Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.     

Differentiation in callus cultures of a tobacco (Nicotiana tabacum cv. White Burley) variant: some biochemical aspects

A slow-growing variant plant with distinct foliar and floral morphology was obtained in tissue cultures ofNicotiana tabacum cv. White Burley. The root and shoot differentiation in the callus derived from normal plants occurred on the 8th and 12th day, respectively, but took 10 and 14 days, respectively, in the variant callus. Amylase and acid phosphatase activities, starch and soluble carbohydrate contents were studied in non-differentiating callus (NDC), root differentiating callus (RDC) and shoot differentiating callus (SDC). The activities of amylase and acid phosphatase were low in the variant as compared to normal. Maximum amylase and acid phosphatase activities coincided with the appearance of roots or shoots. There was more starch accumulation in normal callus on differentiating media, but the variant showed a less pronounced change. The normal callus under differentiating conditions also showed more increase in soluble carbohydrates as compared to the variant. The increase in soluble carbohydrate was maintained till roots and shoot appeared. The increase the variant in differentiating was reflected in slow development of enzyme activities and low starch and sugar concentrations.     

Effect of temperature on carbohydrate content,ADP glucose pyrophosphorylase,and ATP- and PPi-dependent phosphofructokinase activity of potato tuber callus tissue

Callus derived from Lemhi Russet and Russet Burbank tuber tissue was incubated at 20°C and 30°C on a high sucrose medium for starch-formation and subjected to simulated storage and reconditioning treatments at 5°C and 25°C after transfer of the callus to a medium without a carbon source. Percent dry weight of callus from both cultivars averaged about 21% after starch formation and 5% after storage and reconditioning treatments. Total sugars were higher in callus incubated on the starch forming treatment. Lemhi Russet callus contained predominantly reducing sugars, while Russet Burbank callus contained mostly non-reducing sugars. Total sugar content was generally lower for both cultivars after the storage and reconditioning treatment in callus after starch formation at 20°C. Starch content was generally higher in Lemhi Russet tissue. After starch formation at 20°C Lemhi Russet had higher starch after the storage and reconditioning treatment than tissue from 30°C, while the opposite trend was found in Russet Burbank tissue. Total protein declined in Russet Burbank tissue during the storage and reconditioning treatment regardless of prior incubation conditions, while this decline only occurred in Lemhi Russet tissue initially incubated at 30°C during the starch formation treatment. In tissue of both cultivars, ATP- and PPi-dependent phosphofructokinase activities were inversely proportional to total sugar concentrations, while in RB callus ADP glucose pyrophosphorylase activities were proportional to starch content.Research Paper 91B1 of the Idaho Agricultural Experiment Station.     

Carbohydrate Availability and Shoot Formation in Tobacco Callus Cultures

Tobacco callus grown on a shoot-forming medium containing sorbitol or no carbon source survived, but did not produce shoots. Transfer of tissue from a sucrose medium to carbohydrate-deficient media and vice versa suggested that the growth of the tissue was a function of a total period in contact with available carbohydrate. Both starch and free sugars in the tissue were utilized during shoot initiation. Furthermore, it appeared that the continuous availability of carbohydrate was required for shoot primordium growth and/or their development into leafy vegetative shoots in dark-grown cultures.     

Shoot histogenesis in tobacco callus cultures

Summary The earliest histological event observed in light-grown shoot-forming tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus was the deposition of safranin-stainable substances (probably suberin) on cut, exposed cell surfaces. This was followed by the initiation of cell files and the appearance of starch granules. Nodules with lignified tracheary elements also were observed in the upper part of the callus. Pronounced starch accumulation occurred in the lower part of the callus in which protrusions of tissue into the medium occurred. Meristemoids were found in these protrusions as well as elsewhere. In between meristemoids, parenchyma cells with starch granules of varying sizes were observed. Cell strands that connected with the meristemoids also were observed. These strands often terminated at the surface of the protrusion at which point shoot apices originated. The earliest shoots were formed in these protrusions. With time, additiional shoots were formed in other parts of the bottom of the callus and finally in the top part of the callus on prolonged culture. The determination of the loci at which shoot primordia were formed sequentially, was interpreted in relation to the physiological gradient concept. This work was carried out while E. M. was a Visiting Scientist at the University of Calgary under the Scientific Exchange Program between the National Research Council of Canada and the Japan Society for the Promotion of Science. Support for this study was provided by N.R.C. (Canada) Grant A-6467 to T. A. T.     

Studies on Histology and Histochemistry of the Morphogenesis of Stem Segment in Chinese Gooseberry Cultured in vitro

The stem segments of Chinese gooseberry cultured in vitro, 2ip added in the medium would give good effect on the regeneration of the plantlet same as zeatin. The effect of 6-BA was less and kinetin almost had no effect. Inhibition was found in almost all cases, when gibberellie acid (GA3) or GA3 (1,5, 10 ppm) combined with zeatin was used. Calli were originated from cambium and ploem. The primary xylem elements also participated in the formation of callus. The shoot primordium originated from the surface cells of callus and the subepidermal cells also participated in it. In certain conditions, shoot primordium was produced from internal part of callus. During the shoot primordium formed, a positive correlation between the starch accumulation and primordium formation could be obtained. The accumulated starch in the cells gradually disappeared as the shoot formation proceeded.     

甜菊愈伤组织生长、分化与甜菊糖苷积累的关系

耐菊（Steviarebaudiana）愈伤组织中甜菊糖苷的积累与愈伤组织的生长呈负相关、与愈伤组织细胞的组织化及转绿呈正相关。愈伤组织芽的分化并不是积累较高水平甜菊糖苷的必要前提。绿色、质地致密、生长缓慢的愈伤组织,不论有芽分化或无芽分化时,其甜菊糖苷含量均较高。在电镜下观察到,这两种愈伤组织细胞具有类似的超微结构特征：细胞高度液泡化;叶绿体发育成熟,光合膜系统结构发达,基质浓厚且含有质体小球;微体具有典型的晶格结构,常与叶绿体紧密相靠。黄色、质地致密、生长缓慢的愈伤组织中甜菊糖苷含量较低,其细胞内质体富含淀粉粒,只有少量分散的片层结构,有的质体甚至完全被淀粉粒所充塞。黄色、质地疏松、生长快速的愈伤组织中甜菊糖苷含量最低,其细胞内质体结构简单,片层稀少。质体的发育和液泡的分化与甜菊糖苷的积累密切相关。愈伤组织具有较高的甜菊糖苷含量在于愈伤组织细胞的组织化以及细胞的高度液泡化并具有发育成熟的叶绿体。     

Transformed callus does not necessarily regenerate transformed shoots

Agrobacterium tumefaciens carrying a disarmed Ti plasmid vector was used for the transformation of flax tissue. Transformed callus was obtained from inoculated hypocotyl segments and healthy green shoots were regenerated from this callus. Nopaline assays on shoot tissue were positive for nopaline content if carried out soon after removal of the shoot from the callus but negative if carried out 2–3 weeks after removal. All of these shoots gave rise to kanaymcin sensitive progeny and were most likely escapes arising from non-transformed cells protected from the selective agent by transformed cells in the callus. Careful analysis of regenerated shoots from transformed callus is necessary in order to distinguish escapes from true transgenics.     

Biosynthesis and accumulation of a medicinal compound,Picroside-I,in cultures of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> Royle ex Benth

Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non- detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures.     

Starch turnover in shoot-forming tobacco callus

Tobacco callus grown under shoot-forming conditions or in the presence of gibberellic acid, which inhibits shoot formation, was incubated in [¹⁴C]-sucrose at three different periods in culture and then replanted. Evolution of ¹⁴CO₂ occurred during the 10 day post-incubation period. Most of the radioactivity was incorporated into the ethanol-soluble fraction, which lost most of its label after 24 h. Starch was the major ethanol-insoluble component and post-incubation synthesis occurred in this fraction for 24 h or longer. Greater net synthesis of starch occurred in shoot-forming tissue and the loss of label from starch began later than in tissue cultured in the presence of gibbe-rellic acid. Newly synthesized starch was not immediately utilised in the organogenic process, but its utilization could be correlated with the shoot-forming process.     

Callus formation versus differentiation of cultured barley embryos: hormonal and osmotic interactions

The effect of various plant growth substances as single agents was evaluated in a complex tissue culture system: whole embryo culture of early differentiating barley embryos. Callus formation in unsupplemented medium derives from the mesocotyl and is uniquely characteristic of cultures initiated at this stage of embryonic development. This phenomenon could be prevented or reversed by incorporation of gibberellic acid in the medium resulting in plantlet formation. Indoleacetic acid enhanced callus growth, whereas kinetin did not promote either callus or meristematic development. Callus tissue markedly accumulated starch, effectively lowering the cellular osmolarity, while inducing a corresponding rise in the osmolarity of the culture medium. This osmotic pattern was reversed by gibberellic acid induction of shoot formation. These osmotic-hormonal interactions are interpreted relative to in vivo, in situ normal embryogeny or developmental lesions such as tumors.     

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.     

Starch characteristics in cultures of normal and mutant maize endosperm

Summary Vigorously growing suspension cultures of normal, amylose-extender (ae) and waxy (wx) maize endosperm were established from near isogenic lines of maize inbred A636. The recovery of the ability to produce vigorous cultures of ae and wx endosperm by backcrossing demonstrate the genetic control of endosperm growth in vitro. Phenotypic expression of the endosperm mutants in culture was studied by examining the properties of starch accumulated in endosperm cultures and starch from developing and mature kernels of the same genotype. After 9 months in culture, the amylose contents of the starch in normal callus tissue and normal endosperm tissue were not significantly different, 28.2% and 31.7%, respectively. Starch granules from normal cultures and endosperm stained blue-black with iodine and were round to polygonal in shape. The starches of wx endosperm and callus cultures contained no amylose, and wx starch granules stained brown-orange with iodine. Although, wx starch granules were primarily round, a few granules with jagged edges were observed in starch samples isolated from cultures and kernels. The percent amylose in starch from ae callus was significantly lower than the amylose content of starch from ae endosperm tissue, 39.9% and 67.7%, respectively. Starch granules from ae endosperm and cultures were smaller than normal and wx starch granules. Irregular starch granules which are typical of ae endosperm were present in ae callus tissue, but were less frequently observed. We conclude that specific endosperm mutant phenotypes are expressed in vitro.Supported in part by the United States Department of Agriculture Competitive Grant 85-CRCR-1-1740. Contribution No. 94, Department of Horticulture. The Pennsylvania State University. Authorized for publication as paper No. 7373 in the journal series of the Pennsylvania Agricultural Experiment Station     

Callus formation versus differentiation of cultured barley embryos: Hormonal and ostomic interactions

Summary The effect of various plant growth substances as single agents was evaluated in a complex tissue culture system: whole embryo culture of early differentiating barley embryos. Callus formation in unsupplemented medium derives from the mesocotyl and is uniquely characteristic of cultures initiated at this stage of embryonic development. This phenomenon could be prevented or reversed by incorporation of gibberellic acid in the medium resulting in plantlet formation. Indoleacetic acid enhanced callus growth, whereas kinetin did not promote either callus or meristematic development. Callus tissue markedly accumulated starch, effectively lowering the cellular osmolarity, while inducing a corresponding rise in the osmolarity of the culture medium. This osmotic pattern was reversed by gibberellic acid induction of shoot formation. Thise osmotic-hormonal interactions are interpreted relative to in vivo, in situ normal embryogeny or developmental lesions such as tumours. Research supported by Inst. Grant No. 994.     

In Vitro Shoot Regeneration from Callus, Leaf Axils and Petioles of Sugar Beet (Beta vulgaris L.)

Procedures are described for producing axillary shoots fromseedling apices and adventitious shoots from petioles and leaf-derivedcallus of sugar beet cultivars. The rate of adventitious shootregeneration from petioles was influenced by temperature, BAPconcentration of the medium, and the time in culture of theseedling apices from which the petioles were excised. Petiolesectioning confirmed that adventitious shoots originated inthe sub-epidermal parenchyma. Two distinct types of callus wereproduced from leaf explants, but only white friable callus wascapable of shoot development. This callus developed from browntissue and was composed of thin-walled cells with dense cytoplasmand prominent nuclei. Green compact callus with thick-walledlignified cells developed from green tissue, but did not produceshoots. Successful seed sterilization and shoot regenerationfrom petiole explants and callus was cultivar-dependent. Adventitiousshoots were rooted and successfully transplanted to pottingcompost under glasshouse conditions. Key words: Adventitious shoots, axillary shoots, callus, sugar beet (Beta vulgaris L.)     

Changes in the starch content during organogenesis in in vitro cultured Begonia rex stem explants

Stem explants, excised from greenhouse-grown Begonia rex plants, were cultured on basal medium (T. Murashige and F. Skoog, Physiol. Plant. 15: 473–497, 1962) contained in sterile Petri dishes. The medium was supplemented with benzyladenine (0.1 mg 1⁻¹) naphthaleneacetic acid (0.01 mg 1⁻¹) and, according to experimental requirements, with either sucrose (3%) or mannitol (3%). Histochemical and biochemical examination of the starch content of the explant was carried out over several days. There was no starch deposition or organogenesis in tissue cultured on mannitol and carbohydrate-free growth medium. The most dramatic finding was the heavy accumulation of starch in tissue cultured on sucrose medium. This copious accumulation preceded any organ formation and was mainly in regions which ultimately gave rise to shoot primordia. The heavy build-up of starch preceding organogenesis was also observed when explants previously cultured on mannitol medium were transferred to medium containing sucrose. During shoot primordia development there was a decrease in the starch content of the cultured tissue indicating the utilization of the polyglucan in the organogenic process.     

CALLUS FORMATION AND ORGANOGENESIS FROM CULTURED LEAF SEGMENTS OF CRASSULA ARGENTEA: CYTOKININ-INDUCED DEVELOPMENTAL PATTERN CHANGES

Culturing leaf segments on agar medium with a final concentration of 10^–4m isopentenyl adenine inhibits regeneration of plantlets, but stimulates some segments to produce regions of dense callus. The callus is initiated from the basipetal end of a cut vascular bundle, and is therefore the positional equivalent of root initiation during normal regeneration. Once callus has emerged, it forms a sphere of tissue attached to the leaf segment. The cells on the surface of the sphere are smaller than the cells toward the center. Some of these larger cells contain tannins or large starch grains. Shoots are sometimes initiated from organized meristematic regions of surface cells. Roots form endogenously from callus after shoot formation. This regeneration sequence, initially induced by cytokinin treatment, is strikingly dissimilar to the normal pattern, and provides an excellent example of a hormone-induced alteration to a developmental pattern.     

Secondary metabolite content in rhizomes, callus cultures and in vitro regenerated plantlets of Solidago chilensis

An in vitro culture system leading to the formation of callus and plant regeneration, starting from nodal sections and shoot tips, was developed for Solidago chilensis (Asteraceae). The content of the gastroprotective diterpene solidagenone as well as the phenolics chlorogenic acid (CA) and rutin was determined either in rhizomes from wild growing plants and in callus and in in vitro regenerated plantlets by analytical HPLC. Additionally, total phenolic and flavonoid content was assessed in plant samples, callus and cell suspensions. In terms of dry starting material, the percentual solidagenone content in nine S. chilensis samples ranged from 0.5-3.5% for rhizomes from wild growing plants, 0.1-0.3% for callus and 0.3% for an in vitro regenerated plantlet, respectively. The highest solidagenone contents were found in the wild plant during the late summer in the months of March and April (3.5-2.2%) while highest values for chlorogenic acid (0.5%) and rutin (0.4%) were detected in May, before senescence. The callus tissue and cell suspensions contained some 1.8-2.0 and 1.2% of total phenolics, respectively. CA was the main phenolic in the cell suspension while only traces were found in the callus. Rutin was not detected in the callus nor cell culture.     

KT和NAA对知母愈伤组织再分化的影响

以知母愈伤组织为材料,采用单因子试验方法,探讨不同浓度KT和NAA对其再分化的影响。结果表明:知母愈伤组织生根的最佳培养基为MS+KT 2 mg/L;愈伤组织再生芽的最佳培养基为MS+KT 2 mg/L+NAA 1 mg/L;愈伤组织再生苗长高的最佳培养基为MS+KT 2 mg/L+NAA 0.5 mg/L;愈伤组织增殖的最佳培养基为MS+KT 2 mg/L+NAA 1 mg/L。     

Differential Response of Various Tissues4Asparagus officinalis to Sodium Chloride

Tolerance of Asparagus officinalis tissues of different levelsof organization to 0–2% NaCl was studied in a tissue culturesystem. The following tissues (organs) were examined: friablecallus with no organogenesis, compact callus exhibiting organogenesis,one-bud shoot segments, and plantlets. Growth of friable callus,the less organized tissue, was progressively inhibited by risingconcentrations of NaCl, whereas growth of compact callus wassomewhat less sensitive. NaCl at concentrations of 1% or highercaused an increase in the mortality of one-bud shoot segmentsand inhibited growth. Concentrations up to 2% NaCl did not causeany mortality of plantlets, the most organized tissue tested;root production declined progressively in response to salinitywhile shoot growth was inhibited only at 2% NaCl. Moderate concentrationsof NaCl (0.5–1.0%) stimulated growth and induced phyllocoidproduction in both shoot segments and plantlets. With increasedNaCl there was a massive uptake of sodium and chloride, principallyinto the shoots, while uptake and accumulation of potassiumdecreased somewhat in shoots, but not in roots and rhizomes.Rooted and unrooted plantlets exhibited similar levels of tolerance.It is, therefore, inferred that salt tolerance of asparagusin culture is dependent on tissue organization. Key words: Asparagus officinalis, salt tolerance, callus, shoot segment, plantlet