Abstract
Genetic assessment of pear regeneration population is very important for genetic fidelity maintenance in genetic engineering breeding. In this research, a regeneration system for a pear dwarfing rootstock cultivar, ‘Qingzhen D1’, was established using NN69 medium supplemented with different concentrations of thidiazuron (TDZ) and indole-3-butyric acid (IBA). The genetic stability of the regenerated population was evaluated through flow cytometry (FCM) and whole genome resequencing techniques. The highest regeneration rate (93%) was induced on NN69 medium supplemented with 3.0mg·L− 1 TDZ and 2.4mg·L− 1 IBA, with 3.9 adventitious buds per leaf. No phenotypes and ploidy changes were detected in regeneration population. But a high variation frequency (5.0 × 10 − 4) at a single site was detected. The mutations occurred most frequently on chromosome 5, 10 and 15, while least 4. High-impact changes only accounted for 0.065% of single-nucleotide polymorphism (SNPs) and 1.429% of insertions or deletions of nucleotides from a sequence (Indels) of all polymorphisms, most of which caused by frameshift. Functional analysis of genes affected by high-impact variants showed that they were mainly related to adaptation to environment. This study will provide a theoretical basis for the application of pear genetic engineering breeding in the future.
This is a preview of subscription content, log in via an institution to check access.
Data Availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Anil VS, Lobo S, Bennur S (2018) Somaclonal variations for crop improvement: Selection for disease resistant variants in vitro. Plant Sci Today 5:44–54. https://doi.org/10.14719/PST.2018.5.2.382
Araújo LG, Prabhu AS, Filippi MC, Chaves LJ (2001) RAPD analysis of blast resistant somaclones from upland rice cultivar IAC 47 for genetic divergence. Plant Cell Tiss Org 67:165–172. https://doi.org/10.1023/A:1011960225472
Bairu MW, Aremu AO, Van Staden J (2010) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173. https://doi.org/10.1007/s10725-010-9554-x
Balyan HS, Gupta PK, Kumar S, Dhariwal R, Jaiswal V, Tyagi S, Agarwal P, Gahlaut V, Kumari S, Varshney R (2013) Genetic improvement of grain protein content and other health-related constituents of wheat grain. Plant Breeding 132:5. https://doi.org/10.1111/pbr.12047
Batley J, Barker G, O’Sullivan H, Edwards KJ, Edwards D (2003) Mining for single nucleotide polymorphisms and insertions/deletions in maize expressed sequence tagdata. Plant Physiol 132:84–91. https://doi.org/10.1104/pp.102.019422
Beemster GT, Fiorani F, Inzé D (2003) Cell cycle: the key to plant growth control? Trends Plant Sci 8:154–158. https://doi.org/10.1016/S1360-1385(03)00046-3
Cardoso JC, Zanello CA, Chen JT (2020) An overview of orchid protocorm-like bodies: mass propagation, biotechnology, molecular aspects, and breeding. Int J Mol Sci 21:1–32. https://doi.org/10.3390/ijms21030985
Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6:80–92
Dewir YH, Nurmansyah, Naidoo Y, Teixeira da Silva JA (2018) Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep 37:1451–1470
Endo M, Kumagai M, Motoyama R, Sasaki-Yamagata H, Mori HS, Hamada M, Kanamori H, Nagamura Y, Katayose Y, Itoh T, Toki S (2015) Whole-genome analysis of herbicidetolerant mutant rice generated by Agrobacterium-mediated gene targeting. Plant Cell Physiol 56:116–125. https://doi.org/10.1093/pcp/pcu153
Fabienne M, Cyrill G, Adam H, Richaud F, Singh R, Cheah SC, Rival A, Duval Y, Tregear JW (2006) Somaclonal variation in micropropagated oil palm. Characterization of two novel genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin. Tree Physiol 26:585–594. https://doi.org/10.1093/treephys/26.5.585
Fossi M, Amundson K, Kuppu S, Britt A, Comai L (2019) Regeneration of Solanum tuberosum plants from protoplasts induces widespread genome instability. Plant Physiol 180:78–86. https://doi.org/10.1104/pp.18.00906
Ghag SB, Shekhawat UK, Ganapathi TR (2014) Characterization of Fusarium wilt resistant somaclonal variants of banana cv. Rasthali by cDNA-RAPD. Mol Biol Rep 41:7929–7935. https://doi.org/10.1007/s11033-014-3687-3
Grafi G, Barak S (2014) Stress induces cell dedifferentiation in plants. Biochim Biophys Acta 1849:378–384. https://doi.org/10.1016/j.bbagrm.2014.07.015
Haque MS, Nath UK, Iqbal MS, Khatun R, Jani AHAA (2015) Assessment of field performance and genetic diversity analysis of tissue culture variants of strawberry. J Agr Sci Tech-Iran 11:107–125
Hermisson J, Wagner GP (2004) The population genetic theory of hidden variation and genetic robustness. Genet 168:2271–2284. https://doi.org/10.1534/genetics.104.029173
Jain M, Moharan KC, Shankar R, Kumari R, Garg R (2014) Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance. Plant Biotechnol 12:253–264. https://doi.org/10.1111/pbi.12133
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 19:684–690. https://doi.org/10.1007/s002999900177
Jiang C, Mithani A, Gan X, Belfield EJ, Klingler JP, Zhu JK, Ragoussis J, Mott R, Harberd NP (2011) Regenerant Arabidopsis Lineages Display a Distinct Genome-Wide Spectrum of Mutations Conferring Variant Phenotypes. Curr Biol 21:1385–1390. https://doi.org/10.1016/j.cub.2011.07.002
Joyce SM, Cassells AC (2002) Variation in potato microplant morphology in vitro and DNA methylation. Plant Cell Tiss Org 70:125–137. https://doi.org/10.1023/A:1016312303320
Kaeppler SM, Kaeppler F, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188. https://doi.org/10.1023/A:1006423110134
Kim SJ, Park S, Shin YH, Park YD (2021) Identification and Validation of Genetic Variations in Transgenic Chinese Cabbage Plants (Brassica rapa ssp. pekinensis) by Next-Generation resequencing Genes. 12:621–637. https://doi.org/10.3390/genes12050621
Krishna H, Sairam RK, Singh SK, Patel VB, Sharma RR, Grover M, Nain L, Sachdeva A (2008) Mango explants browning: effect of ontogenic age: mycorrhization and pre-treatments. Sci Hortic 118:132–138. https://doi.org/10.1016/j.scienta.2008.05.040
Krishna H, Alizadeh M, Singh D, Singh U, Chauhan N, Eftekhari M, Sadh RK (2016) Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6:1–18
Larkin PJ, Scowcroft WR (1981) Somaclonal variation-a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214
Leblay C, Chevreau E, Raboin LM (1991) Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communis L.). Plant Cell Tiss Org 25:99–105. https://doi.org/10.1007/BF00042180
Leva AR, Petruccelli R (2012) Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISSR markers. Biol Plant 56:373–376. https://doi.org/10.1007/s10535-012-0044-z
Liberatore CM, Rodolfi M, Beghè D, Fabbri A, Chiancone B (2020) In vitro leaf-derived organogenesis and somaclonal variant detection in Humulus lupulus L. vitro Cell Dev-Pl 56:865–874. https://doi.org/10.1007/s11627-020-10088-7
Lilly JW, Bartoszewski G, Malepszy S, Havey MJ (2001) A major deletion in the cucumber mitochondrial genome sorts with the MSC phenotype. Curr Genet 40:144–151. https://doi.org/10.1007/s002940100238
Machczyńska J, Orlowska R, Zimny J, Bednarek PT (2014) Extended metAFLP approach in studies of tissue culture induced variation (TCIV) in triticale. Mol Breed 34:845–854. https://doi.org/10.1007/S11032-014-0079-2
Mançano AP, de Oliveira SB, Rodrigues de Oliveira Garcia R, Atalla Mansur E, Gagliardi RF (2019) Genetic stability among in vitro eggplant clones induced by different plant growth regulators. Biotecnol Veg 19:75–83
Matsuda S, Sato M, Ohno S (2014) Cutting leaves and plant growth regulator application enhance somaclonal variation induced by transposition of VGs1 of Saintpaulia. J Jpn Soc Hortic Sci 83:308–316. https://doi.org/10.2503/jjshs1.MI-009
Miler N, Zalewska M (2014) Somaclonal variation of chrysanthemum propagated in vitro from different explants types. Acta Sci Pol-Hortoru 13:69–82. https://doi.org/10.1080/01140671.2014.891527
Miyao A, Nakagome M, Ohnuma T, Yamagata H, Kanamori H, Katayose Y, Takahashi A, Matsumoto T, Hirochika H (2012) Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome resequencing. Plant Cell Physiol 53:256–264. https://doi.org/10.1093/pcp/pcr172
Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Culture. Physiol Plant 15:473–479. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Neelakandan AK, Wang K (2012) Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep 31:597–620. https://doi.org/10.1007/s00299-011-1202-z
Nehra NS, Kartha KK, Stushnoff C, Giles KL (1992) The influence of plant growth regulator concentrations and callus age on somaclonal variation in callus culture regenerants of strawberry. Plant Cell Tiss Org 29:257–268. https://doi.org/10.1007/BF00034361
Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:854–887. https://doi.org/10.1126/science.163.3862.85
Orłowska R, Machczńska J, Oleszczuk S, Zimny J, Bednarek PT (2016) DNA methylation changes and TE activity induced in tissue cultures of barley (Hordeum vulgare L.). Biol Res 23:19–31. https://doi.org/10.1186/s40709-016-0056-5
Ossowski S, Schneeberger K, Lucas-Lledo JI, Warthmann N, Clark RM, Shaw RG, Weigel D, Lynch M (2010) The Rate and Molecular Spectrum of Spontaneous Mutations in Arabidopsis thaliana. Science 327:92–94
Palombi MA, Lombardo B, Caboni E (2007) In vitro regeneration of wild pear (Pyrus pyraster Burgsd) clones tolerant to Fe-chlorosis and somaclonal variation analysis by RAPD markers. Plant Cell Rep 26:489–496. https://doi.org/10.1007/s00299-011-1202-z
Roy AR, Sajeev S, Pattanayak A, Deka BC (2012) TDZ induced micropropagation in Cymbidium giganteum Wall. Ex Lindl. and assessment of genetic variation in the regenerated plants. Plant Growth Regul 68:435–445. https://doi.org/10.1007/s10725-012-9732-0
Sales EK, Butardo NG (2014) Molecular analysis of somaconal variation in tissue culture derived bananas using MSAP and SSR markers. Int J Biol Vet Agric Food Eng 8:63–610
Sato M, Kawabe T, Hosokawa M, Tatsuzawam F, Doi M (2011) Tissue culture induced flower-color changes in Saintpaulia caused by excision of the transposon inserted in the flavonoid 3’,5’ hydroxylase (F3’5’H) promoter. Plant Cell Rep 30:929–939. https://doi.org/10.1007/s00299-011-1016-z
Skarzyńska A, Pawelkowicz M, Plader W (2020) Genome-wide discovery of DNA variants in cucumber somaclonal lines. Gene 736:1–11. https://doi.org/10.1016/j.gene.2020.144412
Smulders M, Klerk G (2011) Epigenetics in plant tissue culture. Plant Growth Regul 63:137–146. https://doi.org/10.1007/s10725-010-9531-4
Stefano P (2001) Mutation induction and tissue culture in improving fruits. Plant Cell Tiss Org 64:185–210. https://doi.org/10.1023/A:1010623203554
Wagner A (2000) Robustness against mutations in genetic networks of yeast. Nat Genet 24:355–361. https://doi.org/10.1038/74174
Wagner GP, Booth G, Bagheri-Chaichian H (1997) A population genetic theory of canalization. J Evol 51:329–347. https://doi.org/10.1111/j.1558-5646.1997.tb02420.x
Wakeley J (1996) The excess of transitions among nucleotide substitutions: new methods of estimating transition bias underscore its significance. Trends Ecol Evol 11:158–162. https://doi.org/10.1016/0169-5347(96)10009-4
Wang C, Tian M, Zhang Y, Ouyang T (2019) Molecular spectrum of somaclonal variation in PLB-regenerated Oncidium revealed by SLAF-seq. Plant Cell Tiss Org 137:541–552. https://doi.org/10.1007/s11240-019-01589-4
Xin-min F, Jian-ron F, Huan-xi R, Zhu-ye X, Xiu-kang Z, Ying Z (2018) Establishment of regeneration system of leaves in -vitro korla fragrant pear. Xinjiang Agric Sci 55:829–836
Xu M, Li X, Kobrban SS (2004) DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose (Rosa hybrida L.). Theor Appl Genet 109:899–910. https://doi.org/10.1007/s00122-004-1717-6
Yang NJ, Hinner MJ (2015) Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods Mol Biol 1266:29–53. https://doi.org/10.1007/978-1-4939-2272-7_3
Yousefiara M, Jafarkhani Kermani M, Bagheri A, Habashi AA, Abdollahi H (2014) Study of factors affecting direct shoot regeneration of pear (Pyrus communis L.). JPMB 2:21–28. https://doi.org/10.22058/JPMB.2014.8426
Yunqing Y, Huatao J, Chao G, Jun W (2017) Research on the establishment of rapid propagation and leaf regeneration system of pear ‘xuehuali’. Nanjing Agric Univ 40:68–75
Zhang D, Wang Z, Wang N, Gao Y, Liu Y, Wu Y, Bai Y, Zhang Z, Lin X, Dong Y, Ou X, Xu C, Liu B (2014) Tissue culture- induced heritable genomic variation in rice, and their phenotypic implications. PLoS ONE 9:1–10. https://doi.org/10.1371/journal.pone.0096879
Funding
This work was supported by the National Key R&D Program of China [2018YFD1000102]; China Agriculture Research System of MOF and MARA [CARS-28-07]; the Agricultural Variety Improvement Project of Shandong Province [2019LZGC008].
Author information
Authors and Affiliations
Contributions
Q.L, YJY and RW conceived and designed the experiments. QL performed the experiments. QL and YJY wrote the manuscript. JLL, JKS, DLL and RCW helped with experiments. All authors read and approved the final article.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no competing interest.
Additional information
Communicated by Henryk Flachowsky.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Liu, Q., Yang, Y., Liu, J. et al. Establishment of regeneration system of Pyrus and the genetic stability analysis of regenerated population. Plant Cell Tiss Organ Cult 152, 215–228 (2023). https://doi.org/10.1007/s11240-022-02378-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-022-02378-2