Plantlet regeneration from primary callus cultures of Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, a rare bulbous germplasm
- 103 Downloads
Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, an endangered valuable genetic resource, was used to establish and optimize a callus propagation system and to investigate the effects of internal and external phytohormones for the purpose of germplasm conservation. Of the combinations and concentrations of auxins and cytokinins examined, Murashige and Skoog (MS) medium supplemented with 8 g L−1 agar, 30 g L−1 sucrose, 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.69 to 5.37 μM α-naphthaleneacetic acid, and 0.44 μM 6-benzyladenine, 0.45 μM thidiazuron, and 0.28 μM zeatin riboside generated the best results, effectively promoting callus proliferation. Four callus types could be discriminated, of which type A (yellowish, granular) and type B (yellow, medium-granular) were dry, friable, and grew well. Periodic acid-Schiff staining revealed small and regular cells, with numerous starch granules surrounding each nucleus. In culture, callus clumps produced an average of 14.33 shoots under “MS + 7-d-dark–light” treatment with 100% regeneration frequency. Bulblets formed within 60 d after shoot transfer to bulblet formation medium. Type A and B callus was likely to be embryogenic, according to morphology, cytology, and high shoot regenerating capacity. Examination of endogenous phytohormone levels showed that the abscisic acid to indole-3-acetic acid (ABA/IAA) ratio gradually increased with increasing diameter of callus clumps treated with all exogenous phytohormones, except zeatin riboside, leading to the hypothesis that callus induction competence was closely associated with endogenous ABA/IAA ratio. This first report should assist further genetic studies of this rare Lilium and other bulbous plants.
KeywordsLilium Callus status Endogenous phytohormones Morphogenesis Bulblet formation
The authors would thank American Journal Experts for editing and improving the English language of this manuscript.
Authors’ contribution statement
YPX, YW, YDM, YL, and LZ conceived and designed the experiments. YDM, YL, and YW performed the experiments and analyzed the data. YW, YL, and YDM wrote the manuscript. LZ and YPX revised the manuscript. All authors approved the final manuscript.
This work was funded by National Natural Science Foundation of China (No. 31772337).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Bakhshaie M, Babalar M, Mirmasoumi M, Khalighi A (2010) Effects of light, sucrose, and cytokinins on somatic embryogenesis in Lilium ledebourii (Baker) Bioss. via transverse thin cell-layer cultures of bulblet microscales. J Hortic Sci Biotechnol 85:491–496. https://doi.org/10.1080/14620316.2010.11512703 CrossRefGoogle Scholar
- Bhaskaran S, Smith RH (1990) Regeneration in cereal tissue culture: a review. Crop Sci 30:1328–1337. https://doi.org/10.2135/cropsci1990.0011183X003000060034x CrossRefGoogle Scholar
- Du F, Jiang J, Jia HM, Zhao XY, Wang WH, Gao QK, Mao WH, Wu Y, Zhang L, Grierson D, Xia YP, Gao ZS (2015) Selection of generally applicable SSR markers for evaluation of genetic diversity and identity in Lilium. Biochem Syst Ecol 61:278–285. https://doi.org/10.1016/j.bse.2015.05.002 CrossRefGoogle Scholar
- Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 43:27–47. https://doi.org/10.1023/B:GROW.0000038275.29262.fb CrossRefGoogle Scholar
- Kanchanapoom K, Ponpiboon T, Wirakiat W, Kanchanapoom K (2011) Regeneration of lily (Lilium longiflorum ‘Easter lily’) by callus derived from leaf explants cultured in vitro. Scienceasia 37:373–376. https://doi.org/10.2306/scienceasia1513-1874.2011.37.373 CrossRefGoogle Scholar
- Kim SJ, Lee JY, Kin YM, Yang SS, Hwang OJ, Hong NJ, Kim KM, Lee HY, Song PS, Kim JI (2007) Agrobacterium-mediated high-efficiency transformation of creeping bentgrass with herbicide resistance. J Plant Biol 50:577–585. https://doi.org/10.1007/BF03030712
- Kumari A, Baskaran P, Plackova L, Omamikova H, Nisler J, Dolezal K, Van Staden J (2018) Plant growth regulator interactions in physiological processes for controlling plant regeneration and in vitro development of Tulbaghia simmleri. J Plant Physiol 223:65–71. https://doi.org/10.1016/j.jplph.2018.01.005 CrossRefPubMedGoogle Scholar
- Li GY, Chen ZH, Yan FB (2007) Lilium brownii var. giganteum: a new variety of Lilium from Wenling, Zhejiang. J Zhejiang A&F Univ 24:767–768. https://doi.org/10.3969/j.issn.2095-0756.2007.06.023 Google Scholar
- Li HY, Zhang J, Yang Y, Jia NN, Wang CX, Sun HM (2017) miR171 and its target gene SCL6 contribute to embryogenic callus induction and torpedo-shaped embryo formation during somatic embryogenesis in two lily species. Plant Cell Tissue Organ Cult 130:591–600. https://doi.org/10.1007/s11240-017-1249-9 CrossRefGoogle Scholar
- Li XY, Wang CX, Cheng JY, Zhang J, da Silva JAT, Liu XY, Duan X, Li TL, Sum HM (2014) Transcriptome analysis of carbohydrate metabolism during bulblet formation and development in Lilium davidii var. unicolor. BMC Plant Biol 14:358. https://doi.org/10.1186/s12870-014-0358-4 CrossRefPubMedPubMedCentralGoogle Scholar
- Liu Q, Zhu YH, Wu S, Shen GZ, Xiao LT (2007) Studies on the content changes of endogenous phytohormones in rice calli for genetic transformation. Sci Agr Sin 40:2361–2367. https://doi.org/10.3321/j.issn:0578-1752.2007.10.034 Google Scholar
- Magdalena KE, Bach A (2005) Morphogenesis of Lilium martagon L. explants in callus culture. Acta Biol Cracov 47:65–73Google Scholar
- Mowry RW (1963) The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. With revised directions for the colloidal iron stain, the use of Alcian Blue G8X and their combinations with the periodic acid-Schiff reaction. Ann N Y Acad Sci 106:402–423. https://doi.org/10.1111/j.1749-6632.1963.tb16654.x CrossRefGoogle Scholar
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
- Nhut DT, Hanh NTM, Tuan PQ, Nguyet LTM, Tran NTH, Chinh NC, Nguyen NH, Vinh DN (2006) Liquid culture as a positive condition to induce and enhance quality and quantity of somatic embryogenesis of Lilium longiflorum. Sci Hortic 110:93–97. https://doi.org/10.1016/j.scienta.2006.05.015 CrossRefGoogle Scholar
- Wang J, Liu GF, Bao MZ, Huang L (2008) Adjustment of the status of embryogenic callus and plant regeneration of Lilium longiflorum. Acta Hortic Sin 12:1795–1802. https://doi.org/10.3321/j.issn:0513-353X.2008.12.011 Google Scholar
- Wu Y, Xu K, Zhang L, Ma YD, Xia YP (2014) Nutritional analysis of a new variety of Lilium (Lilium brownii var. giganteum). Acta Nutrimenta Sin 36:102–104Google Scholar
- Wu Z, Liang JH, Yang X, He HN, Yi MF (2017) Morpholohical and histological investigation of nodule origination and differentiation in lily (oriental hybrid ‘Siberia’) petiole explants. Propag Ornam Plants 17:111–119Google Scholar
- Yin ZD, 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 49:333–342. https://doi.org/10.1007/sl1627-013-9501-4
- Zhang J, Gai MZ, Li XY, Li TL, Sun HM (2016) Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant. Biosci Biotechnol Biochem 80:1898–1906. https://doi.org/10.1080/09168451.2016.1194178 CrossRefPubMedGoogle Scholar
- Zhang J, Xue BY, Gai MZ, Song SL, Jia NN, Sun HM (2017) Small RNA and transcriptome sequencing reveal a potential miRNA-mediated interaction network that functions during somatic embryogenesis in Lilium pumilum DC. Fisch. Front Plant Sci 8:566. https://doi.org/10.3389/fpls.2017.00566 CrossRefPubMedPubMedCentralGoogle Scholar
- Zhou Y, Zhang JJ, Chen MM, Gu JJ (2013) Development of plant regeneration system via somatic embryogenesis from roots of Lilium hybrid cultivars. Propag Ornam Plants 13:130–137Google Scholar