorghum bicolor (L.) Moench) by amino acids. Maydica 40:153–157Google Scholar
  16. Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JS et al (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80:4803–4807CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gao Z, Jayaraj J, Muthukrishnan S, Claflin L, Liang GH (2005a) Efficient genetic transformation of Sorghum using a visual screening marker. Genome 48(2):321–333CrossRefPubMedGoogle Scholar
  19. Gao Z, Xie X, Ling Y, Muthukrishnan S, Liang GH (2005b) Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system. Plant Biotechnol J 3:591–599CrossRefPubMedGoogle Scholar
  20. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37CrossRefPubMedPubMedCentralGoogle Scholar
  21. Girijashankar V, Sharma HC, Sharma KK, Swathisree V, Prasad LS, Bhat BV, Royer M, Secundo BS, Narasu ML, Altosaar I, Seetharama N (2005) Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus). Plant Cell Rep 24:513–522CrossRefPubMedGoogle Scholar
  22. Grootboom AW, Mkhonza NL, Mbambo Z, O’Kennedy MM, da Silva LS, Taylor J, Taylor JR, Chikwamba R, Mehlo L (2014) Co-suppression of synthesis of major α-kafirin sub-class together with γ-kafirin-1 and γ-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum. Plant Cell Rep 33:521–537CrossRefPubMedGoogle Scholar
  23. Grootboom AW, Mkhonza NL, O’Kennedy MM, Chakauya E, Kunert K, Chikwamba RK (2010) Biolistic mediated sorghum (Sorghum bicolor L. Moench) transformation via mannose and bialaphos based selection systems. Int J Bot 6:89–94CrossRefGoogle Scholar
  24. Guo C, Cui W, Feng X, Zhao J, Lu G (2011) Sorghum insect problems and management. J Integr Plant Biol 53:178–192CrossRefPubMedGoogle Scholar
  25. Gupta S, Khanna VK, Singh R, Garg GK (2006) Strategies for overcoming genotypic limitations of in vitro regeneration and determination of genetic components of variability of plant regeneration traits in sorghum. Plant Cell Tiss Organ Cult 86:379–388CrossRefGoogle Scholar
  26. Gurel S, Gurel E, Miller TI, Lemaux PG (2012) Agrobacterium-mediated transformation of Sorghum bicolor using immature embryos. Methods Mol Biol 47:109–122CrossRefGoogle Scholar
  27. Gurel S, Gurel E, Kaur R, Wong J, Meng L, Tan H-Q, Lemaux PG (2009) Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep 28:429–444CrossRefPubMedGoogle Scholar
  28. Hagio T, Blowers AD, Earle ED (1991) Stable transformation of sorghum cell cultures after bombardment with DNA-coated microprojectiles. Plant Cell Rep 10:260–264CrossRefPubMedGoogle Scholar
  29. Harshavardhan D, Rani TS, Ulaganathan K, Seetarama N (2002) An improved protocol for regeneration of Sorghum bicolor from isolated shoot apices. Plant Biotechnol 19:163–171CrossRefGoogle Scholar
  30. Herrera-Estrella L, DeBlock M, Messens E, Hernalsteens JP, Van Montagu M, Schell J (1983) Chimeric genes as dominant selectable markers in plant cells. EMBO J 2:987–996PubMedPubMedCentralGoogle Scholar
  31. Hill Ambroz KL, Weeks JT (2001) Comparison of constitutive promoters for sorghum transformation. Cereal Res Commun 29:17–24Google Scholar
  32. Howe A, Sato S, Dweikat I, Fromm M, Clemente T (2006) Rapid and reproducible Agrobacterium-mediated transformation of sorghum. Plant Cell Rep 25:784–791CrossRefPubMedGoogle Scholar
  33. Jeoung JM, Krishnaveni S, Muthukrishnan S, Trick HN, Liang GH (2002) Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers. Hereditas 137:20–28CrossRefPubMedGoogle Scholar
  34. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:e188CrossRefPubMedPubMedCentralGoogle Scholar
  35. Joersbo M, Donaldson I, Kreibery J, Petersen SG, Brunstedt J, Okkels FT (1998) Analysis of mannose selection used for transformation of sugar beet. Mol Breed 4:111–117CrossRefGoogle Scholar
  36. Jogeswar G, Ranadheer D, Anjaiah V, KaviKishor PB (2007) High frequency somatic embryogenesis and regeneration in different genotypes of Sorghum bicolor (L.) Moench from immature inflorescence explants. In vitro Cell Dev Biol Plant 43:159–166CrossRefGoogle Scholar
  37. Kaeppler HF, Pedersen JF (1997) Evaluation of 41 elite and exotic inbred Sorghum genotypes for high quality callus production. Plant Cell Tiss Organ Cult 48:71–75CrossRefGoogle Scholar
  38. Kaeppler HF, Pedersen JF (1996) Media effects on phenotype of callus cultures initiated from photoperiod-insensitive elite inbred sorghum lines. Maydica 41:83–89Google Scholar
  39. Kosambo-Ayoo LM, Bader M, Loerz H, Becker D (2011) Transgenic sorghum (Sorghum bicolor L. Moench) developed by transformation with chitinase and chitosanase genes from Trichoderma harzianum expresses tolerance to anthracnose. Afri J Biotechnol 10:3659–3670Google Scholar
  40. Krishnaveni S, Joeung JM, Muthukrishnan S, Liang GH (2001) Transgenic sorghum plants constitutively expressing a rice chitinase gene show improved resistance to stalk rot. J Genet Breed 55:151–158Google Scholar
  41. Kumar T, Dweikat I, Sato S, Ge Z, Nersesian N, Chen H, Elthon T, Bean S, Ioerger BP, Tilley M, Clemente T (2012) Modulation of kernel storage proteins in grain sorghum (Sorghum bicolor (L.) Moench). Plant Biotechnol J 10:533–544CrossRefPubMedGoogle Scholar
  42. Kumaravadivel N, Rangasamy S (1994) Plant regeneration from sorghum anther cultures and field evaluation of progeny. Plant Cell Rep 13:286–290CrossRefPubMedGoogle Scholar
  43. Lipkie TE, De Moura FF, Zhao ZY, Albertsen MC, Che P, Glassman K, Ferruzzi MG (2013) Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum. J Agric Food Chem 61:5764–5771CrossRefPubMedGoogle Scholar
  44. Liu G, Godwin ID (2012) Highly efficient sorghum transformation. Plant Cell Rep 31:999–1007CrossRefPubMedPubMedCentralGoogle Scholar
  45. Liu G, Gilding EK, Godwin ID (2013) Additive effects of three auxins and copper on sorghum in vitro root induction. In vitro Cell Dev Biol Plant 49:191–197CrossRefGoogle Scholar
  46. Lu L, Wu X, Yin X, Morrand J, Chen X, Folk WR, Zhang ZJ (2009) Development of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker. Plant Cell Tissue Organ Cult 99:97–108CrossRefGoogle Scholar
  47. Ma L, Lukasik E, Gawehns F, Takken FL (2012) The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves. Methods Mol Biol 835:61–74CrossRefPubMedGoogle Scholar
  48. MacKinnon C, Gunderson G, Nabors MW (1987) High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Vitro Cell Dev Biol Plant 23:443–448CrossRefGoogle Scholar
  49. Maheswari M, Jyothilakshmi N, Yadav SK, Varalaxmi Y, Vijaya Lakshmi A, Vanaja M, Venkateswarlu B (2006) Efficient plant regeneration from shoot apices of Sorghum. Biol Plant 50:741–744CrossRefGoogle Scholar
  50. Maheswari M, Varalaxmi Y, Vijayalakshmi A, Yadav SK, Sharmila P, Venkateswarlu B, Vanaja M, Saradhi PP (2010) Metabolic engineering using mtlD gene enhances tolerance to water deficit and salinity in sorghum. Biol Plant 54:647–652CrossRefGoogle Scholar
  51. Mall TK, Dweikat I, Sato SJ, Neresian N, Xu K, Ge Z, Wang D, Elthon T, Clemente T (2011) Expression of the rice CDPK-7 in sorghum: molecular and phenotypic analyses. Plant Mol Biol 75:467–479CrossRefPubMedGoogle Scholar
  52. Maralappanavar MS, Kuruvinashetti MS, Harti CC (2000) Regeneration, establishment and evaluation of somaclones in Sorghum bicolor (L.) Moench. Euphytica 115:173–180CrossRefGoogle Scholar
  53. Mok MC, Mok DWS, Armstrong DJ, Shudo K, Isogai Y, Okamoto T (1982) Cytokinin activity of N-phenyl-N’-1, 2, 3-thiadiazol-5-ylurea (Thidiazuron). Phytochemistry 21:1509–1511CrossRefGoogle Scholar
  54. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  55. Nguyen T-V, Thu TT, Claeys M, Angenon G (2007) Agrobacterium-mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system. Plant Cell Tiss Organ Cult 91:155–164CrossRefGoogle Scholar
  56. Nirwan RS, Kothari SL (2003) High copper levels improve callus induction and plant regeneration in Sorghum bicolor (L.) Moench. Vitro Cell Dev Biol Plant 39:161–164CrossRefGoogle Scholar
  57. Nirwan RS, Kothari SL (2004) High frequency shoot organogenesis in Sorghum bicolor (L.). J Plant Biochem Biotechnol 13:149–152CrossRefGoogle Scholar
  58. Oria MP, Hamaker BR, Axtell JD, Huang CP (2000) A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies. Proc Natl Acad Sci USA 97:5065–5070CrossRefPubMedPubMedCentralGoogle Scholar
  59. Ou-Lee T, Turgeon R, Wu R (1986) Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat and sorghum. Proc Natl Acad Sci USA 83:6815–6819CrossRefPubMedPubMedCentralGoogle Scholar
  60. Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H, Wang X, Wicker T, Bharti AK, Chapman J, Feltus FA, Gowik U, Grigoriev IV, Lyons E, Maher CA, Martis M, Narechania A, Otillar RP, Penning BW, Salamov AA, Wang Y, Zhang L, Carpita NC, Freeling M, Gingle AR, Hash CT, Keller B, Klein P, Kresovich S, McCann MC, Ming R, Peterson DG, Mehboob-ur-Rahman Ware D, Westhoff P, Mayer KF, Messing J, Rokhsar DS (2009) The Sorghum bicolor genome and the diversification of grasses. Nature 457:551–556CrossRefPubMedGoogle Scholar
  61. Pola SR, Saradamani N (2006) Somatic embryogenesis and plantlet regeneration in Sorghum bicolor(L.) Moench from leaf segments. J Cell Mol Biol 5:99–107Google Scholar
  62. Sai Kishore N, Visarada KB, Aravinda Lakshmi Y, Pashupatinath E, Rao SV, Seetharama N (2006) In vitro culture methods in sorghum with shoot tip as the explant material. Plant Cell Rep 25:174–182CrossRefPubMedGoogle Scholar
  63. Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K (2000) Overexpression of a single Ca2-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J 23:319–327CrossRefPubMedGoogle Scholar
  64. Sairam RV, Seetharama N, Devi PS, Verma A, Murthy UR, Potrykus I (1999) Plant regeneration from mesophyll protoplasts in sorghum [Sorghum bicolor (L.) Moench]. Plant Cell Rep 18:972–977CrossRefGoogle Scholar
  65. Sato S, Clemente T, Dweikat I (2004) Identifi cation of an elite sorghum genotype with high in vitro performance capacity. In Vitro Cell Dev Biol Plant 40:57–60CrossRefGoogle Scholar
  66. Schmidt M, Bothma G (2006) Risk assessment for transgenic sorghum in Africa: crop-to-crop gene flow in Sorghum bicolor (L.) Moench. Crop Sci 46:790–798Google Scholar
  67. Schulz P, Herde M, Romeis T (2013) Calcium-Dependent Protein Kinases: Hubs in Plant Stress Signaling and Development. Plant Physiol 163:523–530CrossRefPubMedPubMedCentralGoogle Scholar
  68. Scott A, Wyatt S, Tsou PL, Robertson D, Allen NS (1999) Model system for plant cell biology: GFP imaging in living onion epidermal cells. BioTechnique 26:1128–1132Google Scholar
  69. Seetharama N, Sairam RV, Rani TS (2000) Regeneration of sorghum from shoot tip cultures and field performance of the progeny. Plant Cell Tiss Organ Cult 61:169–173CrossRefGoogle Scholar
  70. Tadesse Y, Sagi L, Swennen R, Jacobs M (2003) Optimization of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment. Plant Cell Tissue Organ Cult 75:1–18CrossRefGoogle Scholar
  71. Tesso T, Kapran I, Grenier  C, Snow A, Sweeney P (2008) The potential for crop-to-wild gene flow in sorghum in Ethiopia and Niger: a geographic survey.  Crop Sci 38:1425–1431Google Scholar
  72. The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815CrossRefGoogle Scholar
  73. The International Rice Genome Sequence Project (2005) The map-based sequence of the rice genome. Nature 436:793–800CrossRefGoogle Scholar
  74. Visarada KBRS, Padmaja PG, Saikishore N, Pashupatinath E, Royer M, Seetharama N, Patil JV (2014) Production and evaluation of transgenic sorghum for resistance to stem borer. Vitro Cell Dev Biol Plant 50:176–189CrossRefGoogle Scholar
  75. Wang W, Wang J, Yang C, Li Y, Liu L, Xu J (2007) Pollen-mediated transformation of Sorghum bicolor plants. Biotechnol Appl Biochem 48:79–83CrossRefPubMedGoogle Scholar
  76. Wong JH, Lau T, Cai N, Singh J, Pedersen JF, Vensel WH (2009) Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm. J Cereal Sci 49:73–82Google Scholar
  77. Wu XR, Kenzior A, Willmot D, Scanlon S, Chen Z, Topin A, He SH, Acevedo A, Folk WR (2007) Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine. Plant J 50:627–636CrossRefPubMedGoogle Scholar
  78. Wu Y, Li X, Xiang W, Zhu C, Lin Z, Wu Y, Li J, Pandravada S, Ridder DD, Bai G, Wang ML, Trick HN, Bean SR, Tuinstra MR, Tesso TT, Yu J (2012) Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1. Proc Natl Acad Sci USA 109:10281–10286CrossRefPubMedPubMedCentralGoogle Scholar
  79. Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu U, Cho MJ, Zhao ZY (2014) Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants. Vitro Cell Dev Biol Plant 50:9–18CrossRefPubMedGoogle Scholar
  80. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefPubMedGoogle Scholar
  81. Zhao ZY, Cai TS, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798CrossRefPubMedGoogle Scholar
  82. Zhao Z-Y, Glassman K, Sewalt V, Wang N, Miller M, Chang S, Thompson T, Catron S, Wu E, Bidney D, Kedebe Y, Jung R (2003) Nutritionally improved sorghum transgenic. In: Vasil IK (ed) Plant biotechnology 2002 and beyond. Kluwer Academic Dordrecht, The Netherlands Publishers, pp 413–416Google Scholar
  83. Zhong H, Wang W, Sticklen M (1998) In vitro morphogenesis of Sorghum bicolor (L.) Moench: efficient plant regeneration from shoot apices. J Plant Physiol 153:719–726CrossRefGoogle Scholar
  84. Zhu H, Muthukrishnan S, Krishnaveni S, Wilde G, Jeoung JM, Liang GH (1998a) Biolistic transformation of sorghum using a rice chitinase gene. J Genet Breed 52:243–252Google Scholar
  85. Zhu H, Muthukrishnan S, Krishnaveni S, Wilde G, Jeoung J-M, Liang GH (1998b) Biolistic transformation of sorghum using a rice chitinase gene. J Genet Breed 52:243–252Google Scholar

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© Springer International Publishing AG 2016

Authors and Affiliations

  1. 1.ICAR-National Research Centre on Plant BiotechnologyPusaNew DelhiIndia

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