Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation¹. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years². This capacity allows high frequencies of gene targeting^3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment¹⁰ and PEG-mediated DNA uptake¹¹. Although particle bombardment can produce a high transformation efficiency¹², gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported¹³. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.     

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in ^1,2).To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells ³. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region ^4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation ^6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity ⁵, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level ^3,8.Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal ^3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

Functional identification of AtSKIP as a regulator of the cell cycle signaling pathway in Arabidopsis thaliana

Light is a key environmental cue controlling plant development, which involves meristemic activation by cell proliferation and differentiation. Here, we identify one gene, AtSKIP, associated with cell cycle-regulated root and leaf growth processes in Arabidopsis. The spatial pattern of β-glucuronidase (GUS) activity indicated that AtSKIP is expressed in the leaf primodia, root meristem region and root vascular system, and can be activated by light. Ectopic expression of AtSKIP resulted in enhanced leaf development but suppressed root elongation in Arabidopsis, whereas AtSKIP^DD seedlings displayed retarded leaf growth and normal root growth. Moreover, AtSKIP cells displayed enhanced sensitivity to a cytokinin in a callus induction assay, further demonstrated that AtSKIP expression altered endogenous cell cycle-regulated signaling in plants. Together, these data indicate that AtSKIP participates in cell cycle-mediated growth of leaf and root.     

Bax-induced cell death of Arabidopsisis meditated through reactive oxygen-dependent and -independent processes

An Arabidopsisprotoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsismesophyll protoplasts expressing murine BaxcDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivotargeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N-acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsisnucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of ArabidopsisBax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥（Arabidopsis thaliana）Wer：：GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术（FACS）分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer：：GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

Overexpression of a cytosol-localized rhamnose biosynthesis protein encoded by Arabidopsis RHM1 gene increases rhamnose content in cell wall

l-Rhamnose (Rha) is an important constituent of pectic polysaccharides, a major component of the cell walls of Arabidopsis, which is synthesized by three enzymes encoded by AtRHM1, AtRHM2/AtMUM4, and AtRHM3. Despite the finding that RHM1 is involved in root hair formation in Arabidopsis, experimental evidence is still lacking for the in vivo enzymatic activity and subcellular compartmentation of AtRHM1 protein. AtRHM1 displays high similarity to the other members of RHM family in Arabidopsis and in other plant species such as rice and grape. Expression studies with AtRHM1 promoter-GUS fusion gene showed that AtRHM1 was expressed almost ubiquitously, with stronger expression in roots and cotyledons of young seedlings and inflorescences. GFP::AtRHM1 fusion protein was found to be localized in the cytosol of cotyledon cells and of petiole cells of cotyledon, indicating that AtRHM1 is a cytosol-localized protein. The overexpression of AtRHM1 gene in Arabidopsis resulted in an increase of rhamnose content as much as 40% in the leaf cell wall compared to the wild type as well as an alteration in the contents of galactose and glucose. Fourier-transform infrared analyses revealed that surplus rhamnose upon AtRHM1 overexpression contributes to the construction of rhamnogalacturonan.     

芜菁原生质体的分离及绿色荧光蛋白的瞬时表达

目的：分离芜菁叶片原生质体,建立蛋白质在芜菁原生质体的瞬时表达系统。方法：以津田芜菁成叶为试材,酶解分离原生质体;通过PEG介导的转化,将编码绿色荧光蛋白（GFP）的瞬时表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中GFP的表达情况。结果：分离出大量的津田芜菁原生质体,并获得了较高的转化效率,GFP在整个原生质体中都有表达。结论：建立了津田芜菁原生质体瞬时表达系统。     

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment ¹. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions ^1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion³. Matrix density ⁴, stiffness ^5-6, and structure ^6-7, interstitial fluid pressure ⁸, and interstitial fluid flow ⁸ are all altered during cancer progression.Interstitial fluid flow in particular is higher in tumors compared to normal tissues ^8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 μm s^-1, depending on tumor size and differentiation ^{9, 11}. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability ¹². Interstitial fluid flow has been shown to increase invasion of cancer cells ^13-14, vascular fibroblasts and smooth muscle cells ¹⁵. This invasion may be due to autologous chemotactic gradients created around cells in 3-D ¹⁶ or increased matrix metalloproteinase (MMP) expression ¹⁵, chemokine secretion and cell adhesion molecule expression ¹⁷. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts ¹⁸, (b) transporting of antigens and other soluble factors to lymph nodes ¹⁹, and (c) modulating lymphatic endothelial cell morphogenesis ²⁰.The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion ^{13-15, 17}. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.     

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis^1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA³. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment⁴. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion^5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages⁹. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment¹⁰. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture^11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)^13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs^9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting¹⁶. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K⁺ channel subunit that assembles with different inward-rectifying K⁺ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K⁺ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K⁺ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K⁺ current in Arabidopsis root epidermal protoplasts as well as K⁺ acquisition and growth in seedlings when channel-mediated K⁺ uptake was limiting. That SYP121 should be important for gating of a K⁺ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K⁺ uptake coordinately with membrane expansion for cell growth.     

Uptake and retention of external solutes from the digest medium during preparation of protoplasts

The extent to which solutes present in the digest medium enter cells and are retained during preparation of protoplasts was investigated. When barley (Hordeum vulgare, L. cv. Clipper) leaf slices were incubated in sorbitol there was considerable uptake of sorbitol into the tissue, which continued for up to 6 h and was dependent on the sorbitol concentration in the external medium. Protoplasts prepared by digesting leaf slices in a medium containing [¹⁴C]sorbitol but isolated and purified in media with unlabelled sorbitol contained significant amounts of [¹⁴C]sorbitol. From measurements of the protoplast volume, the internal sorbitol concentration was calculated to be 100 mM, assuming uniform distribution of the sorbitol throughout the protoplasm. The uptake of sorbitol during digestion and its retention by protoplasts was confirmed by measuring sugars in protoplast extracts by gas sucrose or inositol. Vacuoles prepared from the protoplasts contained 83% of the sorbitol present in protoplasts. It is concluded that considerable uptake of solutes from the external medium occurs during digestion of leaf tissue and that these solutes are retained within the protoplasts during isolation and purification. The solutes appear to be uniformly distributed throughout the subcellular compartments of the protoplast.     

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)^1,2. However, understanding the biosynthesis and function of these components remains challenging.Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall³. Furthermore, their composition is also modified during development¹, or in response to environmental cues⁴.In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis⁵. However, relatively few of the biosynthetic genes have been functionally characterized ^4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types⁶. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained⁷. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.Monoclonal antibodies (mAbs)^8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously^9,10,11.Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)^10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems¹² and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 10⁶–10⁸ peach mesophyll cells and protoplasts · gfw^-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml^-1. Purification of protoplasts on a sucrose gradient yielded about 10⁷ protoplasts · gfw^-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - FDA fluorescein diacetate - MES 2-M-morpholinoethane sulphonic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PVP polyvinylpyrrolidone