Izvestiya of Saratov University.

Chemistry. Biology. Ecology

ISSN 1816-9775 (Print)
ISSN 2541-8971 (Online)


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Krasova Y. V., Fadeev V. V., Моисеева Е. М., Gusev Y. С., Чумаков М. И. Optimization of the technique for maize protoplast isolation and their nativity after electroporation. Izvestiya of Saratov University. Chemistry. Biology. Ecology, 2022, vol. 22, iss. 4, pp. 445-454. DOI: 10.18500/1816-9775-2022-22-4-445-454, EDN: UTPGJS

This is an open access article distributed under the terms of Creative Commons Attribution 4.0 International License (CC-BY 4.0).
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Russian
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Article
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576.08.572.22
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UTPGJS

Optimization of the technique for maize protoplast isolation and their nativity after electroporation

Autors: 
Krasova Yuliya Viktorovna, Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal State Budgetary Research Institution Saratov Federal Scientifi c Centre of the Russian Academy of Sciences (IBPPM RAS)
Моисеева Елизавета Михайловна, Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal State Budgetary Research Institution Saratov Federal Scientifi c Centre of the Russian Academy of Sciences (IBPPM RAS)
Gusev Yuri Сергеевич, Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal State Budgetary Research Institution Saratov Federal Scientifi c Centre of the Russian Academy of Sciences (IBPPM RAS)
Чумаков Михаил Иосифович, Institute of Biochemistry and Physiology of Plants and Microorganisms – Subdivision of the Federal State Budgetary Research Institution Saratov Federal Scientifi c Centre of the Russian Academy of Sciences (IBPPM RAS)
Abstract: 

We optimizes the composition and concentration of the enzymes, the time for enzymatic treatment and the volume of the enzyme mixture. We also optimized the concentration osmotic of agent, the centrifugation mode, and fi lter pore size for protoplasts isolating from epidermal cells of maize roots (Zea mays L.) of the Brown Marker (BM) line. It was found that 150 minutes is the optimal time for 150 mg root tissue maceration. The yield of intact protoplasts was ~ 4.4 ± 0.2 × 105 cells/mL at the following concentrations of enzymes and osmotic stabilizer: cellulase – 17,4, pectolase – 1.2, hemicellulase – 0.07, D-mannitol – 9.3%. The the concentration of protoplasts was to 23 times higher (p < 0.05) in 800 μl, compared with 200 μl of the enzyme mixture with equal concentrations of enzymes and osmotic stabilizer. It was found that fi ltration of 800 μl protoplast suspension through a fi lter with a pore size of 15 × 15 microns increases the yield of protoplasts up to 3.3 times, compared with a fi lter with a pore size of 15×39. Fractional centrifugation without preliminary fi ltration of the solution and the fl otation method did not produce an increase in the yield of protoplasts. The residual number and protoplast wholeness after ~ 20 hours at +3 °C incubation was evaluated. The protoplast number decreased up to 2 times (p < 0.05) after electroporation.

Reference: 
  1. Davey M. R., Anthony P., Patel D., Power J. B. Plant protoplasts: isolation, culture and plant regeneration // Plant Cell Culture: Essential Methods. New York : WileyBlackwell, 2010. P. 153–173.
  2. Bailey-Serres J., Dawe R. K. Both 5’ and 3’ sequences of maize adh1 mRNA are required for enhanced translation under low-oxygen conditions // Plant Physiol. 1996. Vol. 112. P. 685–695. https://doi.org/10.1104/pp.112.2.685
  3. Кудряшов А. П., Шапчиц М. П. Оценка нативности протопластов и клеток растений // Труды Белорусского государственного университета: научный журнал. 2009. Т. 4, № 2. С. 1–6.
  4. Maccarrone M., Veldink G. A., Agro A. F., Vliegenthart J. F. G. Lentil root protoplasts, a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules // Biochem. Biophys. Acta. (BBA). 1995. Vol. 1243. P. 136–142. https://doi. org/10.1016/0304-4165(94)00124-G
  5. Jang J.-C., Sheen J. Sugar sensing in higher plants // Plant Cell. 1994. Vol. 6. P. 1665–1679. https://doi.org/10.1105/ tpc.6.11.1665
  6. Ziv M., Altman A. Tissue culture General Principles // Encyclopedia of Applied Plant Sciences Elsevier Science Ltd. 2003. P. 1341–1353. https://doi.org/10.1016/B0-12- 227050-9/00213-1
  7. Reed K. M., Bargmann B. O. R. Protoplast regeneration and its use in new plant breeding technologies // Frontiers in Genome Editing. 2021. Vol. 3. P. 1–26. https://doi. org/10.3389/fgeed.2021.734951
  8. Скапцов M. В., Смирнов С. В., Куцев М. Г. Получение протопластов облепихи (Hippophae rhamnoides L.) // Turczaninowia. 2013. Т. 16, № 3. С. 152–156. https://doi.org/10.14258/turczaninowia.16.3.20
  9. Papadakis A., Reustle G., Roubelakis-Angelakis K. A. Protoplast technology in grapevine // Molecular Biology & Biotechnology of the Grapevine. Springer, Dordrecht, 2001. Ch. 14. P. 353–392. https://doi.org/10.1007/978-94-017-2308-4_14
  10. Cocking E. A. Method for the isolation of plant protoplasts and vacuoles // Nature. 1960. Vol. 187. P. 962–963.
  11. Bhojwani S. S., Razdan M. K. Protoplast isolation and culture // Studies in Plant Science. 1996. Vol. 5, ch. 12. P. 337–372.
  12. Кузнецова Е. А., Парамонов И. Н., Зомитев В. Ю. Оценка использования ферментов целлюлазного комплекса при получении протопластов растений // Технология и товароведение инновационных пищевых продуктов. 2013. № 5. С. 9–12.
  13. Gronwald J. W., Leonard R. T. Isolation and transport properties of protoplasts from cortical cells of corn roots // Plant Physiology. 1982. Vol. 70, № 5. P. 1391–1395.
  14. Perlin D. S., Spanswick R. M. Labeling and isolation of plasma membranes from corn leaf protoplasts // Plant Physiology. 1980. Vol. 65, № 6. P. 1053–1057.
  15. Senn A., Pilet P. E. Isolation and some morphological properties of maize root protoplasts // Zeitschrift für Pfl anzenphysiologie. 1980. Bd. 100, № 4. S. 299–310.
  16. Скапцов М. В., Куцев М. Г. Получение мезофильных протопластов Rumex aquaticus и Rumex acetosa сорт «широколистный» // Проблемы ботаники Южной Сибири и Монголии. 2011. № 10. С. 170–173.
  17. Ochatt S. J., Caso O. H. Shoot regeneration from leaf mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L.) // Journal of Plant Physiology. 1986. Vol. 122, № 3. P. 243–249. https://doi.org/10.1016/S0176-1617(86)80123-7
  18. Молчан О. В., Ромашко С. Н., Кенькова М. А., Юрин В. М. Иммобилизация протопластов мезофилла листа Catharanthus roseus // Известия Национальной академии наук Беларуси. Серия биологических наук. 2010. № 4. С. 45–49.
  19. Rao K. S., Prakash A. H. A simple method for the isolation of plant protoplasts // Journal of Biosciences. 1995. Vol. 20. P. 645–655. https://doi.org/10.1007/BF02703304
  20. Evans D. A., Bravo J. E. Plant protoplast isolation and culture // Intern. Rev. Cytol. Suppl. 2013. Vol. 16. P. 33–53.
  21. Прилепский А. Ю., Дроздов А. С., Богатырев В. А., Староверов С. А. Методы работы с клеточными культурами и определение токсичности наноматериалов. СПб. : Университет ИТМО, 2019. 43 с.
  22. Kanchiswamy C. N. DNA-free genome editing methods for targeted crop improvement // Plant Cell Rep. 2016. Vol. 35. P. 1469–1474.
  23. Woo J. W., Kim J., Kwon S. I., Corvalán C., Cho S. W., Kim H., Kim S.-G., Kim S.-T., Choe S., Kim J.-S. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins // Nat. Biotechnol. 2015. Vol. 33. P. 1162–1164. https://doi.org/10.1038/nbt.3389
  24. Malnoy M., Viola R., Jung M. H., Koo O.-J., Kim S.,Kim J.-S., Velasco R., Kanchiswamy C. N. DNA-free genetically edited grapevine and apple protoplast using Crispr/Cas9 ribonucleoproteins // Frontiers Plant Sci. 2016. Vol. 7, № 1904. P. 1–9. https://doi.org/10.3389/ fpls.2016.01904
  25. Andersson M., Turesson H., Olsson N., Fält A.-S., Ohlsson P., Gonzalez M. N., Samuelsson M., Hofvander P. Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery // Physiol. Plant. 2018. Vol. 164, № 4. P. 378–384. https://doi.org/10.1111/ppl.12731
  26. Liang Z., Chen K., Gao C. Biolistic delivery of CRISPR/ Cas9 with ribonucleoprotein complex in wheat // Methods Mol. Biol. 2019. Vol. 1917. P. 327–335. https:// doi.org/10.1007/978-1-4939-8991-1_24
  27. Kim H., Kim S. T., Ryu J., Kang B.-C., Kim J.-S., Kim S.-G. CRISPR/Cpf1-mediated DNA-free plant genome editing // Nat. Commun. 2017. Vol. 8, № 14406. P. 1–7. https://doi.org/ 10.1038/ncomms14406
  28. Данилова С. А. Методы генетической трансформации зерновых культур // Физиология растений. 2007. Т. 54, № 5. С. 645–658.
  29. Чесноков Ю. В. Проблемы генетической трансформации растений. Методические подходы (обзор) // Сельскохозяйственная биология. 2004. Т. 39, № 1. С. 26–40.
  30. Fromm M., Taylor L. P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation // Proceedings of the National Academy of Sciences USA. 1985. Vol. 82, № 17. P. 5824–5828.
  31. Bates G. W. Plant transformation via protoplast electroporation // Plant Cell Culture Protocols. 1999. Vol. 111. P. 359–366. https://doi.org/10.1385/1-59259- 583-9:359
  32. Rhodes C. A., Pierce D. A., Mettler I. J., Mascarenhas D., Detmer J. J. Genetically transformed maize plants from protoplasts // Science. 1988. Vol. 240, № 4849. P. 204–207. https://doi.org/10.1126/science.283294
  33. Gomez-Cano L., Yang F., Grotewold E. Isolation and effi cient maize protoplast transformation // Bio-protocol. 2019. P. e3346–e3346. https://doi.org/10.21769/ BioProtoc.3346
  34. Lyznik L. A., Kamo K. K., Grimes H. D., Ryan R., Chang K.-L. Hodges T. K. Stable transformation of maize: The impact of feeder cells on protoplast growth and transformation effi ciency // Plant Cell Reports. 1989. Vol. 8. P. 292–295. https://doi.org/ 10.1007/ BF00274133
  35. Chase S. S. Monoploids and monoploid-derivatives of maize (Zea mays L.) // The Botanical Review. 1969. Vol. 35, № 2. P. 117–168. https://doi.org/10.1007/BF02858912
  36. Ortiz-Ramírez C., Arevalo E. D., Xu X., Jackson D. P., Birnbaum K. D. An effi cient cell sorting protocol for maize protoplasts // Current Protocols in Plant Biology. 2018. Vol. 3, № 3. P. e20072. https://doi.org/10.1002/cppb.20072
  37. Пескова Н. Н., Балалаева И. В., Брилкина А. А., Шилягина Н. Ю., Масленникова А. В., Мысягин С. А. Оценка жизнеспособности клеток in vitro: учеб.- метод. пособие. Нижний Новгород : Нижегородский госуниверситет, 2020. 25 с.
Received: 
03.08.2022
Accepted: 
11.08.2022
Published: 
23.12.2022
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