Stability of rRNA genes during germination of Vicia faba seeds

Germinating Vicia embryos and the roots of 4-day-old seedlingswere assayed for the proportion of DNA that is complementaryto Vicia rRNA. Hybridization experiments which were performedusing DNA purified by ordinary procedures including RNase digestionshowed a higher level of saturation only for the DNA from theembryos. The embryo DNA showed a Tm of 74?C in 0.1?SSC, whereasthe Tm of the root DNA was 70?C. However, our final conclusionis that there is no gross amplification of rRNA genes for Viciafaba, since the saturation level for the embryo DNA did notexceed that for the root DNA when the DNAs were used after additionalpurification on CsCl gradients. ¹ Present address: Department of Botany, Faculty of Education,Utsunomiya University, Utsunomiya 320, Japan. (Received November 25, 1974; )     

Heritability and Variability in Ribosomal RNA Genes of Vicia faba

We have compared the restriction patterns and copy numbers of ribosomal RNA genes (rDNA) between and within individuals of Vicia faba . While the EcoRI blot-hybridization patterns changed only after one to two generations, copy number changes were found among different tissues of the same plant. Copy number differences among individuals in the population were as great as 95-fold, whereas as much as a 12-fold variation was seen among tissues of the same plant. Among individual F(1) progeny from genetic crosses, nearly an 8-fold variation was seen, and among individuals of the F(2) generation a spread of 22-fold was measured. Among individual siblings of self-pollinated parents, up to 7-fold variation was observed. However, changes in copy number did not necessarily indicate changes in rDNA EcoRI blot-hybridization pattern, and vice versa. Furthermore, nearest neighbor analysis of R-loop experiments showed that the arrangement of members of the "nontranscribed" spacer (NTS) size classes along the chromosome was not random, but some clustering was indicated. The data are consistent with the hypothesis that sister chromatid exchange in somatic cells of V. faba is the primary mechanism for altering the rDNA copy number as well as causing the extreme variation observed in the NTS. Variation among individuals in rDNA blot-hybridization pattern was also observed for Vicia villosa, Vicia dasycarpa, Vicia benghalensis and Vicia pannonica.     

Altered features in the secondary structure of Vicia faba 5.8s rRNA.

We have re-examined the nucleotide sequence of Vicia faba (broad bean) 5.8S rRNA using partial chemical degradation and a new approach to high temperature (65-80 degrees C) sequencing gels. The results indicate that the secondary structure was not completely disrupted in previous studies (Tanaka, Y., Dyer, T.A. and Brownlee, G.G. (1980) Nucleic Acid Res. 8, 1259-1272) and explain ambiguities between the nucleotide sequence and T1 ribonuclease digests. Despite this revision, estimates in the secondary structure suggest that this 5.8S rRNA differs from previously examined examples in two respects, more open conformations in both the "GC-rich" and "AU-rich" stems. The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques. These studies and theoretical considerations generally supported the "burp gun" model previously proposed for all 5.8S rRNAs and were inconsistent with the recently suggested "cloverleaf" configuration. More importantly, they were also consistent with more open stem structures in this higher plant.     

Apical Dominance in Vicia faba

Apical dominance phenomena have been studied in seedlings ofVicia faba particularly in relation to the movement about theplant of uracil-2-¹⁴C applied to the cotyledons. Decapitationjust below the second node releases the growth of the lowermostlateral bud and inhibition is completely reimposed by applicationof indole-3-acetic acid (IAA) to the cut surface. Uracil-2-¹⁴Capplied in solution to the cotyledons is distributed in thestems of all experimental seedlings with no consistent differencesdue to decapitation or IAA application. On the other hand, decapitationresults in a rapid increase in uracil-2-¹⁴C content in the lateralbuds which far exceeds their promoted growth. This uptake iscompletely suppressed by IAA application. A ring of tri-iodobenzoicacid (TIBA)-lanolin paste around the stem above the bud suppressesIAA action both in bud growth and on uracil-2-¹⁴C uptake, andalso on the movement of IAA-1-¹⁴C down the stem. TIBA-application to the base of the bud does not prevent IAAaction on bud growth, but also does not prevent the movementof IAA-1-¹⁴C (or a water soluble product of its metabolism)into the bud. Direct application of kinetin to the lateral bud of intact plantscauses a short-lived release of growth. Gibberellic acid producesa smaller and scarcely significant increase which is additiveto the kinetin effect. Neither has any effect on uracil-2-¹⁴Cmovement into the bud. The implications of these findings are discussed in relationto various existing theories of the mode of auxin action inapical dominance and it is concluded that their strongest supportis for a mechanism involving the suppression of phloem differentiationin the vascular supply to the bud.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Sister chromatid exchanges in Vicia faba

Evolutionary loss of the Y chromosome has occurred in Climacia areolaris (Hagen) of the neuropteran family Sisyridae. The diploid set comprises 6 pairs of autosomes, plus 2 X chromosomes in the female and 1 X in the male. The Y is retained in Sisyra vicaria (Walker) of the same family: its chromosome number is 14 in both sexes including 2X chromosomes in the female and 1X plus Y in the male. Two alternative pathways for the segregation of the sex chromosomes-distance segregation and sex bivalent formation-co-exist in the latter species in a ratio of approximately 1 to 6; the possible phylogenetic significance of this feature is discussed.     

蚕豆染色体的复制带显带技术

本文采用作者首创的双周期BrdU二次标记法研究了蚕豆根尖细胞染色体的复制带,得到了分布在整条染色体上的清晰稳定的多条带纹.这是复制带首次在植物染色体上取得的具有实用意义的带型.为进一步制定植物染色体的标准带型和研究植物染色体的复制方式提供了一条途径.     

Sister chromatid exchanges in Vicia faba

The relationship between chromosomal aberrations and sister chromatid exchanges (SCE's) after treatment of Vicia faba root tips with thiotepa, caffeine and 8-ethoxycaffeine (EOC) was studied by using a modified fluorescent plus Giemsa (EPG) technique. At concentrations which had little effect on the frequency of chromosomal aberrations, thiotepa strongly increased the frequency of SCE's, provided that the chromosomes were allowed to replicate between treatment and fixation. Frequently, the size of the exchanged material was smaller than the diameter of the chromatid. Post-treatments with caffeine of roots previously exposed to thiotepa strongly increased the frequency of aberrations, but had little effect on the frequency of SCE's. In contrast to thiotepa, EOC caused only a slight increase in the frequency of SCE's even at concentrations which produced a high frequency of chromosomal aberrations. Thus, there was not a close correlation between SCE's and chromosomal aberrations. Single-strand exchanges between the DNA double helices in sister chromatids were not detected.     

The effect of Actinomycin-D on chiasma frequency in Vicia faba

The effect of Actinomycin-D was studied on chiasma frequency in Vicia faba (var. Weibull's Akerabona). A significant decrease in chiasma frequency over control was observed in all cases irrespective of the time of fixation and chemical concentration. However, the maximum decrease was found in the material fixed after a recovery period of 24 hours. The decrease in chiasma frequency showed a linear relationship with time of recovery. Besides reduction in the chiasma frequency, cells showing complete failure of homologous pairing were also observed. These findings are discussed in the light of actinomycin-D action.     

The control of chiasma frequency in Vicia faba L.

Summary InVicia faba two groups of chromosomes can be distinguished and consequently correlations of chiasma frequency can be calculated between the groups and also within the group of small chromosomes. A negative correlation was demonstrated during some analyses while positive ones occurred in others, in fact a wide range of coefficients could be calculated by both methods. It is suggested that negative correlation of chiasma frequency occurs only when reproduction of the chromosomes is precipitated or pairing delayed, so that the degree of pairing may be affected in plants where many chromosomes are present or where the chromosomes are large in size. Such correlations can have no influence on recombination, but rather they are the occasional outcome of the mechanism controlling chiasma frequency.     

The Initiation and Maintenance of Vicia faba Tissue Cultures

Explants obtained by removing the radicle tip and the plumule from embryos of Vicia faba have been induced to form callus in culture. Of a range of agar-solidified culture media tested, only that of Schenk and Hildebrandt (1972) was consistently successful. Improved growth, measured as increasing fresh weight was obtained by increasing the nitrogen content of the medium, either as potassium nitrate or as ammonium nitrate. A kinetin concentration of 0.01 mg/1 (5 × 10⁻⁸M) and a 2,4-dichlorophenoxyacetic acid (2,4-D) concentration of 0.5 mg/1 (2.3 × 10⁻⁶M) allowed optimum initial callus growth. A 2,4-D concentration of 2.3 × 10⁻⁸M, while insufficient to induce callus formation was able to inhibit lateral root development which occurred from embryo explants cultured without added 2,4-D. Subcultured tissue grew well on media supplemented with casein hydrolysate or a mixture of the eight most common amino acids in casein hydrolysate. Growth in subcultures was inhibited by two other amino acid mixtures used by other workers for different species.     

The effect of diepoxybutane on chromosomes of Vicia faba

Summary The frequency of aberrations induced by DEB depends on the temperature and concentration of the agent. The effect of DEB is delayed. Pretreatment with EDTA increased the frequency of aberrations. The frequency of aberrations was also increased after simultaneous treatment with DEB and chloramphenicol or streptomycin. Pre- and posttreatment with protein synthesis inhibitors had a reverse effect, the frequency of aberrations was decreased.