Large Numbers Of Viable Protoplasts Research Articles

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

BackgroundCotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton.ResultsWe established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 μg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein–protein interaction studies.ConclusionsThe protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

In this issue

AbstractBioluminescent screeningBacart et al.: Biotechnol. J. 2008, 3, 311–324The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light‐emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been presented using different substrates and energy donor/acceptor couples. Today, BRET is considered one of the most versatile techniques for studying the dynamics of protein‐protein interactions in living cells. Various studies have applied BRET‐based assays to screen new receptor ligands and inhibitors of disease‐related‐proteases. Inhibitors of proteinprotein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET‐based methodology, more recent variants, and potential applications to drug screening.Switchgrass transformationMazarei et al.: Biotechnol. J. 2008, 3, 354–359Transient assay systems using protoplasts have been utilized in several plant species and are a powerful tool for rapid functional gene analysis and biochemical manipulations. A protoplast system has not been used in switchgrass (Panicum virgatum L.), even though it is a bioenergy crop that has received considerable attention. Here researchers from Knoxville (TN, USA) and Palestine report the first protocol to isolate large numbers of viable protoplasts from both leaves and roots of two switchgrass genotypes. They show that protoplast transformation with either a 35S or a ubiquitin promoter resulted in an increase in GUS activity compared to the untransformed controls. Their results indicate an efficient protoplast isolation and a transient assay system that can be used to facilitate gene expression studies in switchgrass.Stem cells on chipsJu et al.: Biotechnol. J. 2008, 3, 383–391Directional induction and differentiation of mesenchymal stem cells (MSCs) is very important to clinical therapy, but the mechanisms that govern differentiation are not well understood. Traditional plate culture cannot precisely control cellular behavior because cells take up substances while secreting cytokines and wastes. Here, Chinese researchers used a microfluidic device to culture MSCs inside a microchamber. Hepatic differentiation medium was perfused to evaluate the ability of MSCs to differentiate toward hepatic cells on the chip. Parallel differentiation on 96‐well plates was used to provide a detailed comparison of the differences between the two culturing methods. Their results indicate that a microfluidic platform might be a potential tool for cost‐effective and automated cell culture, and has potential applications in reliable cell‐based screens and assays.

Protoplast isolation and transient gene expression in switchgrass, Panicum virgatum L.

Transient assay systems using protoplasts have been utilized in several plant species and are a powerful tool for rapid functional gene analysis and biochemical manipulations. A protoplast system has not been used in switchgrass (Panicum virgatum L.), even though it is a bioenergy crop that has received considerable attention. Here we report the first protocol to isolate large numbers of viable protoplasts from both leaves and roots of two switchgrass genotypes. Furthermore, we demonstrate transient expression of PEG-mediated DNA uptake in the isolated protoplasts by measuring the activity of beta-glucuronidase (GUS) reporter gene driven by either the Cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin 1 promoter. Protoplast transformation with either the 35S or the ubiquitin promoter resulted in an increase in GUS activity compared to the untransformed controls; however, the extent of GUS activity was considerably higher for the ubiquitin promoter than for the 35S promoter. Collectively, our results indicate an efficient protoplast isolation and transient assay system that can be used to facilitate gene expression studies in switchgrass.

Fertile pea plants regenerate from protoplasts when calluses have not undergone endoreduplication

Large numbers of viable protoplasts were isolated and cultured from five pea genotypes. Calluses obtained (percent final plating efficiency (% FPE)=0.65–2.82% of initially plated protoplasts) exhibited great differences in proliferation and regeneration competence between and within genotypes. Flow cytometric analyses showed the occurrence of endoreduplication processes correlated with such differences, and could serve as a tool for the early prediction of plant regeneration competence from protoplasts. Fertile plants were produced only from calluses with a normal DNA level.

Efficient intergeneric fusion of pea ( Pisum sativum L.) and grass pea ( Lathyrus sativus L.) protoplasts

Large numbers of viable protoplasts of pea ( Pisum sativum ) and grass pea ( Lathyrus sativus ) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm −1 ), or on the use of macro or micro‐methods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.

Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts

Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm(-1)), or on the use of macro or micromethods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.

Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium

Efficient synthesis of a large number of viable protoplasts from Trichoderma longibrachiatum (Trichoderma reesei) mycelium has been found to be influenced by a number of factors. In this case, Trichoderma harzianum NCIM 1185 culture filtrate has been used to prepare protoplasts from Trichoderma longibrachiatum QM 9414. A method has been devised to isolate a large number of viable protoplasts from the mycelium. Detailed analysis of different factors affecting the synthesis of protoplasts from T. longibrachiatum has not yet been reported. The most suitable conditions for protoplasting were as follows: age of the organism in slant, 3 days; mycelium age, 20 h; volume of lytic enzymes, 190 ml; mycelial weight (dry equivalent), 1.66 g; time of contact with lytic enzymes, 2 h; temperature of protoplasting, 30°C; phosphate buffer, 25 m m , pH 6.5; KCl as osmotic stabilizer, 0.7 m . Proper spreading of the mycelium in contact with the lytic enzymes was also found to be necessary.

像走査法による細胞の分級技術に関する研究

Increasing the efficiency of hybrid generation of biological cell using a fusion apparatus requires a large number of viable protoplasts, which are classified beforehand from suspension-included viable and disintegrated biological cells. A cytometric classifier has been developed for protoplasts whose diameters are more than about ten micrometers. This paper introduces a new classification method. Protoplasts are supplied through a sheath flow in a flow cell and their images are focused on four slits arranged at different angles to streamline and scan the moving images relatively. Through these slits, three direction length of a protoplast are measured and the protoplast is classified. The classified protoplasts are sorted into microchambers in isotonic solution. Cytometric resolution of the present classifier is ±10 μm. From the experimental result, knowledge is obtained on the improvement of cytometric resolution and classifying efficiency.

Plant regeneration from callus protoplasts of adult Japanese persimmon ( Diospyros kaki L.)

A large number of viable protoplasts were isolated from calli derived from leaf primordia excised from dormant winter buds of adult Japanese persimmon ( Diospyros kaki L. cv. Jiro). The first cell division occurred within 1 week in KM8p agarose medium with an initial plating density at more than 1 × 10 5 protoplasts/ml. After 2 weeks of culture, the agarose plate was cut into blocks and cultured in bead-type culture. Microcalli, 1–2 mm in diameter, were produced after 12 weeks of culture. When they were subcultured onto KM8 agar medium, half of them developed into callus masses. A high frequency of adventitious bud formation occurred on Murashige and Skoog's (MS) agar medium containing 10 μM zeatin and 0.1 μM 3-indoleacetic acid (IAA). After shoot growth, rooting was induced by 3-indolebutyric acid (IBA) treatment. Plantlets regenerated were then raised in vermiculite.

Plant Regeneration from Leaf Mesophyll Protoplasts of in vitro Cultured Shoots of a Columnar Apple

By culturing shoot cultures of apple in dim light (1 μEm−2s−1) for 6 days before digestion of the leaves and by using an enzyme mixture containing pectolyase (0.01 % w/v), a large number of viable protoplasts were obtained (2–4 × 106 protoplasts/g leaf tissue). Addition of ABA (10−4M) to the plasmolyticum increased the number of initial dividing protoplasts from 8 to 12 % in a modified PPP medium including 1 mg zeatin/1 and 2 mg/1 2,4-D. Sucrose as plasmolyticum in the protoplast growth medium improved survival and increased the divisions during the initial growth phase. Thidiazuron (1 mg/l) combined with 1 mg/l 2,4-D improved the yield of protoplast divisions in ABA-treated cells to 15 %. Regeneration of shoots, initially cultured in 0.5 mg/1 BAP and 2 mg/1 2,4-D, was induced by thidiazuron (1 or 2 mg/1) in MS-medium with 0.5 mg NAA/1. The protoplast-derived shoots were diploid as were the original columnar apple shoots.

Plant Regeneration from Protoplasts of Apple Rootstocks and Scion Varieties ( Malus X domestica Borkh.)

Large numbers of viable protoplasts were isolated from leaves of apple rootstocks M.9, MM. 106 and the scion variety Spartan and also from leaf tissues, callus and cell suspensions of Bramley's Seedling apple. Upon culture, using modified MS-based media (for rootstocks M.9 and MM.106) or modified KM media (for the scions Spartan and Bramley's Seedling), protoplasts entered division to give callus. Shoot buds were regenerated, from the protoplast-derived calli of M.9, MM.106 and Spartan with the production of rooted, autotrophic plants, for M.9 and Spartan apple.

Isolation, culture and plant regeneration of colt cherry ( Prunus avium × pseudocerasus) protoplasts

Large numbers of viable protoplasts were isolated from leaf mesophyll tissues and cell suspension cultures of Colt cherry using 1% (w/v) Onozuka R-10, 0.2% (w/v) Macerozyme R-10, 0.1% (w/v) Driselase, 1% (w/v) polyvinylpirrolidone (av. MW 10 000) (PVP-10) and 2% (w/v) Meicelase, 2% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. Culture media, based on Murashige and Skoog's (MS) salts, supplemented with 9% (w/v) mannitol and various combinations of α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and zeatin (Z) promoted cell wall regeneration followed by cell colony and callus formation. Protoplasts of both sources were compared in relation to their cultural requirements. Protoplast-derived callus underwent organogenesis.

Improved isolation and culture methods for cucumber mesophyll protoplasts

Improved methods for the isolation of large numbers of viable protoplasts from cotyledons and first leaves of two cultivars of cucumber, Cucumis sativus L. cv. Ashley and China are described. Short-term isolation methods (STM), and long-term isolation methods (LTM), using a variety of commercial enzymes are outlined. Liquid culture and subsequent solid culture of isolated first leaf protoplasts resulted in sustained cell division and eventual compact callus formation, which has been induced to form roots. No shoot or whole plant regeneration has, as yet, been demonstrated.

The isolation and culture of cucumber mesophyll protoplasts

Large numbers of viable protoplasts were obtained from the first leaves of two cultivars of cucumber, Cucumis sativus L. cv. Ashley and China. Cell wall regeneration was observed in liquid culture within 3 days of isolation, and division within 5 days.