Growth and Development of Pelargonium Pith Cells in vitro

Pelargonium pith callus cultured in media containing auxin and cytokinin will not differentiate formed organs if monthly subcultures are made into fresh medium. Left on semisolid medium for prolonged periods, such callus gives rise to nodules containing lignified cells. Such nodules can be stimulated to produce shoots and roots if cultured for one month in shaken liquid medium or for longer times on semisolid mediium lacking both auxin and cytokinin. The subsequent development of organs is best on semisolid medium. Entire plants have heen produced in this way from initial pith explants containing several hundred cells.     

Growth and tissue formation from single geranium cells from virus-infected geraniums

Summary Single geranium (Pelargonium hortorum, Bailey) cells from callus isolated originally from stem tips of virus-infected plants were grown in microculture chambers in liquid Murashige and Skoog medium supplemented with 0.1 mg per liter of α-naphthalane acetic acid and 10.0 mg per liter of kinetin. Four of 1,000 of these single cells divided in the microculture chamber and produced colonies of 15 to 20 cells in 9 to 16 days. In all of the cases the plane of the first few cell division was at right angles to the long axis of the cells. Subsequently, one of the masses of cells obtained from a single cell, when transferred to solid Murashige and Skoog medium, established itself as a clone of callus tissue. Although the yield was low, the results were encouraging for the ultimate production of plants. These results suggested that, just as virus-free tobacco plants have been induced from single cell clones from certain tobacco species, virus-free single geranium cells may be useful to establish single cell clones from which pathogen-free geranium plants may be induced to differentiate. This work was supported by funds from USDA Hatch Project 981 and the NASA Institutional Grant. Published with the approval of the Director, Wisconsin Agricultural Experiment Station. The authors are indebted to Mr. E. H. Herrling and Mr. Steve Vicen for preparation of illustrations.     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Isolation of paraquat-tolerant mutants from tomato cell cultures

Summary Tomato callus clones selected for the ability to grow at paraquat concentrations lethal to wild-type cells were found at an approximate frequency of 5×10^–8 per viable cell. Diploid plants were regenerated from nine of the nineteen paraquat-tolerant callus clones isolated. Although some of these plants appeared normal, others had altered morphology and reduced vigor and fertility. New callus cultures initiated from these regenerated plants typically had at least a 30-fold increase over the wild type in tolerance to paraquat. Tests on callus from sexual progeny showed that the paraquat-tolerant phenotypes of clones PQT⁴, PQT⁶, and probably also PQT¹³ resulted from dominant nuclear mutations, but the number of loci involved is not yet known. Paraquat spray experiments indicated that slight paraquat-tolerance was expressed at the plant level in PQT¹³, but not in any of the other clones tested.     

Determination of the Cellular Mechanisms Regulating Thermo-Induced Stem Growth in Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a species with a cold requirement for the initiation of reproductive development (thermoinduction). Work in this laboratory has been focused on elucidating the biochemical and molecular mechanisms underlying the bolting or rapid stem elongation response that is an intricate part of reproductive development in this species. In the present paper the cellular basis for thermo-induced stem growth was determined. Evidence is presented indicating that bolting results from the production of new cells that elongate to their original length before thermoinduction. This increase in cell division occurs in the pith and cortex approximately 0.5 to 5.0 millimeters below the stem apex. For at least the early stages of thermo-induced stem growth, enhanced cell elongation does not appear to be a factor because average lengths of pith cells from stems of thermo-induced plants were similar or less than noninduced controls. In addition, both the amount of increase in the production of new pith cells and stem growth were positively correlated with the length of the cold treatment. Two other lines of evidence are presented corroborating previous assertions (JD Metzger [1985] Plant Physiol 78: 8-13) that gibberellins mediate thermo-induced stem growth in field pennycress. First, treatment of noninduced plants with gibberellin A₃ completely mimicked the effects of a 4-week cold treatment on mitotic activity in the pith and cortex. Second, very little increase in the production of new cells was observed in the pith and cortex of thermo-induced plants of a gibberellin-deficient dwarf mutant of field pennycress. It is also shown that the influence of photoperiod on stem growth is mediated by an effect on the final length that cells ultimately attain.     

Cytokinin Habituation in Juvenile and Flowering Tobacco

The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38.Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.     

Cytokinesis, cell expansion, and the potential for cytokinin-autonomous growth in tobacco pith

Pith tissue from Nicotiana tabacum L. cv `Maryland Mammoth' or `Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center.     

The Role of Auxins in Root-Knot Nematode-Induced Growth on Excised Tobacco Stem Segments

Root-knot nematodes, Meloidogyne incognita, induced lumps of callus tissue on the cambial surfaces of peeled tobacco stem segments cultured in vitro. Except for a layer 1 to 3 cells thick, callus was limited to the basal ends of control segments. Indole-3-acetic acid (IAA) applied in agar blocks to the centers of stem segments, when it had any effect on the cambial surface, induced streaks of callus extending from the blocks toward the basal ends of the segments. IAA in agar blocks also increased callus growth at the basal ends of the segments, increased the growth of pith on the undersides of the segments, promoted root initiation, but inhibited bud initiation. Nematodes produced none of these effects, nor did they change the type of organs induced by various concentrations of IAA in the medium. Callus tissue did grow on the cambial surface of stem segments surrounding agar blocks containing 2,3,5-triiodobenzoic acid, an inhibitor of polar auxin transport. Paraffin sections showed that the nematodes were confined to the callus tissue on the cambial surfaces of the segments. Except for occasional syncytia and areas of cell division, nematode-induced callus was composed of thin-walled, irregularly shaped cells arising from the cambium. Differences between the responses of tobacco stem segments to root-knot nematodes and IAA-agar blocks indicate that auxins were not freed from the plant tissue nor secreted by the nematodes. Instead, it is suggested that nematodes enabled the tissue to retain and use endogenous auxins that otherwise would have been transported to the basal ends of the segments.     

八角莲组织培养的器官发生途径研究

为探究小檗科植物八角莲组织培养的器官发生方式,该研究以八角莲离体叶片、叶柄在MS培养基上诱导产生的愈伤组织、不定芽、不定根为对象,用连续石蜡切片技术分析八角莲组织培养的器官发生途径。结果表明:八角莲愈伤组织形成的解剖学特征是靠近表皮的薄壁细胞经激素刺激恢复分裂能力,继续培养形成拟分生组织。拟分生组织可形成许多分化中心。通过对八角莲组织培养产生的不定芽细胞组织学观察发现芽原基起源于愈伤组织外侧的几层薄壁细胞,芽原基背离愈伤组织中央生长形成不定芽,故八角莲脱分化形成的芽起源方式为外起源。而八角莲的根原基起源于组织深处髓部薄壁细胞和部分维管形成层细胞,进而形成类似球形或楔形并朝韧皮部突起的根原基轮廓,根原基继续发育会突破表皮生成不定根,起源方式为内起源。八角莲离体再生途径为器官发生型,在组培苗生长过程中先诱导形成不定芽,再诱导形成不定根,在愈伤组织上形成维管组织将不定芽和不定根连接成完整植株。     

FORMATION AND DISSOCIATION OF CELL AGGREGATES IN SUSPENSION CULTURES OF PAUL'S SCARLET ROSE

In liquid culture stem tissue of Paul's Scarlet rose produces a suspension containing cell aggregates of extremely variable dimensions. There is, however, a definite pattern of change in the degree of cell aggregation over time. During the period of most rapid cell division large aggregates form as the result of a minimal separation of the proliferating cells. As the rate of cell division slows, the average number of cells per aggregate decreases. The dissociation of cell aggregates continues at a uniform rate after cell division has stopped. Cell separation is inhibited at low (0.1 mg/1) auxin (NAA) concentrations and by substitution of sucrose for glucose in the culture medium. Cell separation is delayed (but not greatly inhibited) by kinetin. The presence of casein hydrolysate prevents the formation of the large cell aggregates normally occurring in the early stages of the culture cycle. A variant strain which shows a much higher degree of cell separation has been isolated from stock callus tissue grown on solid medium.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

翅果油树茎段愈伤组织和芽发生的组织学研究

本文对翅果油树大宫灯型茎段培养在ＭＳ附加６－ＢＡ较高、ＮＡＡ较低浓度的培养基上培养０～３０ｄ的组织学变化进行了研究。创伤对其愈伤组织的形成有明显的刺激作用,培养３～４ｄ切口处的皮层细胞、形成层细胞、韧皮部薄壁细胞以及髓组织细胞,甚至表皮细胞均脱分化开始分裂;培养８～１１ｄ,切口明显膨大,起源于髓及维管组织周围薄壁细胞的愈伤组织突起大;培养１２～２０ｄ愈伤组织块中出现了分生组织和维管组织结节;培养２１～３０ｄ,愈伤组织表层和近表层细胞分化出芽原基,但与维管组织结节无直接联系。     

The frequency of marker changes in sugarcane plants regenerated from callus culture

Leaf tissue from five sugarcane clones with distinctive markers was cultured on a medium favoring callus growth. Transferred to a differentiation medium, calli produced over 5000 plants. Plants differentiated from two clones with stem markers exhibited a high rate of remission of the marker, but the marker reappeared in the vegetative progeny of these plants, and remission was, therefore, transient. Plants differentiated from callus from two clones with leaf markers showed a low rate of remission (2 or 3 per thousand) of the marker and the vegetative progeny was stable. A clone with variegated leaves produced plants with the majority having green leaves, some were albino, and some variegated, suggesting that plant differentiation may start with more than one cell. Permanent phenotypic change may result from tissue culture, but the results suggest that such changes are not frequent and may be confounded by temporary alterations or by chimeras formed in the process of differentiation.     

Cytohistological Studies on the Tissue Culture of Olea europaea L. I. Development of Callus in Vitro

The development of callus in vitro was investigated using the isolated explants from the autumn twig of Olea europaea. The callus developed on the cut surface of the stem-segment originate, d from the living cells of different tissues. Two distinct morphological types of the calli were observed. The highly friable callus was generally obtained while a non-friable form might develop occasionally. The change from one form to another was achieved by changing the level of NAA in the culture medium. These results obtainecl from above may be used to explain the possible role of the auxin playing a part in the form of callus. However, another distinct type of the callus in addition was also obtained in the same culture medium. This type of the callus developed in the form of a knob. It originated from cortical parenchyma cells distal from the cut surface of the stem The divisions of the cortex cells induced only were restricted to the inner layers near by the pericyele. The present observation suggests that the interpretation for the variations of the callus forms may be elaborated further when the roles of callus of the different sources will have been explored. There were three stages in the course of callus development from the cut sur- face of stem-segments, namely, activation, division and formation. These three dovelopmental stages were characterized by changes in cell morphology of the population as well as in the cell division, grawth and the relative RNA accomulation in the callus tissue. The results are shown in table 1. The formation stage proposed in this paper differs with the differentiation phase (Yeoman, 1970, 1973) on the differen- tiation of the tissues and it is suggested that appearance of wound-cambinm and differentiation of the parenchyma in this stage may be considered as the characteristics of callus development. The general conceptions of activation, dedifferentiation and differentiation are also briefly discussed.     

Control of cell proliferation and differentiation by regulating polyamine biosynthesis in cultures of Brassica and its correlation with glyoxalase-I activity

Differentiation in Brussica cultures could be induced on basal medium lacking hormones, while addition of hormones (NAA, BA) resulted in profuse callusing without any differentiation. Supplementing the hormone medium with spermidine resulted in increase in the fresh weight and glyoxalase-I activity by 330% and 8-fold, respectively. Omission of hormones caused spermidine to be less effective in inducing either cell proliferation or differentiation. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, had a retarding effect on callus induction and division of cells in suspension cultures but lead to differentiation and inhibited glyoxalase-I activity. The ability of spermidine to overcome MGBG enhanced differentiation was probably through the breaking of cell cycle arrest. Addition of glutathione, a coenzyme for glyoxalase-I enzyme, promoted cell division and enzyme activity both in callus and suspension cultures. pH emerged as an important factor in controlling glyoxalase-I activity and cell division. Results indicate involvement of spermidine in cell proliferation and differentiation and its correlation with glyoxalase-I activity.     

甘蔗茎尖原生分生组织区域化

甘蔗茎尖原生分生组织是甘蔗地上部分一切形态组织的发源中心,通过对6个不同茎径甘蔗品种4个不同营养发育时期的茎尖原生分生组织显微和超微结构观察研究发现:甘蔗茎尖原生分生组织呈半卵型结构,明显分为原套原始细胞区、原体原始细胞区、周缘分生细胞区、髓分生区,其区域化符合原套-原体学说。原套原始细胞区为最外一层细胞,原套细胞之间胞间连丝丰富,而原套与原体细胞之间胞间连丝极少,细胞以垂周分裂为主,扩大原生分生组织表面积;原体位于原套下的分生组织的中央区域,细胞可以进行各个方向的分裂,不断增加体积,原体原始细胞区呈一个球体;周缘分生区位于原套、原体下方两侧,细胞活跃产生叶原基和原形成层细胞;髓分生区细胞位于原体下方周缘分生区内侧,细胞横向分裂纵向排列,使甘蔗茎伸长。     

In Vitro Selection for Salt Tolerance in Brassica juncea L. Using Cotyledon Explants, Callus and Cell Suspension Cultures

Salt-tolerant Brassica juncea L. cell lines or plants have beenselected by screening callus pieces, cell suspension culturesand cotyledon explants in vitro on high concentrations of NaCl.Callus-based selection was unsatisfactory, as only two out ofseven isolated clones retained tolerance after 3 months of subcultureon NaCl-free medium. Selections made via plated cell suspensionswere found to be more stable for salt-tolerance. AH selectedtolerant cell lines, however, failed to regenerate plantlets.A third selection method, employing cotyledon explants was basedon their high potential for regenerating multiple shoots. Outof a total of 2620 explants cultured on high salt media, threesurvived, showed sustained callus proliferation and each regeneratedone shoot. The salt-selected shoots withstood the stabilitytest after 3 months of growth and axillary bud multiplicationon NaCl-free medium. While one of these somaclones was morphologicallyabnormal and sterile, the other two could be reared to maturitywith normal seed set. Brassica juncea, tissue culture, in vitro selection, salt-tolerance, plant regeneration     

Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia, Viviani

Summary Several amino acid requiring auxotrophs have been isolated from unsupplemented protoplast cultures of haploid Nicotiana plumbaginifolia following incubation with BUdR (1-5x10^-5, 2 days) and recovery on complete medium. The auxotrophic lines required the following amino acid(s) for growth: his, ile, leu, ile+val, met or try. Met^– is a new type isolated in higher plants. The same absolute amino acid requirement was observed in plants regenerated from auxotrophic cultures. Precursor feeding tests, enzyme assays, and/or metabolic complementation through protoplast fusion were used to identify the genetic lesion leading to auxotrophy. Mutant seeds were obtained from supplemented Met^– plants. Seeds were also collected from selfed plants regenerated from various complementing fusion products, and a His^– revertant. Genetic analysis indicated that under natural conditions of seed formation amino acid auxotrophy-in contrast to NR deficiency-failed to segregate in progeny tests.Abbreviations and definitions BUdR and FUdR 5-bromo- and fluoro-deoxyuridine respectively - AP imidazole acetol phosphate - IGP imidazole glycerol phosphate - NR nitrate reductase - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - TIP total isolation procedure - ER Escape rate—the proportion of the selected cell population surviving the BUdR treatment - BR Recovery rate—the proportion of clones identified as amino acid auxotrophs from total escaping clones - TS Total surviving colonies—the number of inoculated protoplasts/variant x plating efficiency - TST Total starvation time—the number of days on minimal medium (preincubation time+BUdR incubation time). The relationship days vs. number of divisions is as follows: 3- to 4-day-old protoplasts, 1 division; 5–6 days, 2 divisions; 7–8 days, 3 divisions Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal