Elsevier

Scientia Horticulturae

Volume 271, 20 September 2020, 109404
Scientia Horticulturae

Effect of ‘Chloroxynil’ on Agrobacterium-mediated transformation efficiency of Lilium cv ‘Manissa’

https://doi.org/10.1016/j.scienta.2020.109404Get rights and content

Abstract

As a powerful technique, gene transformation can produce new lily cultivars with specific characteristics such as new flower colors and resistance to pathogens. However, it is difficult to use this technique in monocot plants like lilies. A new phenolic compound called ‘chloroxynil’ has been introduced to replace ‘acetosyringone’ and is considered to improve transformation efficiency of Lilium. Our results showed that the addition of chloroxynil into the inoculation and co-cultivation media significantly improved the transformation efficiency. At 4 μM, chloroxynil-mediated callus regeneration increased 7 days after it was transferred to the regeneration medium. Transformation efficiency was 6.6 % at 100 μM acetosyringone, while 11.1 % at 4 μM CX. Finally, 11 kanamycin-resistant plants were successfully produced at 4 μM CX, and gene transformation in the plantlets was validated by PCR and RT-PCR techniques.

Introduction

Lily is an important cut flower in the floriculture industry. It plays a crucial role in the flower market economy. North America and Southeast Asia are the main origins of this flower (Anderson, 2006). About 100 lily species have been identified and recorded. These 100 species have been divided into seven sections including Lilium, Martagon, Pseudolirium, Archelirion, Sinomartagon, Leucolirion, and Oxypetalum (De Jong, 1974). After tulips, lily is the second ornamental geophytes, with about $84,776,000 annual turnover in the USA (USDA Statistics Floriculture Crops, 2015). According to Kamenetsky and Okubo (2012), the most applicable strategies for breeding of new lily cultivars are (1) interspecific and intraspecific crossings; (2) meiotic and mitotic polyploidization; (3) mutation; and (4) molecular breeding. A molecular breeding section has been divided into two individual techniques: utilization of molecular markers and genetic engineering. Because Lilium belongs to monocotyledons, scientists have used the particle bombardment or (biolistic) technique in gene manipulation studies. This method has some disadvantages, including the requirement of specific equipment and personal skills, high copy number, and the tendency of DNA sequences to undergo complex rearrangements prior to or during integration (Oksman-Caldentey and Braz, 2002a, b).

The first step for the successful production of genetically-modified plants is the establishment of a simple, efficient, and practical protocol for the transfer and integration of recombinant DNA molecules to host cells, after which the second step is the regeneration of the recombinant cells to mature plants. Many modifications in the inoculation, co-cultivation, and regeneration media have been tested by researchers to reach an efficient gene transformation protocol. Ogaki et al. (2008) reported that pH adjustment in co-cultivation media had a critical role in the transformation of Lilium × formolongi. In this study, by adding MES (2-morpholinoethane-sulfonic acid) to co-cultivation media, the pH was stopped from rising, thus improving the transformation efficiency (Ogaki et al., 2008). By removing some main elements such as CaCl2 (Montoro et al., 2000) and NH4NO3 (Hoshi et al., 2004) from the basal salt mix of MS medium, the transformation rate increased. A highly efficient Agrobacterium-mediated transformation system has been established by removing macro-elements such as CaCl2, KH2PO4, NH4NO3, and KNO3 from inoculation and co-cultivation media (Azadi et al., 2010). The addition of acetosyringone (3, 5-dimethoxy-4-hydroxyacetophenone) increased the rate of transformation in Eustoma grandiflora by up-regulating the virulence genes of Agrobacterium (Nakano, 2017). Although acetosyringone has long been used to improve transformation efficiency, it is wise to test other chemical compounds to improve the transformation protocol. An interesting phenolic compound called chloroxynil has shown more effective results compared to acetosyrigone. It significantly increased the rate of transformation in Lotus japonicus (Kimura et al., 2015). Moreover, chloroxynil has been reported to have similar results on genetic transformation of rice (Kimura et al., 2015). In the present study, we used chloroxynil for the first time in the Agrobacterium-mediated transformation of Lilium.

Section snippets

Plant materials

In this experiment, we used meristematic nodular calli (NOD) from ‘Mannisa’ cultivar by using the successful protocol previously reported by Mii et al. (1994), with some modifications. Firstly, healthy bulbs were collected from a commercial greenhouse, washed with tap water, and immersed in fungicide for 30 min. The scales were sterilized with 70 % v/v ethanol for 1 min and then with 0.1 % w/v HgCl2 for 10 min. They were then washed with sterile distilled water three times. The scales were cut

Results

Callus multiplication was found to be affected by different treatments. One week after the calli were transferred to the selection media, the highest multiplication rate was recorded for 4μM CX (68.4 %) treatment, and the lowest for the negative control (10 %) (Fig. 2). Callus growth rate at 4μM CX treatment was 2 times as that of the control treatment (34. 57 %). The effects of CX and AS on callus growth differed significantly (p ≤ 0.05). Callus multiplication rate increased with CX during the

Discussion

Agrobacterium-mediated transformation, as a powerful technique, is able to integrate several copies of the interesting gene into the host plant genome (Men et al., 2003; Remblière et al., 2018), but successful transformation depends on a lot of things such as the host plant; Agrobacterium strain; vector type; and inoculation, selection, and regeneration media (Lacroix and Citovsky, 2013). In this study, we observed lower callus multiplication in control treatment than in CX treatments, which

Conclusion

In this work, we have observed transformation efficiency of Lilium cv Manissa to be improved by the addition of 4 μM chloroxynil to inculcation and co-cultivation media. This chemical compound can be used to improve lily transformation protocol. Although the benefit of using chloroxynil to increase transformation efficiency of Lilium was approved by our study, we safely conclude that this protocol needs to be tested with other Lilium cultivars.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

CRediT authorship contribution statement

Hojatollah Abbasi: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Visualization. Roohangiz Naderi: Supervision, Conceptualization, Project administration, Validation. Mohsen Kafi: Conceptualization, Supervision. Pejman Azadi: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - review & editing, Visualization. Morteza Shakh-Asadi: Conceptualization,

Declaration of Competing Interest

The authors whose named above certified that they have not any financial interest in the subject matter or materials discussed in this manuscript.

References (22)

  • M. Guo et al.

    Is there any crosstalk between the chemotaxis and virulence induction signaling in Agrobacterium tumefaciens?

    Biotechnol. Adv.

    (2017)
  • M. Mii et al.

    Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium ×formolongi by utilizing meristematic nodular cell clumps

    Plant Sci.

    (1994)
  • A. Anderson

    Flower Breeding and Genetics; Issues, Challenges, and Opportunities for the 21st Century

    (2006)
  • P. Azadi et al.

    Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium

    Plant Cell Tissue Organ Cult.

    (2010)
  • P.C. De Jong

    some notes on the evolution of lilies. Lily Yearbook

    North Am. Lily Soc.

    (1974)
  • Y. Hoshi et al.

    Production of transgenic lily plants by Agrobacterium-mediated transformation

    Plant Cell Rep.

    (2004)
  • R.A. Jefferson

    Assaying chimeric genes in plants: the GUS gene fusion system

    Plant Mol. Biol. Rep.

    (1987)
  • R. Kamenetsky et al.

    Ornamental Geophytes: From Basic Science to Sustainable Production

    (2012)
  • M. Kimura et al.

    A novel phenolic compound, Chloroxynil, improves agrobacterium-mediated transient transformation in Lotus japonicus

    PLoS One

    (2015)
  • B. Lacroix et al.

    The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation

    Int. J. Dev. Biol.

    (2013)
  • C.A. McCullen et al.

    Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer

    Annu. Rev. Cell Dev. Biol.

    (2006)
  • Cited by (10)

    • An efficient and novel method to screen Botrytis cinerea resistance genes based on TRV-induced gene silencing with lily petal discs

      2022, Physiological and Molecular Plant Pathology
      Citation Excerpt :

      Fisch, Lilium ‘Sorbonne’ and L. longiflorum [11–13]. Generally, the stable transformation process of lilies includes callus induction, Agrobacterium-mediated transformation, co-culture, resistance screening, and tissue regeneration [14,15]. Although many improvements, including optimizing co-cultivation medium pH, re-suspension solution, and tissue growth conditions, have been tried to improve transformation efficiency [11,15], the whole process is still time-consuming, labor-intensive, and inefficient.

    • Regeneration and Agrobacterium-mediated genetic transformation in Dianthus chinensis

      2021, Scientia Horticulturae
      Citation Excerpt :

      After 3 days, the infected cotyledons were moved into differentiation and regeneration medium for adventitious bud differentiation (Fig. 4d). After 3–4 weeks, a mass of Kana-resistant plantlets formed, the same result had been showed in a previous study, which adding kanamycin will affect the regeneration shoots during the transformation process (Abbasi et al., 2020). Researchers have found that when the kanamycin selection pressure is too high, the transformed cell or tissue will not grow, and may even die.

    View all citing articles on Scopus
    View full text