Factors Influencing the Growth and Division of Pea Mesophyll Protoplasts

High yields of viable protoplasts were produced from pea leaves provided that only leaves of the same age were used in each preparation. The conditions under which the pea plants were grown and the age of the plants were also important. The protoplasts were cultured in a medium supplemented with 1 mg/1 2iP and 1 mg/1 2,4-D. They were able to regenerate cell walls within two days. After 5 days cell divisions were apparent and sustained divisions led to callus formation. Special emphasis has been given in this paper to the choice of leaf material for protoplast isolation.     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

烟草花粉原生质体与叶肉原生质体的融合及培养早期变化

用ＰＥＧ—高Ｃａ高ＰＨ法诱导抗卡那霉素的烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ）品系Ｎ３６４＋Ｋｍ＋花粉原生质体和黄花烟草（Ｎｉｃｏｔｉａｒｕｓｔｉｃａ）叶肉原生质体融合。幼嫩花粉原生质体和叶肉原生质体之间的融合体培养启动胚胎发生分裂,经卡那霉素筛选后,少数多细胞团存活并形成小愈伤组织。成熟花粉原生质体与叶肉原生质体之间的融合体则仅产生管状结构。这一结果表明,作为融合一方的花粉原生质体的发育时期对融合产物的发育途径有重要影响。     

Effects of Culture Conditions on Cell Division and Composition of Regenerated Cell Walls in Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere grown in a liquid medium, and the effects of osmolarityand growth regulators on cell division and on the compositionof regenerated cell walls were investigated. The concentrationof mannitol optimal for cell division was 0.3–0.4 M. Thepresence of 2,4-D was essential for cell division, and 6-benzylaminopurine(BAP) enhanced cell division at concentrations of 0.03–0.1ppm. However, the composition of regenerated cell walls wasabnormal under suspension culture; the predominant sugar wasglucose, indicating that the regenerated cell walls consistedmostly of glucans, and that the other cell-wall components werereleased into the medium. Mannitol, 2,4-D, and BAP at variousconcentrations did not significantly affect the sugar compositionof the regenerated cell walls. Compared with liquid culture,cell division was stimulated when protoplasts were culturedon agar, and their regenerated cell walls had a compositionsimilar to that of the original culture. The importance of thephysical environment for the deposition of polysaccharide componentsin cell walls and the interrelationship between cell divisionand the regeneration of cell walls by protoplasts are discussed. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan. (Received June 10, 1981; Accepted December 12, 1981)     

Pressure Effects on Membrane Potentials of Mesophyll Cell Protoplasts and Epidermal Cell Protoplasts of Commelina communis L.

Pantoja, O. and Willmer, C. M. 1986. Pressure effects on membranepotentials of mesophyll cell protoplasts and epidermal cellprotoplasts of Commelina communis L.—J. exp. Bot. 37:315–320. Membrane potentials of epidermal cell protoplasts and mesophyllcell protopiasts of Comnelina communis were measured when theprotoplasts were immobilized in a suction micropipette. Whenzero suction was employed, membrane potentials of both protoplasttypes were near to zero. As suction pressure was increased,membrane potentials became increasingly more negative with gradientsof 14·3 mV/kPa and 10·5 mV/kPa for mesophyll cellprotoplasts and epidermal cell protoplasts, respectively. Theplasma membrane is stretched when suction pressure is appliedto protoplasts and it is considered that this simulates cellturgor pressure which is associated with negative membrane potentialsof intact cells. The results help to explain why some investigatorsobtain positive membrane potentials for protoplasts while othersobtain negative values. The results also indicate that considerablecaution is needed in the interpretation of ion flux data whenprotoplasts are used. Key words: Commelina communis, membrane potentials, pressure, protoplasts     

Isolation and Regeneration of Tobacco Mesophyll Cell Protoplasts under Low Osmotic Conditions

A method is described for the isolation of large numbers of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyll cell protoplasts under relatively low external osmotic conditions. The procedure utilized 0.2 m sucrose as the primary osmoticum and a mixture of 0.5% macerozyme, 4% cellulase, and 2% polyvinylpyrrolidone, pH 5.4. The viability of resultant protoplasts was confirmed through regeneration of fertile plants. Plating and regeneration studies revealed, however, that qualitative and quantitative modifications in plating and differentiation media were necessary for protoplasts prepared in this manner. Over-all, the procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration. In addition, the system should permit studies related to the influence of differing osmoticum levels on a variety of cell functions.     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

The Roles of Cell Division and Cell Expansion in the Growth of Alfalfa Leaf Mesophyll

Leaf mesophyll of Medicago sativa (L.) was investigated to determinethe roles of cell division and cell expansion in tissue growth.Samples of leaf tissue were macerated, stained, and squashed.The slides were studied under a phase microscope to determinethe percentage of recently divided cells and the average celldiameter for leaflets of varying lengths. Cell division wasgreatest in young leaflets and virtually ceased as a leaf lengthof 12 mm was attained. For leaflets less than 12 mm in length,the rate of increase in cell size appeared to be inversely associatedto the degree of cell division. For alfalfa leaflets greaterthan 12 mm in length, the mean cell size increased in proportionto leaf length since cell division had virtually ceased.     

叶绿体S S R标记:柑橘体细胞杂种胞质遗传分析的一种新方法

从水稻(Oryza sativa L.)、烟草(Nicotiana tabacum L.)和黑松(Pinus thunbergiiParl.)等植物的22对叶绿体SSR引物中筛选出 5对能用于柑橘叶绿体SSR分析的引物,应用这5对引物对9个组合的柑橘体细胞杂种的叶绿体遗传进行了分析.结果表明:这些组合再生的杂种中叶绿体都呈现随机分离,该现象与以前报道的RFLP分析结果一致,而且其可靠性已被CAPS分析所证实.表明柑橘叶绿体SSR同RFLP及CAPS一样可靠,并且更简单高效、易于操作,特别适合对柑橘等植物体细胞杂种进行早期胞质遗传组成分析.     

叶绿体SSR标记：柑橘体细胞杂种胞质遗传分析的一种新方法

从水稻(Oryza sativa L．)、烟草(Nicotiana tabacum L．)和黑松(Pinus thunbergii Parl．)等植物的22对叶绿体SSR引物中筛选出5对能用于柑橘叶绿体SSR分析的引物,应用这5对引物对9个组合的柑橘体细胞杂种的叶绿体遗传进行了分析。结果表明：这些组合再生的杂种中叶绿体都呈现随机分离,该现象与以前报道的RFLP分析结果一致,而且其可靠性已被CAPS分析所证实。表明柑橘叶绿体SSR同RFLP及CAPS一样可靠,并且更简单高效、易于操作,特别适合对柑橘等植物体细胞杂种进行早期胞质遗传组成分析。     

Genetic Dissection of Neural Properties using Somatic Cell Hybrids

A set of neuronal genes can be expressed in neuroblastoma × L cell hybrids and hybrid cell lines with specific defects in neural function can be generated in high yield.     

Interaction between Pseudomonas syringae pv. Pisi and Isolated Pea Mesophyll Protoplasts

The interaction between five races of Pseudomonas syringae pv. pisi (PSP) and isolated mesophyll protoplasts obtained from five pea cultivars was studied. There was no trend in the attachment of bacterial cells to surfaces of compatible and incompatible host protoplasts. The viability of protoplasts from compatible and incompatible host-pathogen interactions did not differ significantly; however, changes in the viability of cultivar Kelvedon Wonder protoplasts, compatible with all five races, was relatively more stable following inoculation with bacteria than those of cultivar Fortune, incompatible with all of the five races. Protoplast cell wall regeneration did not take place until 24–48 h after isolation. It is concluded that the use of pea protoplasts as a model system for studying the pea-PSP interaction appears to have considerable potential for the future, but more basic research is required.     

Direct Somatic Embryogenesis, Plant Regeneration and Evaluation of Plants Obtained from Mesophyll Protoplasts of Brassica juncea)

Isolated mesophyll protoplasts of Brassica Juncea directly developedinto somatic embryos on medium supplemented with an auxin anda cytokinin. The somatic embryos germinated into plantlets whenmedium was supplemented with GA₂, and ABA. The plantlets weretransferred to the field and the second generation (P₂) wasevaluated for various agronomic characters. Some of the P₂ linesshowed variation in height and time required for flowering.Only two out of 53 lines were comparable to the control in grainyield Brassica Juncea, Indian mustard, direct embryogenesis, protoclonal variation, yield     

Photoperiodic Regulation of Cell Division and Chloroplast Replication in Heterosigma akashiwo

Cell division and chloroplast replication in Heterosigma akashiwo(Hada) Hada occurred as separate synchronous events during thecell cycle when cells were subjected to light-dark regimes.Under three different photoperiodic cycles of 10L/14D (10 hlight/14 h dark), 12L/12D or 16L/8D, cell division began athour 19–20 and finished at hour 23–26 after theonset of the light period, while chloroplast replication beganat hour 20–22 after the onset of the dark period. Almostall the cells divided only once in the 12L/12D cycle. The rateof increase in chloroplast number during one light-anddark cyclewas always equal to that in cell number in every photoperiodexamined. Light was essential for both cell division and chloroplast replication,but the minimum light period necessary for each event differed.When the light period was shorter than 6 h, no cell divisionoccurred; when it was shorter than 3 h, no chloroplast replicationoccurred. (Received February 26, 1987; Accepted June 17, 1987)     

The Effects of Extracellular Calmodulin on Cell Wall Regeneration of Protoplasts and Cell Division

The effects of anti-calmodulin (CaM) serum, CaM antagonist W7-agaroseand exogenous pure CaM on cell wall regeneration of protoplastsand cell division for Angelica dahurica and other plants werestudied. Anti-CaM serum inhibited cell wall regeneration ofprotoplasts and the first cell division in dose-dependent manner,while the same amount of preimmune serum had a much less inhibitoryeffect than anti-CaM serum. The first cell division was alsoinhibited by CaM antagonist W7-agarose. The addition of exogenouspure CaM enhanced cell wall regeneration of protoplasts andthe cell division for several species of plants, while the sameamount of bovine serum albumin had no obvious effect. CaM wasdetected in the normal culture medium by means of enzyme-linkedimmunosorbent assay. Its content increased with the culturetime. The results suggest that extracellular CaM plays an importantrole in promoting cell wall regeneration of protoplasts andcell division. The possible mechanisms by which extracellularCaM achieves its effects are discussed. (Received February 24, 1994; Accepted November 14, 1994)     

Hormonal Control of Gene Expression During Reactivation of the Cell Cycle in Tobacco Mesophyll Protoplasts

The roles of auxin and cytokinin in cell cycle reactivation were studied during the first 48 h of culture of mesophyll protoplasts of Nicotiana tabacum. Using hormone delay and withdrawal studies we found that auxin was required by 0–4 h of culture, whereas cytokinin was not required until hour 10–12, which is 6–10 h before S phase. Cycloheximide blocks division, indicating that protein synthesis is required. In an effort to detect a molecular response to either hormone, we examined the expression of the cell cycle marker, cdc2. Cdc2 expression was detected by 12 h of culture, coincident with the timing of the cytokinin requirement and well before the entry into S. However, cdc2 was partially induced by either auxin or cytokinin alone, suggesting that cdc2 expression is not the primary target of either hormone. Our hormone delay experiments suggest that there are separate signal transduction pathways leading from auxin and from cytokinin to reactivation of the cell cycle and that these pathways converge before S. The underlying mechanisms for these distinct pathways remain to be elucidated. Received November 4, 1997; accepted October 7, 1998