بهینه‌سازی و ارائه دستورالعمل استخراج و باززایی پروتوپلاست سیب‌زمینی و تراریختی آن

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری رشته بیوتکنولوژی، دانشکده کشاورزی دانشگاه شهید مدنی آذربایجان

2 گروه بیوتکنولوژی، دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان، تبریز، ایران

3 بیوتکنولوژی گیاهی-بیولوژی مولکولی، دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان

چکیده

هدف: ویژگی‌های منحصر بفرد پروتوپلاست‌های گیاهی، آن‌ها را به ابزاری قدرتمند برای محققین، جهت مطالعه تغییرات ژنتیکی سلولی تبدیل کرده است. مهمترین پیش­نیاز استفاده از پروتوپلاست‌ها، توانایی استخراج ساده در مقادیر بالا، کشت و باززایی آن‌ها جهت تشکیل کلونی سلولی و تولید گیاه است. معمول‌ترین روش استخراج پروتوپلاست از بافت‌های گیاهی، روش آنزیمی است. غلظت و ترکیب آنزیم‌ها و مراحل جداسازی و کشت، از عوامل کلیدی استخراج پروتوپلاست­ می‌باشد. هدف این تحقیق، بررسی اثر فاکتورهای مذکور بر روی پروتوپلاست‌های گیاهان درون‌شیشه­ای سیب‌زمینی (Solanum tuberosum L.) و معرفی روشی کارا و مناسب برای استخراج، تراریختی و کالوس‌زایی/باززایی از این گیاه است.
مواد و روش‌ها: برای جداسازی پروتوپلاست سیب‌زمینی رقم رایج تجاری آگریا، تیمارهای مختلف غلظت آنزیم‌های سلولاز و مسروزیم، مدت نگهداری در محلول آنزیمی و مراحل جداسازی بر روی بافت­ برگ، ساقه و بخش‌های هوایی گیاهچه‌های درون‌شیشه­ای و اثر غلظت هورمن‌های مختلف برکشت و باززایی پروتوپلاست مورد بررسی قرار گرفت. پس از جداسازی، کارآیی تراریختی پروتوپلاست‌ها به‌واسطه­ی PEG 4000 با استفاده از ناقل pBin61-GFP ارزیابی شد.
نتایج: بهترین نتایج از ریزنمونه‌های برگی و بالاترین تعداد پروتوپلاست (106 × 2 پروتوپلاست بر میلی­لیتر)، پس از 16 ساعت نگهداری در محلول آنزیمی (حاوی 1 درصد سلولاز و 2/0 درصد مسروزیم) از بافت‌های مزوفیل برگ حاصل شد. کارآیی تراریختی پروتوپلاست‌ها به­واسطه­ی PEG با غلظت 40 درصد و مدت زمان 30 دقیقه ،50 درصد بود. بهترین تیمار محیط کشت برای القای کالوس‌زایی، محیط MS حاوی 5 میلی‌گرم بر لیتر NAA و 1/0 میلی‌گرم بر لیتر BAP بدست آمد که در این تیمار هورمونی، 100 درصد پروتوپلاست‌ها بعد از حدود یک ماه کالوس تشکیل دادند. بهترین تیمار باززایی از کالوس ترکیب هورمونی 2 میلی‌گرم برلیتر Zeatin با 1/0 میلی‌گرم بر لیتر GA3 به­همراه 01/0 میلی‌گرم بر لیتر NAA با راندمان 86 درصد بود.
نتیجه‌گیری: بر اساس نتایج حاصل، دستورالعمل استخراج پروتوپلاست و انتقال ژن توصیف شده در این تحقیق برای مهندسی ژنتیک، انتقال و بیان ژن در گیاه سیب‌زمینی کارآیی لازم را دارد.

کلیدواژه‌ها


عنوان مقاله [English]

Optimization and Presentation of a Protocol for Protoplast Isolation, Regeneration and Gene Transfer in Potato

نویسندگان [English]

  • Esmat Ashaar Ghadim 1
  • Maghsoud Pazhouhandeh 2
  • Mohammad Ahmadabadi 3
1 Ph.D. student in plant Biotechnology, Department of Biotechnology, Faculty of Agriculture, University of Shahid Madani Azarbayejan
2 Biotechnology Dept. Agriculture Fac. Azarbaijan Shahid Madani University, Tabriz, Iran
3 Assistant professor, Azarbaijan Shahid Madani University, Tabriz, Iran
چکیده [English]

Objective
The unique characteristics of plant protoplasts have made them a powerful tool for researchers to study the genetic modification of plant cells. The most important prerequisite for the use of protoplasts is the ability to extract them easily and in large quantities, and to cultivate and regenerate them for cell colony formation and plant production. The most common method of extracting protoplasts from plant tissues is the enzymatic method. Concentration and composition of enzymes and isolation and culture steps are key factors in protoplast extraction. The aim of this study was to investigate the effect of these factors on protoplasts of in vitro potato (Solanum tuberosum L.) and to develop an efficient and suitable method for their extraction, transfection, callus formation and regeneration.
 
Materials and methods
For protoplast isolation from commercial and common Agria cultivar of potato, different treatments of Cellulase and Macerozyme enzymes concentration, incubation time in enzyme solution and isolation steps on leaf tissue, stem and aerial part of in vitro plantlets were investigated. The effects of different hormone concentration on culture and regeneration of protoplasts were studied. After protoplast isolation, the efficiency of transfection by PEG 4000 using pBin61-GFP plasmid was evaluated.
 
Results
The best results were obtained from leaf explants and the highest number of protoplasts (2×106 protoplasts per ml) were extracted from leaf mesophilic tissues after 16 hours' incubation in enzyme solution (1% Cellulase and 0.2% Macerozyme). The transfection efficiency of protoplasts by PEG with 40% concentration and duration of 30 minutes was 50%. The MS medium containing 5 mg/l NAA and 0.1 mg/l BAP was selected as the best culture medium for callus induction. In this hormonal treatment, 100% of callus induction from protoplasts was obtained after about one month. The best medium for plant regeneration and shooting from callus was the combination of 2 mg/l Zeatin with 0.1 mg/l GA3 and 0.01 mg/l NAA with 86% efficiency.
 
Conclusions
Based on the results, the described protocol for protoplast extraction and gene transfer in this study provides necessary steps for genetic engineering, transformation and gene expression in the potato plant.

کلیدواژه‌ها [English]

  • Cellulose
  • Genetic Engineering
  • Macerozyme
  • Polyethylene glycol
  • Protoplast
 
Al-Maarri K, Al-Qabbani N, Al-Biski F (2014) In vitro protoplast isolation and regeneration of Solanum tuberosum cv. Binella and Burren. J Agric Sci (Belihuloya) 10, 32-44.
Andersson M, Turesson H, Nicolia A et al. (2017) Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant cell Rep 36, 117-128.
Anjum MA (1998) Effect of protoplast source and media on growth and regenerability of protoplast-derived calluses of Solanum tuberosum L. Acta Physiol Plant 20, 129-133.
Baltes NJ, Gil-Humanes J, Voytas DF (2017) Genome engineering and agriculture: opportunities and challenges. Prog Mol Biol Transl Sci 149, 1-26.
Binding H, Zuba M, Rudnick J et al. (1988) Protoplast gel fusion. J Plant Physiol 133, 409-413.
Bajaj Y (1974) Potentials of protoplast culture work in agriculture. Euphytica 23, 633-649.
Baek K, Kim DH, Jeong J et al. (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6, 1-7.
Bokelmann G, Roest S (1983) Plant regeneration from protoplasts of potato (Solarium tuberosum cv. Bintje). Z Pflanzenphysiol 109, 259-265.
Cao Y, Li H, Pham AQ et al. (2016) An improved transient expression system using Arabidopsis protoplasts. Curr Protoc Plant Biol 1, 285-291.
Castelblanque L, García-Sogo B et al. (2010). Efficient Plant Regeneration from Protoplasts of Kalanchoe Blossfeldiana via Organogenesis. Plant Cel. Tiss. Organ. Cult. 100, 107–112.
Cardi T, Puite KJ, Ramulu KS (1990) Plant regeneration from mesophyll protoplasts of Solanum commersonii Dun. Plant Sci 70, 215-221.
Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles Nature 187:962-963.
Collins AG (2016) Comparison of mesophyll protoplast isolation and transformation between Panicum virgatum and Panicum hallii.
Cosgrove DJ (1998) Cell walls: structure, biogenesis, and expansion. Plant Physiology” by Taiz Lincoln and Zeiger Eduardo, Sinauer Associates, Inc, Publishers, 409-443.
Craig W, Gargano D, Scotti N et al. (2005) Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts. Plant Cell Rep 24, 603-611.
Creissen GP (1984) Protoplast regeneration and selection for toxin resistance in Solanum.
Davey MR, Anthony P, Power JB et al. (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23, 131-171.
Davey MR, Anthony P, Patel D et al. (2010) Plant protoplasts: isolation, culture and plant regeneration. Plant cell culture essential methods Wiley-Blackwell, New York, 153-173.
Devaux A, Kromann P, Ortiz O (2014) Potatoes for sustainable global food security. Potato Res 57, 185-199.
Dlugosz EM, Lenaghan SC, Stewart Jr CN (2016) A robotic platform for high-throughput protoplast isolation and transformation. J Vis Exp, e54300.
Echeverri D, Romo J, Giraldo N et al. (2019) Microalgae protoplasts isolation and fusion for biotechnology research. Rev Colomb Biotecnol 21, 101-112.
Eeckhaut T, Lakshmanan PS, Deryckere D et al. (2013) Progress in plant protoplast research. Planta 238, 991-1003.
Fateri Rezvani S, Pazhouhandeh M, Shirzad A et al. (2016) Production of potato resistant plant to PVX using an RNA silencing mechanism. Genet Eng Biosaf J 5, 1-14.
Fowke LC, Constabel F (1985) Plant protoplasts. CRC Press Boca Raton.
Gamborg O, Phillips GC (2013) Plant cell, tissue and organ culture: fundamental methods. Springer Science & Business Media.
Guangyu C, Conner A, Christey M et al. (1997) Protoplast isolation from shoots of asparagus cultures. Int J Plant Sci 537-542,158.
Hameed A, Zaidi SS-e-A, Shakir S et al. (2018) Applications of new breeding technologies for potato improvement. Front Plant Sci 9, 925.
Ikeuchi M, Ogawa Y, Iwase A et al. (2016) Plant regeneration: cellular origins and molecular mechanisms. Development 143, 1442-1451.
Jiang F, Zhu J, Liu H-L (2013) Protoplasts: a useful research system for plant cell biology, especially dedifferentiation. Protoplasma 250, 1231-1238.
Jones H, Karp A, Jones MG (1989) Isolation, culture, and regeneration of plants from potato protoplasts. Plant cell Rep 8, 307-311.
Kang HH, Naing AH, Kim CK (2020) Protoplast isolation and shoot regeneration from protoplast-derived callus of Petunia hybrida cv. Mirage rose. Biology 9, 228.
Kikuta Y, Fujino K, Saito W et al. (1986) Protoplast culture of potato: An improved procedure for isolating viable protoplasts. Hokkaidō Daigaku Nōgakubu 62, 429-439.
Kim H, Kim S-T, Ryu J et al. (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8, 1-7.
Khromov A, Gushchin V, Timerbaev V et al. (2018) Guide RNA design for CRISPR/Cas9-mediated potato genome editing. Dokl Biochem Biophys. Springer. pp. 90-94.
Liang Z, Chen K, Li T et al. (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8, 1-5.
Ling APK, Ong CPL, Tee CS et al. (2009) Establishment of protoplast isolation protocols of Orthosiphon staminues. Int J Agric Sustain 3, 587â.
Malnoy M, Viola R, Jung M-H et al. (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7, 1904.
Masani MYA, Noll GA, Parveez GKA et al. (2014) Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. PLoS One 9, e96831.
Mastuti R, Rosyidah M (2018) In vitro enzymatic isolation of protoplasts from tissues of the medicinal plant Physalis angulata L. In: AIP Conf Proc. AIP Publishing LLC. pp. 020002.
Mazarei M, Al‐Ahmad H, Rudis MR et al. (2008) Protoplast isolation and transient gene expression in switchgrass, Panicum virgatum L. Wiley Online Library.
Muthoni J, Kabira J, Shimelis H et al. (2015) Tetrasomic inheritance in cultivated potato and implications in conventional breeding. Aust J Crop Sci 9, 185-190.
Nadakuduti SS, Buell CR, Voytas DF et al. (2018) Genome editing for crop improvement–applications in clonally propagated polyploids with a focus on potato (Solanum tuberosum L.). Front Plant Sci 9, 1607.
Nanjareddy K, Arthikala M-K, Blanco L et al. (2016) Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis. BMC Biotechnol 16, 1-14.
Navrátilová B (2004) Protoplast cultures and protoplast fusion focused on Brassicaceae: A review. Hortic Sci 31, 140.
Navrátilová B, Skálová D, Ondřej V et al. (2011) Biotechnological methods utilized in Cucumis research-A review. Hortic Sci 38, 150-158.
Nicolia A, Proux-Wéra E, Åhman I et al. (2015) Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts. J Biotechnol 204, 17-24.
Pazhouhandeh M, Karvan G, Razavi A-S et al. (2017) A review on potato genetic engineering researches yet. Genet Eng Biosaf J 6, 175-188.
Potrykus I, Shillito RD (1986) Protoplasts: isolation, culture, plant regeneration. In: Methods Enzymol. Elsevier. pp. 549-578.
Prasertsongskun S (2004) Isolation and culture of suspension protoplasts of vetiver. Journal of Science and Technology 26, 411-416.
Puite K, Roest S, Pijnacker L (1986) Somatic hybrid potato plants after electrofusion of diploid Solanum tuberosum and Solanum phureja. Plant Cell Rep 5, 262-265.
Ren R, Gao J, Lu C et al. (2020) Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in cymbidium orchids. Int J Mol Sci 21, 2264.
Roest S, Gilissen L (1993) Regeneration from protoplasts—a supplementary literature review. Acta botanica neerlandica 42, 1-23.
Selga L (2017) Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre.
Silva Júnior JMd, Paiva R, Campos ACAL et al. (2012) Protoplast production and isolation from Etlingera elatior. Acta Sci Agron 34, 45-50.
Shen Y, Meng D, McGrouther K et al. (2017) Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1. Plant Methods 13, 1-10.
Shepard JF, Totten RE (1977) Mesophyll cell protoplasts of potato: isolation, proliferation, and plant regeneration. Plant Physiol 60, 313-316.
Song J, Sorensen EL, Liang GH (1990) Direct embryogenesis from single mesophyll protoplasts in alfalfa (Medicago sativa L.). Plant cell Rep 9, 21-25.
Svitashev S, Schwartz C, Lenderts B et al. (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7, 1-7.
Subburaj S, Chung SJ, Lee C et al. (2016) Site-directed mutagenesis in Petunia× hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant cell Rep 35, 1535-1544.
Sun B, Yuan Q, Zheng H et al. (2019) An efficient and economical protocol for isolating, purifying and PEG-mediated transient gene expression of Chinese kale hypocotyl protoplasts. Plants 8, 385.
Verma N, Bansal M, Kumar V (2008) Protoplast fusion technology and its biotechnological applications. Chem Eng Trans 14, 113-120.
 Vasil IK, Vasil V (1961) Isolation and culture of protoplasts. In: Int Rev Cytol. Elsevier. pp. 1-19.
Wang Q, Yu G, Chen Z et al. (2021) Optimization of protoplast isolation, transformation and its application in sugarcane (Saccharum spontaneum L). Crop J 9, 133-142.
Wang Y, Sonntag K, Rudloff E et al. (2005) Production of fertile transgenic Brassica napus by Agrobacterium‐mediated transformation of protoplasts. Plant breed 124, 1-4.
Wiszniewska A, Pindel A (2010) Protoplast culture utilization in studies on legume crops. Acta Agric Scand B Soil Plant Sci 60, 389-399.
Zanello LP, Curvetto NR, Barrantes FJ (1988) Rapid method for isolation and purification of protoplasts from epidermal tissue ofVicia faba L. leaves. MIRCEN J Appl Microbiol Biotechnol 4, 275-283.
Zhang X, Wang L, He C et al. (2016) An efficient transient mesophyll protoplast system for investigation of the innate immunity responses in the rubber tree (Hevea brasiliensis). Plant Cell Tissue Organ Cult 126, 281-290.