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High-frequency direct shoot organogenesis from garlic (Allium sativum L.) inflorescence and clonal fidelity assessment in regenerants

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Abstract

Garlic (Allium sativum L.) is an important bulb vegetable with high culinary and medicinal values. It is cultivated by exclusively vegetative propagation which leads to complex virus infection, biodiversity reduction, inhibition of multiple breeding strategies. An efficient and widely applicable shoot regeneration system was established from garlic inflorescence as explant. By optimizing the basal medium type, pH value and explant size, a mean shoot regeneration rate of 97% and mean shoot number of 23.4 per explant were achieved in 14 commercial cultivars with various characteristics and origins. Histological observation revealed that shoots were regenerated through direct organogenesis when meristemoid initiated from subepidermal cells without callus formation followed by periclinal and anticlinal division of epidermal and subepidermal cells. No polymorphic bands were detected by simple sequence repeat (SSR) analysis between regenerants and donor plants. Furthermore, flow cytometry analysis indicated that there was no significant variability of genome size, and all plants maintained their ploidy. These results confirmed the clonal fidelity of regenerants. In conclusion, the present study provides a shoot regeneration system with great potential in micropropagation, germplasm preservation, genetic transformation and ploidy manipulation of garlic.

Key message

Our study developed a widely applicable procedure for direct shoot regeneration via inflorescence of garlic and ascertained the clonal fidelity of regenerants by using simple sequence repeat (SSR) and flow cytometry.

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Abbreviations

BA:

6-Benzyladenine

CTAB:

Cetyltrimethyl ammonium bromide

FCM:

Flow cytometry

NAA:

α-Naphthalene acetic acid

PI:

Propidium iodide

SSR:

Simple sequence repeat

References

  • Al-Zahim MA, Ford-Lloyd BV, Newbury HJ (1999) Detection of somaclonal variation in garlic (Allium sativum L.) using RAPD and cytological analysis. Plant Cell Rep 18(6):473–477

    CAS  Google Scholar 

  • Ayabe M, Sumi S (1998) Establishment of a novel tissue culture method, stem disc culture, and its practical application to micropropagation of garlic (Allium sativum L). Plant Cell Rep 17(10):773–779

    CAS  PubMed  Google Scholar 

  • Ayabe M, Sumi S (2001) A novel and efficient tissue culture method - "stem-disc dome culture" - for producing virus-free garlic (Allium sativum L.). Plant Cell Rep 20(6):503–507

    CAS  Google Scholar 

  • Barandiaran X, Martin N, Alba C, Rodriguez-Conde MF, Di Pietro A, Martin J (1999) An efficient method for the in vitro management of multiple garlic accessions. In Vitro Cell Dev Biol Plant 35(6):466–469

    Google Scholar 

  • Barker AV, Ready KM (1994) Ethylene Evolution by Tomatoes Stressed by Ammonium Nutrition. American Society for Horticultural Science 119(4):706–710

    CAS  Google Scholar 

  • Bhojwani SS (1980) In vitro propagation of garlic by shoot proliferation. Sci Hortic 13(1):47–52

    CAS  Google Scholar 

  • Chani E, Veilleux RE, Boluarte-Medina T (2000) Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants. Plant Cell Tiss Organ Cult 60(2):101–112

    CAS  Google Scholar 

  • Chaturvedi AK, Shalom SR, Faigenboim-Doron A, Teper-Bamnolker P, Salam BB, Daus A, Kamenetsky R, Eshel D (2018) Differential carbohydrate gene expression during preplanting temperature treatments controls meristem termination and bulbing in garlic. Environmental Experimental Botany 150:280–291

    CAS  Google Scholar 

  • De Klerk G-J (1990) How to measure somaclonal variation. Acta Botanica Neerlandica 39(2):129–144

    Google Scholar 

  • Dijk PV (1993) Carlaviruses isolated from cultivated Allium species represent three viruses. Neth J Plant Pathol 99(6):233–257

    Google Scholar 

  • Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2(9):2233–2244

    CAS  PubMed  Google Scholar 

  • Doyle J (1991) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Ebi M, Kasai N, Masuda K (2000) Small inflorescence bulbils are best for micropropagation and virus elimination in garlic. Hortscience 35(4):735–737

    Google Scholar 

  • Etienne H, Bertrand B (2003) Somaclonal variation in Coffea arabica: effects of genotype and embryogenic cell suspension age on frequency and phenotype of variants. Tree Physiol 23(6):419–426

    CAS  PubMed  Google Scholar 

  • Fan B, He R, Shang Y, Xu L, Wang N, Gao H, Liu X, Wang Z (2017) System construction of virus-free and rapid-propagation technology of Baodi garlic (Allium sativum L.). Sci Hortic 225(Supplement C):498–504

    CAS  Google Scholar 

  • Fereol L, Chovelon V, Causse S, Michaux-Ferriere N, Kahane R (2002) Evidence of a somatic embryogenesis process for plant regeneration in garlic (Allium sativum L.). Plant Cell Rep 21(3):197–203

    CAS  Google Scholar 

  • Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220(4601):1049

    CAS  PubMed  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158

    CAS  PubMed  Google Scholar 

  • Gantait S, Mandal N, Das PK (2010) An overview on in vitro culture of genus Allium. American Journal of Plant Physiology 5(6):325–337

    CAS  Google Scholar 

  • Haque MS, Wada T, Hattori K (1997) High frequency shoot regeneration and plantlet formation from root tip of garlic. Plant Cell Tiss Organ Cult 50(2):83–89

    Google Scholar 

  • Hitchcock J, Schäfer G, Katz A, Kaschula CH (2014) The immunomodulating and anti-inflammatory effects of garlic organosulfur compounds in cancer prevention. Eur J Cancer 50(2):S238–S239

    Google Scholar 

  • Ipek M, Sahin N, Ipek A, Cansev A, Simon PW, Ipek M, Sahin N, Ipek A, Cansev A, Simon PW (2015) Development and validation of new SSR markers from expressed regions in the garlic genome. Scientia Agricola 72(1):41–46

    CAS  Google Scholar 

  • Izquierdo-Oviedo H, Disotuar R, González MC, González SJ (2016) Micropropagation of garlic (Allium sativum L.) and determination of the genetic stability of the plantlets obtained by AFLP markers. Biotecnol Apl 33(4):4211–4218

    Google Scholar 

  • Jain SM (2001) Tissue culture-derived variation in crop improvement. Euphytica 118(2):153–166

    CAS  Google Scholar 

  • Jaligot E, Rival A, Beulé T, Dussert S, Verdeil JL (2000) Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Rep 19(7):684–690

    CAS  PubMed  Google Scholar 

  • Jea S (1994) In vitro propagation and bulb formation of garlic. Can J Plant Sci 74(1):155–158

    Google Scholar 

  • Jiang W, Hua S, Zhou X, Han P, Lu Q, Qiu Y (2018) Assessment of genetic stability and analysis of alkaloids potential in micropropagated plants of Croomia japonica Miquel, an endangered, medicinal plant in China and Japan. Plant Cell Tiss Organ Cult 135(1):1–12

    CAS  Google Scholar 

  • Kamenetsky R, Faigenboim A, Mayer ES, Michael TB, Chen G, Kimhi S, Esquira I, Shalom SR, Eshel D, Rabinowitch HD (2015) Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.). BMC Genom 16(1):12–26

    CAS  Google Scholar 

  • Kenel F, Eady C, Brinch S (2010) Efficient Agrobacterium tumefaciens-mediated transformation and regeneration of garlic (Allium sativum L.) immature leaf tissue. Plant Cell Rep 29(3):223–230

    CAS  PubMed  Google Scholar 

  • Khan N, Chaudhary MF, Abbasi AM, Khan SA, Nazir A (2017) Development of an Efficient Callus Derived Regeneration System for Garlic (Allium sativum L.) from Root Explant. Journal of Plant Biology Agriculture Sciences 1(3):1–12

    CAS  Google Scholar 

  • Khatua TN, Borkar RM, Mohammed SA, Dinda AK, Srinivas R, Banerjee SK (2017) Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression. Front Pharmacol 8:18–28

    PubMed  PubMed Central  Google Scholar 

  • Kim EK, Hahn EJ, Murthy HN, Paek KY (2003) High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures. Plant Cell Tiss Organ Cult 73(3):231–236

    Google Scholar 

  • Klerk GJD, Hanecakova J, Jásik J (2008) Effect of medium-pH and MES on adventitious root formation from stem disks of apple. Plant Cell Tiss Organ Cult 95(3):285

    Google Scholar 

  • Krens FA (2003) The Development of an Efficient Cultivar-Independent Plant Regeneration System from Callus Derived from Both Apical and Non-Apical Root Segments of Garlic (Allium sativum L.). In Vitro Cell Dev Biol Plant 39(3):288–292

    Google Scholar 

  • Lakshmanan P, Geijskes RJ, Wang L, Elliott A, Grof CP, Berding N, Smith GR (2006) Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture. Plant Cell Rep 25(10):1007–1015

    CAS  PubMed  Google Scholar 

  • Lattoo SK, Bamotra S, Dhar RS, Khan S, Dhar AK (2006) Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—an endangered medicinal herb. Plant Cell Rep 25(6):499–506

    CAS  PubMed  Google Scholar 

  • Linsmaier EM, Skoog F (1965) Organic Growth Factor Requirements of Tobacco Tissue Cultures. Physiol Plant 18(1):100–127

    CAS  Google Scholar 

  • Liu XX, Wen YB, Cheng ZH, Mou SW (2017) Establishment of a garlic cryopreservation protocol for shoot apices from adventitious buds in vitro. Sci Hortic 226:10–18

    CAS  Google Scholar 

  • Loureiro J, Pinto G, Lopes T, Doležel J, Santos C (2005) Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry. Planta 221(6):815–822

    CAS  PubMed  Google Scholar 

  • Loureiro J, Rodriguez E, Dolezel J, Santos C (2006) Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Ann Bot 98(3):515–527

    CAS  PubMed  PubMed Central  Google Scholar 

  • Luciani GF, Marinangeli PA, Curvetto NR (2001) Increasing nitrate/ammonium ratio for improvement of garlic micropropagation. Sci Hortic 87(1):11–20

    CAS  Google Scholar 

  • Luciani GF, Mary AK, Pellegrini C, Curvetto NR (2006) Effects of explants and growth regulators in garlic callus formation and plant regeneration. Plant Cell Tiss Organ Cult 87(2):139–143

    CAS  Google Scholar 

  • Ma Y, Wang HL, Zhang CJ, Kang YQ (1994) High rate of virus-free plantlet regeneration via garlic scape-tip culture. Plant Cell Rep 14(1):65–68

    CAS  PubMed  Google Scholar 

  • Ma KH, Kwag JG, Zhao W, Dixit A, Lee GA, Kim HH, Chung IM, Kim NS, Lee JS, Ji JJ (2009) Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic ( Allium sativum L.). Sci Hortic 122(3):355–361

    CAS  Google Scholar 

  • Martin-Urdiroz N, Garrido-Gala J, Martin J, Barandiaran X (2004) Effect of light on the organogenic ability of garlic roots using a one-step in vitro system. Plant Cell Rep 22(10):721–724

    CAS  PubMed  Google Scholar 

  • Mohamedyasseen Y, Splittstoesser WE, Litz RE (1994) In vitro shoot proliferation and production of sets from garlic and shallot. Plant Cell Tiss Organ Cult 36(2):243–247

    Google Scholar 

  • Morris DA (2000) Transmembrane auxin carrier systems–dynamic regulators of polar auxin transport. Plant Growth Regul 32(2–3):161–172

    CAS  PubMed  Google Scholar 

  • Mott RL, Cordts JM, Larson AM (1985) Nitrogen and Growth Regulator Effects on Shoot and Root Growth of Soybean in vitro. Basic Life Sci 32:336–337

    Google Scholar 

  • Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol Plant 15(3):473–497

    CAS  Google Scholar 

  • Myers JM, Simon PW (1999) Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time. Plant Cell Rep 19(1):32–36

    CAS  PubMed  Google Scholar 

  • Nagakubo T, Nagasawa A, Ohkawa H (1993) Micropropagation of garlic through in vitro bulblet formation. Plant Cell Tiss Organ Cult 32(2):175–183

    CAS  Google Scholar 

  • Naseer S, Mahmood T (2014) Tissue culture and genetic analysis of somaclonal variations of Solanum melongena L. cv. Nirrala Central European Journal of Biology 9(12):1182–1195

    CAS  Google Scholar 

  • Neta R, David-Schwartz R, Peretz Y, Sela I, Rabinowitch HD, Flaishman M, Kamenetsky R (2011) Flower development in garlic: the ups and downs of gaLFY expression. Planta 233(5):1063–1072

    CAS  PubMed  Google Scholar 

  • Nhut DT, Silva JATD, Aswath CR (2003) The importance of the explant on regeneration in thin cell layer technology. In Vitro Cell Dev Biol Plant 39(3):266–276

    Google Scholar 

  • Novak FJ (1980) Phenotype and cytological status of plants regenerated from callus cultures of Allium sativum L. Zeitschrift Fur Pflanzenzuchtung 84(3):250–260

    Google Scholar 

  • Paiva Neto VBD, Mota TRD, Otoni WC (2003) Direct organogenesis from hypocotyl-derived explants of annatto (Bixa orellana). Plant Cell Tiss Organ Cult 75(2):159–167

    Google Scholar 

  • Palombi M, Damiano C (2002) Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev). Plant Cell Rep 20(11):1061–1066

    CAS  Google Scholar 

  • Peat G, Jones M (2012) A Protocol for Rapid, Measurable Plant Tissue Culture Using Stem Disc Meristem Micropropagation of Garlic. Sch Sci Rev 93(4):93–98

    Google Scholar 

  • Petropoulos S, Fernandes Â, Barros L, Ciric A, Sokovic M, Ferreira I (2018) Antimicrobial and antioxidant properties of various Greek garlic genotypes. Food Chem 245:7–12

    CAS  PubMed  Google Scholar 

  • Phillips RL, Kaeppler SM, Olhoft P (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. Proc Natl Acad Sci USA 91(12):5222–5226

    CAS  PubMed  Google Scholar 

  • Ramakrishnan M, Ceasar SA, Duraipandiyan V, Ignacimuthu S (2014) Efficient plant regeneration from shoot apex explants of maize (Zea mays) and analysis of genetic fidelity of regenerated plants by ISSR markers. Plant Cell Tiss Organ Cult 119(1):183–196

    CAS  Google Scholar 

  • Robledo-Paz A, Villalobos-Arambula VM, Jofre-Garfias AE (2000) Efficient plant regeneration of garlic (Allium sativum L.) by root-tip culture. In Vitro Cell Dev Biol Plant 36(5):416–419

    CAS  Google Scholar 

  • Roy J, Banerjee N (2003) Induction of callus and plant regeneration from shoot-tip explants of Dendrobium fimbriatum Lindl. var. oculatum Hk. f. Sci Hortic 97(3):333–340

    CAS  Google Scholar 

  • Samarina L, Gvasaliya M, Koninskaya N et al (2019) A comparison of genetic stability in tea [Camellia sinensis (L.) Kuntze] plantlets derived from callus with plantlets from long-term in vitro propagation. Plant Cell Tissue Organ Cult 138(3):467–474

    CAS  Google Scholar 

  • Scholten HJ, Pierik RLM (1997) Agar as a gelling agent: chemical and physical analysis. Plant Cell Rep 17(3):230–235

    Google Scholar 

  • Shanjani PS (2003) Nitrogen effect on callus induction and plant regeneration of juniperus excelsa. International Journal of Agriculture Biology 5(4):419–422

    CAS  Google Scholar 

  • Shemesh-Mayer E, Ben-Michael T, Rotem N, Rabinowitch HD, Doron-Faigenboim A, Kosmala A, Perlikowski D, Sherman A, Kamenetsky R (2015) Garlic (Allium sativum L.) fertility: transcriptome and proteome analyses provide insight into flower and pollen development. Front Plant Sci 6:271. https://doi.org/10.3389/fpls.2015.00271

    Article  PubMed  PubMed Central  Google Scholar 

  • Shinohara N, Sugiyama M, Fukuda H (2006) Higher extracellular pH suppresses tracheary element differentiation by affecting auxin uptake. Planta 224(2):394–404

    CAS  PubMed  Google Scholar 

  • Takaichi M, Nagakubo T, Oeda K (2001) Mixed virus infections of garlic determined by a multivalent polyclonal antiserum and virus effects on disease symptoms. Plant Dis 85:71–75

    PubMed  Google Scholar 

  • Teixeira da Silva JA, Gulyás A, Magyar-Tábori K, Wang MR, Wang QC, Dobránszki J (2019) In vitro tissue culture of apple and other Malus species: recent advances and applications. Planta 249(4):975–1006

    CAS  PubMed  Google Scholar 

  • Van Winkle SC, Johnson S, Pullman GS (2003) The impact of Gelrite and activated carbon on the elemental composition of two conifer embryogenic tissue initiation media. Plant Cell Rep 21(12):1175–1182

    PubMed  Google Scholar 

  • Vega-Hissi EG, Andrada MF, Díaz MG, Martinez JCG (2019) Computational study of the hydrogen peroxide scavenging mechanism of allyl methyl disulfide, an antioxidant compound from garlic. Mol Divers.https://doi.org/10.1007/s11030-019-09927-6

    Article  PubMed  Google Scholar 

  • Walkey DGA, Webb MJW, Bolland CJ, Miller A (1987) Production of virus-free garlic (Allium sativum L.) and shallot (A. ascalonicum L.) by meristem-tip culture. Journal of Pomology Horticultural Science 62(2):211–220

    Google Scholar 

  • Wang LX, Li HB, Gu TC, Liu LH, Pang BS, Qiu J, Zhao CP (2014) Assessment of wheat variety stability using SSR markers. Euphytica 195(3):435–452

    CAS  Google Scholar 

  • Xing LD (2009) Regeneration system in vitro of Chive (Allium schoenoprasum L.). Dissertation, Huazhong Agricultural University

  • Xiong ZQ, Li SJ, Liu GQ, Huang BJ (2000) Studies on garlic (Allium sativum L.) rachis in vitro culture. Journal of Nanjing Agricultural University 23(3):25–28 (in Chinese)

    CAS  Google Scholar 

  • Yin ZF, Zhao B, Bi WL, Chen L, Wang QC (2013) Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers. In Vitro Cell Dev Biol Plant 49(3):333–342

    CAS  Google Scholar 

  • Zhang SZ, Li JR (2007) Establishment of a novel rapid propagation system-adventitious shoots regeneration system in the inflorescence of garlic (Allium sativum). J Shandong Agric Univ (Nat Sci) 38(2):159–162 (in Chinese)

    Google Scholar 

  • Zhang YM, Li X, Chen Z, Li JF, Lu JY, Zhou WZ (2013) Shoot organogenesis and plant regeneration in Agave hybrid. Sci Hortic 161(2):30–34

    CAS  Google Scholar 

  • Zhang Y, Wang B, Guo L, Xu W, Wang Z, Li B, Zhang J (2017) Factors influencing direct shoot regeneration from leaves, petioles, and plantlet roots of triploid hybrid Populus sect. Tacamahaca. J For Res 29(6):1533–1545

    Google Scholar 

  • Zheng SJ, Henken B, Krens FA, Kik C (2003) The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (Allium sativum L.). In Vitro Cell Dev Biol Plant 39(3):288–292

    CAS  Google Scholar 

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Acknowledgements

This study was supported by the Education Development Fund project of Northwest A&F University (2017) and National Public Welfare Industry (Agriculture) Special Project (200903018-7).

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YBW conceived the idea, designed and performed the experiment and prepared the draft manuscript. XXL participated in the design of the study, carried out the adventitious shoots regeneration test experiments and reviewed the manuscript. HJL, CNW and HWM contributed to analysis of the data and participated in the sample preparation for histological investigation. This work was conducted under the supervision of ZHC who provided significant intelligence to the paper revision. All authors read and approved the final version of this manuscript.

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Correspondence to Zhi-Hui Cheng.

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Communicated by Qiao-Chun Wang.

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Wen, YB., Liu, XX., Liu, HJ. et al. High-frequency direct shoot organogenesis from garlic (Allium sativum L.) inflorescence and clonal fidelity assessment in regenerants. Plant Cell Tiss Organ Cult 141, 275–287 (2020). https://doi.org/10.1007/s11240-020-01785-7

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