Xanthium pith and hypocotyl tissue in culture

A rapid method is described of obtaining callus tissue cultures from hypocotyls of vegetative and flowering Xanthium strumarium L. seedlings. The tissue is grown on Murashige and Skoog medium modified with 1 g/l casein hydrolysate and 5 mg/l each of kinetin and -napthaleneacetic acid.     

The influence of genomes on autonomous growth of pith cultures of Nicotiana glauca-Langsdorffii hybrids

Summary A differential influence of the two parental genomes on cell proliferation and morphogenesis in pith tissue explants can be observed among the various tumorous hybrid combinations between Nicotiana glauca Grah. and N. langsdorffii Weinm.: the F₁ hybrid (GL), its amphiploid (GGLL), and two different triploids (GGL and GLL). This influence was evident when the explants were cultured in the presence of exogenous auxin (indole-3-acetic acid, 2.5 M), supplied either continuously or for a brief period of time. Compared with the F₁ and the amphiploid, the higher proportion of N. glauca genomes in GGL cells resulted in greater growth, the higher proportion of N. langsdorffii genomes in GLL cells in lesser growth. In addition, shoots are produced on the GGL callus, while only roots are formed on calli of the other types in the same medium. When, in addition to auxin, a cytokinin [6-(3-methyl-2-butenyl-amino)purine] was added to the culture medium, the differential growth of the different tissue types was less pronounced; at 1.0 M of the cytokinin, all tissues grew at about the same rate and remained undifferentiated, regardless of their genomic composition.     

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. Havana 425 are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

Production of Nicotiana tabacum x Nicotiana acuminata hybrid by ovule culture

Using a conventional sexual crossing technique, Nicotiana tabacum x N. acuminata was not produced. After the fertilized ovules were cultured for 20 days in a liquid Nitsch H medium, germination was observed. The roots grew rapidly but leaves did not. However, plantlets were produced in an H medium containing Benzyladenine or Kinetin (0.01–0.1 mg/l). The plantlets grew and flowered in a greenhouse. The chromosome number of the hybrid was 36 and its morphological characteristics were intermediate between those of parental species.Abbreviations BA Benzyl adenine - K Kinetin     

Effect of copper on callus growth and gene expression of in vitro-cultured pith explants of Nicotiana glauca

Abstract

Copper is a vital component of electron transfer reactions mediated by proteins such as superoxide dismutase, cytochrome c oxidase and plastocyanin, but its concentrations in the cells needs to be maintained at low levels. In fact, the same ability of this essential metal ion to transfer electrons can also make it toxic to cells when present in excess. In vitro cultured explants of Nicotiana have been extensively used as a model to analyse metal-DNA interactions. In this report, we examined the effect of copper (1, 10 and 100 μM CuSO₄) on callus growth and protein synthesis of in vitro-cultured pith explants of Nicotiana glauca. In addition, a N. glauca cDNA library from Cu-treated (100 μM CuSO₄) pith explants cultured in vitro for 24 h was analysed by mRNA differential screening. The copper treatments inhibited callus growth of pith explants. The extent of inhibition was directly correlated to metal concentration. One and 10 μM CuSO₄ induced a notable increase of proteins synthesis relative to control explants. By contrast, 100 μM CuSO₄ inhibited protein synthesis relative to control extracts. The SDS-PAGE fluorography of pith proteins revealed, in Cu-treated extracts qualitative and/or quantitative differences in the synthesis of some polypeptides compared with control explants. Copper-modulated patterns of gene expression were also analysed by mRNA differential screening. The N. glauca genes isolated from Cu-treated pith explants shared common identities with other genes known to be elicited by diverse stresses, including pathogenesis and abiotic stress. In particular, the cDNAs were homologues to genes encoding cell wall proteins (i.e., extensin, and arabinogalactan-protein) and pathogenesis-related proteins (i.e., osmotin, endochitinase and a member of the Systemic Acquired Resistance gene family). In addition, an MD-2-related lipid-recognition (ML) domain protein and the enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase appeared involved in the response to copper stress. In animal cells, AdoHcy hydrolase is a copper binding protein in vivo, which suggests that, also in plant tissues, this enzyme may play an important role in regulating the levels and intracellular distribution of copper.     

Single cell protein from bagasse pith by a mixed bacterial culture

A spontaneous association of Cellulomomas sp. with another bacterial strain was studied for its capabilities for single cell protein (SCP) production from bagasse pith. The associated strain was identified as Pseudomonas sp. and further characterized for its physiological properties. The effect of the initial proportions of both strains, the way of propagation, and the effect of pH on the growth of the mixed culture on bagasse pith was studied. Separate propagation of both strains before the fermentation step (“controlled mixed culture”), a range of proportions Cellulomonas-Pseudomonas from 4:1 to 100: 1, and pH 7.0, were found to be the most appropriate conditions of growth. A mutualistic symbiotic relationship was demonstrated to take place between both strains during the mixed growth on bagasse pith, the Cellulomonas supplying the carbon source (glucose produced from bagasse degradation) to the Pseudomonas, and the latter producing the vitamin supplements necessary for the Cellulomonas growth, allowing the growth of the mixed culture in a minimal medium, without any growth factor supplement. Fed-batch cultivation of the mixed culture on this substrate was successful, giving rise to high biomass production (19.4 g/l), thus increasing the productivity of the system. Due to its improved productivity, high biomass production, inexpensiveness of the culture medium, (without any vitamin supplement), and good stability, this culture presents economical advantages and constitutes an attractive choice for lignocellulosic substrate utilization.     

Nicotiana tabacum DNA: Isolation from cell culture

The adaptation of Marmur’s method, suitable for DNA isolation from plant cell culture, is described.     

Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion

Summary Somatic hybrids were produced between Nicotiana tabacum and N. nesophila, two species incapable of conventional sexual hybridization. Sexual hybrids, though, could be produced between these two species by using ovule culture only when N. nesophila was female. Clones of somatic hybrids were compared with sexual hybrids. Statistically significant variation was observed between clones, but not between sexual hybrids, for pollen viability, flower morphology, leaf morphology, and trichome density. As all clones of somatic hybrids have 96 chromosomes, the variability could not be explained by interclonal variation in chromosome number. Variation between somatic hybrids could be the result of cytoplasmic segregation or recombination, mitotic recombination or small chromosomal rearrangements prior to plant regeneration. Variation between clones could be exploited as these interspecies hybrids are now being used to incorporate disease resistance into cultivated tobacco.     

Growth, peroxidase activity, and protein content in stem pith and callus tissues from haploid and diploid Nicotiana tabacum plants

Pith segments isolated from haploid and diploid Nicotiana tabacum cv. IAC-70 plants from five different heights in the stem, were cultured in vitro on MS-62 medium supplied with 0.5 mg/l IAA and 0.02 mg/l kinetin. Pith tissues of haploid plants showed greater growth potential than those of diploids; peroxidase activity was higher in the calluses of diploid tissues whereas protein content was higher in those of haploids. After three subcultures the growth pattern was inversed, as were the results for peroxidase activity and protein content. After the onset of culture, peroxidase activity in haploid explants dropped sharply, reaching values lower than in diploid tissues 40 d later, but after three subcultures these values were higher than those for diploid calluses. The results are discussed from the viewpoint of a possible relationship between greater polyploidization in haploid tissues on the one hand and growth decrease and peroxidase activity increase on the other.     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (≤ 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (≤ 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

Phloem differentiation in Coleus pith explants

A simple tissue culture protocol was used to examine phloem differentiation. Sieve tube elements differentiated from Coleus blumei Benth. pith parenchyma when pith blocks were explanted on to an agar medium containing sucrose and indole acetic acid. Growth regulator concentration had surprisingly little influence on the production of phloem cells. Increasing the availability of sucrose, however, resulted in an enhancement of phloem differentiation. Evidently phloem and xylem differentiation began at the same time in this system, and the concentration of sucrose determined the relative production of phloem cells.     

烟草叶片组织培养及植株再生(简报)

烟草叶片外植体在MS 2,4-D0．5mg／L 6-BA1．0mg,L的培养基上培养15d后,产生絮状疏松的浅黄绿色的愈伤组织,30d后开始从愈伤组织表面分化产生幼芽,60d后平均每块愈伤组织产生30棵幼芽。将幼芽转至含0．2mg/L NAA的MS培养基上形成根,并得到完整植株。     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.