Cotyledon Protoplasts Research Articles

Evaluation of Isoflavones as Allelochemicals with Strong Allelopathic Activities of Kudzu Using Protoplast Co-Culture Method with Digital Image Analysis

The inhibitory allelopathic activity of Pueraria montana (Kudzu), and activities of two putative allelochemical isoflavones, puerarin and daidzein, were evaluated using the protoplast co-culture method with digital image analysis using lettuce as a recipient (DIA-PP method). Cotyledon protoplasts of Kudzu were isolated using Cellulase R10 and Driselase 20 in 0.6 M mannitol solution. Optimal hormonal condition and density for growth of Kudzu protoplasts were surveyed. Medium for co-culture of Kudzu or isoflavones with lettuce protoplasts was 50 μl liquid MS basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose, and 0.4 M or 0.6 M mannitol. Protoplast division of lettuce was strongly inhibited by Kudzu at a low density (104/ml). Slightly less inhibition by Kudzu on cell wall formation and yellow pigment accumulation stages of lettuce growth was also observed. Puerarin did not inhibit the growth of lettuce protoplasts at three growth stages but slightly stimulated growth at high concentrations. By contrast, daidzein, aglycon of puerarin, inhibited growth at three stages of lettuce protoplast growth and strongly inhibited cell division at 100 μM. Daidzein might be one cause of the strong inhibitory allelopathic activity of Kudzu. Grade of inhibitory activities was compared with that of other allelopathic plants including an invader plant and their allelochemicals studied using the DIA-PP method.

A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins.

Calli protoplasts isolated from three soybean cultivars are useful tools to evaluate guide RNAs for clustered regularly interspaced short palindromic repeats (CRISPR)-based precise gene editing. A type V CRISPR effector, LbCpf1(Cas12a) from Lachnospiraceae bacterium ND 2006, has been used for precision editing of the plant genome. We report that callus-derived protoplasts from three soybeans, including Glycine Max var. Williams 82 and two Korean cultivars (Kwangan and Daewon) represent efficient systems for the screening of active crRNA for CRISPR/LbCpf1. CRISPR/LbCpf1 ribonucleoproteins (RNPs) were delivered as complexes of purified endonucleases mixed with designed crRNA to simultaneously edit target genes of GlymaFAD2-1A and GlymaFAD2-1B transfected into three soybean protoplasts including genome-sequenced Williams 82 with cultivars, Kwangan and Daewon. Previously, we reported that nine crRNAs designed for LbCpf1 exhibited varying degrees of editing efficacy for two FAD2 genes. Among the nine crRNAs, the LbCpf1-crRNA3 complexes showed the highest efficiency in soybean cotyledon protoplasts. The new screening systems of callus protoplasts from three soybeans have been successfully used to transfect GFP-tagged markers and CRISPR/LbCpf1 RNPs. The callus protoplasts confirm that the LbCpf1-crRNA3 complex is an active crRNA for LbCpf1 to edit two FAD2 genes similar to cotyledon protoplasts. These results demonstrate that soybean callus protoplast-based CRISPR/crRNA selection is a new and practical tool to screen the efficacy of crRNAs and a prerequisite for progressive regeneration of the edited soybean.

5-Azacytidine promotes shoot regeneration during Agrobacterium-mediated soybean transformation

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl2, MgSO4, and CaCl2, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl2 at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl2 at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl2 and MgSO4 were observed at a wide range of concentrations up to 400 mM. CaCl2 was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.

Characterization of upstream sequences from the 8S globulin gene of Vigna radiata

Seeds rich in protein in nature, are ideal bioreactors for economic production and storage of valueadded recombinant proteins and enzymes for industries. The upstream region of the seed storage protein gene is able to provide an attractive promoter for seed-specific expression of heterologous genes. Our previous research showed that 8S globulin occupied the majority of total soluble protein stored in seeds of mung bean ( Vigna radiata ), a rich source of protein, indicating that the promoter of this gene could be a seed-specific promoter with high activity. To improve the expression of heterologous proteins in plants to act as a bioreactor, the putative seed-specific promoter of 8S globulin gene was characterized in this study. Hence, this potential promoter of beta subunit gene of 8S globulin (8SGb) was isolated by genome walking. Analysis with various promoter prediction softwares showed that the promoter sequence possessed many common motifs related to gene transcription in the seed (such as W-box, ABRE element, E-box, etc.). The putative promoter was subsequently cloned into the binary vector pBI121-GFP by replacing the CaMV 35S promoter. The resultant construct designated as pBI-8SGb-GFP was transformed to mung bean cotyledon mesophyll protoplasts. Reporter gene GFP was expressed high in cotyledon protoplasts, which was detected by confocal microscopy, demonstrating the specific activity of 8SGb promoter in driving gene expression. This study also proved that the 8SGb promoter is an efficient regulatory element for plant seeds to act as a bioreactor. Key words : Seed-specific, promoter, genome walking, Vigna radiata.

A breakthrough in lupin biotechnology: prolific protocolonisation in recalcitrant white lupin (Lupinus albus) triggered by bovine serum albumin

AbstractSix nutrient formulations were studied for their efficacy in inducing mitosis in white lupin seedling cotyledon protoplasts of which the formulations of Schäfer‐Menuhr & Stürmer (AS) and Kao (K8p) were found to be superior over the other four when supplemented with 6‐benzylaminopurine and α‐naphthaleneacetic acid (α‐NAA). An ultrafiltration treatment of K8p increased mitotic frequency by 130% when compared with the untreated control. Medium enrichment with 0.2% bovine serum albumin (BSA) brought about a dramatic 1341% rise in protoplast division in comparison with BSA‐free medium but only when the enrichment was carried out in Kao and Michayluk (KM8p) background containing 2,4‐dichlorophenoxyacetic acid, α‐NAA and zeatin. A higher number of protocolonies (each proliferating from single protoplast following multiple divisions) were seen in 0.4% BSA. With this breakthrough in white lupin protoplast research, it is now possible to reproducibly obtain protocolonies that was hitherto not possible.

Fertile somatic hybrids between Solanum integrifolium and S. sanitwongsei (syn. S. kurzii) as candidates for bacterial wilt-resistant rootstock of eggplant

UV-irradiated cotyledonary protoplasts of Solanum integrifolium and iodoacetamide-treated cotyledonary protoplasts of S. sanitwongsei were electrofused and cultured. Regenerated plants were classified into three groups based on morphology and genomic in situ hybridization data. Morphology of the first group was intermediate between those of parental species. The plants bore fruits with viable seeds and had a chromosome number of 2n� 48, the sum of the parental chromosome numbers, suggesting that they were symmetric fusion hybrids. Morphology of the plants in the second group was more S. integrifolium-like than that of the first group, and had two sets of S. integrifolium chromosomes and one set of S. sanitwongsei chromosomes. In contrast, plants in the third group had one set of S. integrifolium chromosomes and two sets of S. sanitwongsei chromosomes. Plants in the second and third groups were less vigorous than those in the first group, and bore few fruits. Electrophoretic analysis of the isozymes shikimate dehydrogenase, isocitrate dehydrogenase, and phosphoglucomutase, as well as random amplified polymorphic DNA analysis, demonstrated that 23 of regenerated plants from the three groups were somatic hybrids. The plants in the first group grew more vigorously than the parental plants and produced more than 5000 seeds per plant. The fertile somatic hybrids obtained in this study may be suitable candidates for eggplant rootstocks.

Efficient plant regeneration from protoplasts of eggplant rootstock cultivar and its wild relatives

Protoplast culture and shoot regeneration from protoplast-derived calli were compared among different organs of Solanum integrifolium and three of its wild relatives (S. abutiloides, S. scabrum, and S. toxicarium). Leaves, cotyledons, and hypocotyls were used as sources of protoplast preparation. After one month of culture, a high frequency of visible colony formation was obtained from cotyledon protoplasts of S. integrifolium and S. scabrum, hypocotyl protoplasts of S. integrifolium and S. abutiloides, and leaf protoplasts of S. integrifolium and S. toxicarium. In addition, when the primary culture was started at a density of 2.5−5×104 protoplasts ml−1, the highest frequency of colony formation was obtained. Moreover, when the colonies were subcultured for 7 days on solid callus-proliferation medium before being transferred to shoot-induction medium, the plant regeneration frequency improved to between 91.8 and 98.8%.

Enhanced protoplast division by encapsulation in droplets: An advance towards somatic hybridisation in recalcitrant white lupin

SummaryLupins are highly nutritious fodder and pulse crops but the greatest challenge in their genetic enhancement is the difficulty in obtaining hybrids through conventional sexual approaches. To bypass this, a procedure for the culture of hitherto recalcitrant lupin protoplasts is now being developed so that the somatic hybrids can be regenerated. This study provides a basis for a regime to culture lupin protoplasts. Cotyledonary protoplasts of white lupin (Lupinus albus) were plated in two diverse media for the evaluation of various plating regimes. The protoplasts divided in agarose as well as in Gelrite™ but embedding in agarose at 6 g L−1 concentration resulted in a higher rate of mitosis. Sodium alginate embedding inhibited protoplast division. Protoplast plating in the form of liquid suspension was significantly inferior to embedding. A filter paper substratum was clearly noxious to protoplast division. Vis‐à‐vis other designs of plating, a 400% improvement in protoplast elongation and division was achieved by plating in the form of 25 μL droplets at the base of 60 mm × 15 mm Nunclon™ dish and overlaying with liquid medium. Better results in terms of protoplast elongation and division were obtained with K8p medium as compared to the AS medium. This report on lupin protoplast culture represents a significant breakthrough in the genus in which morphogenesis has not been described to date.

WATER PERMEABILITY CHANGES OF THE CELL MEMBRANE AS A FUNCTION OF TIME

Water loss and microscopic water migration inside tissues or cells are major causes of vegetable deterioration and wilting during the postharvest period. Water loss and water migration caused by respiration and transpiration are influenced by the condition of the cell membrane or other biomembranes. Therefore, for the studying on postharvest technology, it is essential to understand the changes in the properties of biomembranes of the agricultural products during the storage. The change in cell membrane water permeability coefficient (L p ) and osmotic tolerance of barley cotyledon protoplast was measured and the change in dynamic state of water in the cotyledons was estimated by 1 H-NMR during the storage. The barley cotyledons were cut from the plants and stored in test tubes at 20°C in dark. The storage periods were 0, 24, 48 and 72 hours. After the storage, protoplasts were separated from the cotyledons and the Lp of the cell membranes were measured by Two Laminar Flow (TLF) Method.[...]

Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein.

Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato (Lycopersicon esculentum, cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco (Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.

Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana

An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of alpha-naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.

Somatic Hybrids between Lycopersicon esculentum and Lycopersicon chmielewskii.

Interspecific somatic hybrids were obtained by electric protoplast fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. chmielewskii (LA1330). A multiple-step selection procedure was used in selecting the hybrids depending on IOA treatments and a culture medium that only allows cotyledon protoplasts to regenerate. Plants were regenerated on the calli derived from fused protoplasts. After acclimation, the hybrid plants with fruits through self-pollination were obtained. Identification of the regenerated putative hybrid plants was carried out by morphological, chromosomal and random amplified polymorphic DNA (RAPD) analysis, as well as restriction fragment length polymorphism (RFLP) comparison of parental and hybrid plants. The hybrids showed unique morphologies in leaf, flower and stem compared with that of two fusion partners, 2n = 4x = 48, 47, 69-72 chromosomes, both of the parental-specific RAPD bands, and genomic recombination and elimination occurred in mitochondria level. Cell fusion technique shows its new potential in foreign gene transformation between L. esculentum and L. chmielewskii.

TransientuidAgene expression in electroporated cotyledonary protoplasts ofPinus nigrassp.salzmanniiand in bombarded cotyledons

We have developed a method for the routine isolation of Pinus nigra Arn. ssp. salzmannii (Dunal) Franco protoplasts. The optimized electroporation conditions for uidA gene (gene for beta-glucuronidase) expression in protoplasts from cotyledons excised from 8-day-old seedlings were determined using an exponential pulse wave generator for gene transfer. The protoplasts were electroporated with plasmids containing the chimeric uidA gene under the control of several promoters, and in parallel, cotyledons were bombarded with the same constructs using a biolistic gun. Both techniques confirmed that gene expression was higher when controlled by the sunflower polyubiquitin gene promoter than by the cauliflower mosaic virus CaMV35S promoter, whereas the rice actin and maize alcohol dehydrogenase promoters resulted in lower uidA expression levels, as determined fluorometrically. In this study, the electroporation procedure has been more effective than the particle bombardment procedure to determine the promoter strength on transient gene expression in P. nigra.

Comparative investigations on protoplast culture of some Brazilian and Hungarian sweet pepper cultivars and hybrids

Cotyledon protoplasts were isolated from 16-18-day-old in vitro grown seedlings of 9 Brazilian and 3 Hungarian pepper varieties and hybrids. Large numbers (average 9.59 X 106 protoplasts g 14 fresh weight) of highly viable (average 87.0%) protoplasts were released using a pectocellulolytic enzyme mixture. Protoplasts were cultured in K8p mediuni using an alginate disc embedding method. The osmotic pressure of the medium surrounding the alginate-embedded protoplasts was reduced by replenishing the liquid medium at K8p:K8 ratios of 1:0. 2:1, 1:1 in the first. second, and third week, respectively. Initial plating efficiency (IPE) average was 38.5% and after 21 days protoplasts reached microcolonies (15-20 cells) stages. Microcolonies were transferred after 3-4 weeks to a MS-based medium supplemented with 1.0 mg I-1 zeatin, 3.0% (w/v) sucrose, 0.24% (w/v) phytagel and pH 5.8, whereupon they formed callus. Final plating efficiency (FPE) average was 0.29% at a plating density of 1.0 x 105 protoplasts Protoplast-derived calli were cultured on a range of MS-based media supplemented with either BAP, IAA, TDZ; and zeatin. No morphogenic response was observed in any genotype investigated.

Asymmetric somatic hybridisation between the annual legumes Medicago truncatula and Medicago scutellata

Interspecific asymmetric somatic hybrids have been produced between the sexually incompatible annual Medicago species Medicago truncatula and M. scutellata using only one regenerable parent and improved protoplast techniques. Regenerable M. truncatula leaf protoplasts were inactivated by lethal doses of iodoacetamide. Cotyledon protoplasts of non-regenerable M. scutellata were produced from 9 to 13 day old γ-irradiated cotyledons. Polyethylene glycol was used for protoplast fusion, and its successful use depended on a strategy to eliminate toxic effects. More than 50 mature regenerated plants were obtained from the fusion products and amplified fragment length polymorphism and chromosome analysis of a sample of 20 plants allowed the identification of two asymmetric hybrid plants. This technique is suitable for transferring characters from incompatible annual Medicago species to Medicago truncatula.

Protoplast fusion between Lycopersicon esculentum and L. peruvianum -complex: somatic embryogenesis, plant regeneration and morphology

Somatic hybrids were obtained by polyethylene glycol fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. peruvianum (PI270435) or L. chilense (PI128652). The hybrids were selected by a multiple-step selection procedure relying on the different colors of the fusion partners, IOA treatment of cotyledon protoplasts, and the use of a culture medium which only allowed cotyledon protoplasts to regenerate. The somatic embryos were derived from greenish calli that formed from the fusion mixtures, developed progressively through the globular, heart, and torpedo stages, and finally formed complete plantlets. The excised torpedo-stage embryos could be propagated on a modified medium. The morphology of the somatic hybrids were intermediate to their donor partners, and chromosome observations indicated that the hybrids were tetraploid, hexaploid, and aneuploid.

Cotyledon-derived diploid and haploid protoplast culture and diploid plant regeneration in Brassica napus cv. ' Topas '

The present paper describes a simple and reliable protocol for the successful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L. cv. 'Topas'. Various protoplast isolation media, nutrient media, subculture procedures, and protoplast sources were tested under two culture temperatures. Protoplast viability, cell wall regeneration, and cell division were monitored. Single cotyledon-derived protoplasts formed calli in liquid protoplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plants regenerated either by shoot formation or embryogenesis. Continuous culture at 32°C instead of 25°C favoured the initiation of cell division and cell proliferation but prevented regeneration, although calli maintained regeneration capacity. Viable haploid protoplasts were isolated from cotyledons of heat-shock-induced, microspore-derived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were triggered in the two types of haploid protoplast cultures, and microcalli were formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.Key words: cotyledon protoplast culture, haploid culture, plant regeneration.

Regeneration and characterisation of Cucumis melo L. (+) Cucumis anguria L. var. longipes (Hook. fil.) Meeuse somatic hybrids

Cotyledon protoplasts of an albino Cucumis melo L. ‘Cantaloup Charentais’ somaclonal variant were electrofused with protoplasts of the wild species Cucumis anguria L. var. longipes (Hook. fil.) Meeuse. Selection of putative somatic hybrids was based on competence of the albino melon to grow and regenerate shoots together with the ability of the wild species to synthesise chlorophyll. The ITS region of C. anguria ribosomal DNA was sequenced to design species-specific primers, which allowed us to distinguish between parental lines and fusion products by PCR amplification. By using this method, all the organogenic lines characterised proved to be somatic hybrids. Three of sixteen selected lines produced shoots with albino and green sectors. Eleven lines remained green but shoots developed abnormally and did not produce roots in vitro. Two hybrid lines regenerated normal shoots but with a limited ability to produce roots in vitro and in vivo. Applicability of molecular characterisation to optimise the quick recovery of fusion products is discussed.

Production of Intergeneric Hybrid Calli from C3 and C4 Species of Amaranthaceae through Protoplast Fusion.

Intergeneric somatic hybrid calli were obtained through electrofusion between protoplasts of Celosia cristata L. (C3 species) cell suspension and Amaranthus tricolor L. (C4 species) cotyledon. The highest binary fusion frequency, 4∼5%, was attained when the protoplasts were suspended in fusion solution containing 0.4 M sorbitol and 2.0 mM CaCl2, aligned by an AC field (200 V/cm, 0.5 MHz) for 30 s and fused by a single DC square pulse at 1.6 kV/cm for 40 μs. Fusion products were identified by characteristics of both parents in the hybrid cells : they possessed green chloroplasts or pink plastids from cotyledonary protoplasts as well as a cytoplasm-rich colorless sector derived from cell suspension protoplasts. The occurrence of two types of calli, compact and friable, obtained after culturing on proliferation medium lead to the isolation of putative hybrid calli without any artificial selection because C. cristata protoplasts regenerated only friable calli and A. tricolor protoplasts did not produce calli. A total of 14 lines of putative hybrid compact calli were obtained by screening on the basis of callus morphology. An isozyme analysis of acid phosphatase confirmed the hybridity of 14 lines of compact calli. Callus morphology was also studied by scanning electron microscopy.