The 13kDa prolamins, one of the rice storage proteins, consist of complex mixtures of polypeptides encoded by multigene family that show heterogeneity both in size and solubility. Although many researchers have isolated prolamin cDNA clones, it has not been possible to correlate most of these cDNA clones with individual 13kDa prolamin mature polypeptides. We isolated three new prolamin cDNA clones, λRM1, λRM4 and λRM9. Further more, we purified six 13kDa prolamin polypeptides from rice type I protein bodies, and determined these amino acid sequences. Here we demonstrate a classification for the 13kDa prolamin polypeptides which can be divided four classes, 13-I, 13-IIa, 13-IIb and 13-III. Cysteine labeling of the prolamin polypeptides indicated that 13-I contains cysteine residues, but 13-IIa or 13-IIb have no Cysteine residues. The 13-I polypeptide was soluble in nonreducing solution when their cysteine residues form inteamolecular disulfide bonds, but not soluble at intermolecular bonding.
This work was undertaken to determine the growth parameters of Lockhart's equation for finding which component was predominantly contributing to the cell expansion rates of tissue-cultured carnation plants (Dianthus caryophyllus L.). The water potential of the culture media ranged from -0.02 to -0.51MPa so that water stress conditions could be applied. Cell expansion could be inhibited completely by adding 2, 4-dichlorophenoxyacetic acid (2, 4-D) and benzylaminopurine (BA) to the culture media to form callus tissue. The sizes of the water potential gradient between the water source and the elongating cells correlated to the speed of growth rates under nutrient deficiency and growth retardation induced by the plant hormones, indicating that cell expansion rates were mainly associated with how much water could be absorbed by the elongating cells regardless of changes in growth under osmotic stress and growth retardation induced by addition of 2, 4-D and BA.
Shoot organogenesis from mulberry leaves was examined by using a two-step culture system consisting of seedling culture for obtaining leaf explants and leaf culture for inducing shoot organogenesis. Successful induction of shoots was achieved with an almost perfect induction rate, only when both cultures were carried out in the medium supplemented with 2, 3, 5-triiodobenzoic acid (TIBA) and cytokinin such as 6-benzylaminopurine (BA) and thidiazuron (TDZ). The site of shoot formation was confined to the basal part of a leaf, particularly the surface of midrib near the cut-end of the petiole. Complete whole plantlets were produced from the regenerated shoots. The stimulative effect of TIBA, inhibitor of auxin transport, on shoot organogenesis is discussed in relation to the level of endogenous auxin contained in the used leaf explants.
Cell suspension cultures of Saxifraga stolonifera Meerb. were established in MS liquid medium containing 1μM NAA by using the callus induced from adventitious roots of cultured plantlets, and the production of (-)-epicatechin 3-O-gallate (ECG) was confirmed (0.6% of dry weight). The effects of the concentration of major inorganic salts and plant growth regulators in the medium on ECG production were examined. Reduction of KH2PO4 or MgSO4 concentration in MS medium enhanced ECG production. Maximum ECG contents, 1.6% and 1.2%, were obtained at half the original concentration for KH2PO4 and at one-eighth that for MgSO4, respectively. In the modified MS medium, where KH2PO4 and MgSO4 concentration were reduced, increased NAA levels suppressed ECG production, while the addition of BAP stimulated it. Maximum ECG production (2.5%) was achieved in the modified MS medium containing 1μM NAA and 1μM BAP.
Arabidopsis RAV1 and RAV2 are unique transcription factors that contain two distinct DNA binding domains, namely AP2 and B3-like domains, both of which have been identified only in higher plants. We characterized the DNA binding properties and expression of RAV2. RAV2 was shown to bind to the same sequences recognized by RAV1. Expression of RAV2 mRNA was found to be modulated by mechanical stimuli. Developmental regulation of RAV2 was investigated using transgenic plants carrying a RAV2 promoter-GUS construct. The analyses revealed complex developmental regulation of RAV2 expression. In particular, the spatial pattern of GUS expression in roots suggested a possible function of RAV2 related to lateral root formation.
Histological analysis was carried out during the sequence leading to the formation of embryogenic callus and somatic embryos from cotyledon explants of African marigold (Tagetes erecta). Tissues at various developmental stages were excised and observed by light, fluorescent and scanning electron microscopy. Meristematic activity started on cotyledon procambium cells leading to formation of nodular structures. Friable callus was composed by narrow strands of meristematic cells interspersed with large intercellular space, and embedded in a mucilaginous substance. Somatic embryos showed abnormal shape with poorly developed cotyledons and no shoot apex. The origin of the somatic embryos is discussed.
Partial Wiv-1 cDNA for a cell-wall-bound acid invertase isolated from wounded leaves of tomato (Lycopersicon esculentum) was introduced into tomato plants in an antisense orientation. The enzyme activity was markedly decreased in wounded leaves of 3 of 9 transformants. The soluble sugars and starch contents in the source leaves of these 3 transformants were reduced compared with control plants. These results suggest that the cell-wall-bound acid invertase encoded by Wiv-1 regulates the carbohydrate content in source leaves of tomato. Most of the transformants showed low fertility. It is possible that the enzyme encoded by Wiv-1 participates also in sink metabolism in tomato flowers.
Transformed hairy root cultures of chilli pepper (Capsicum frutescens cv. cayenne) containing the CaMV 35S promoter linked to the parsley PAL-2 cDNA were generated. These transformants showed increased PAL activity at early and late stages of culture. The morphology, colour and growth rate of these transformants were quite different from the control hairy roots. Measurements of cell dry weight, content of fibre and lignin-like material, suggested that the altered characteristics of the PAL transformants might be linked to lignification. HPLC analyses of phenolic compounds in transgenic roots revealed the accumulation of several substances that were not found in the controls. In response to the addition of salicylic acid or phenylalanine, lignification of the control hairy roots increased. However, the content of lignin-like substances in the PAL transformants was not increased by these treatments. These results indicate that lignification of the PAL transformants had reached a level that could not be further increased.
Eugenia uniflora Linn. is one of the useful trees in tropical areas. In vitro propagation of E. uniflora was achieved by using newly elongating shoots as material. MS medium supplemented with 0.2mgl-1 of 6BA was suitable for shoot regeneration and proliferation. Shoot elongation, root induction and root elongation were successfully carried out by transplanting to 1/2 MSHF medium. By this method more than fifty thousand regenerated plants can be obtained from one stem segment within one year. In vitro propagation of E. uniflora would be applicable not only for nursery production, but also for the conservation of genetic resources.
A convenient method for producing shoot regeneration has been developed for the Chinese cabbage (Brassica campestris L.). Internodes of seedstalks which were developed from the sterilized seeds by low temperature treatment in vitro were used to induce shoot regeneration. The shoot was stably induced when the seedstalk internode sections were incubated in MS medium containing 1mgl-1 naphthaleneacetic acid (NAA) and 5mgl-1 benzyladenine (BA), though the frequency was about 16%. Similar results were also obtained from the explants grown in a greenhouse as well as in a plant box. These results suggest that this culture system may be available for micropropagetion, genetic transformation and mutation breeding.
Variations in the gliadin composition in the endosperm were studied in R4 somaclonal lines that were derived from immature embryo callus cultures of durum wheat (Triticum durum Desf.). Electrophoregrams of gliadin polypeptides were analyzed in 10 somaclonal lines of cv. ‘Zagorka’ and seven lines of cv. ‘Progress’ and compared with the two parental cultivars. The analysis showed that the parental cultivars were both composed of individuals with different genotypes. Among the somaclonal lines, very high frequencies of variants which showed polypeptide patterns different from those of the parental cultivars were detected. The variations were characterized by the appearance and/or disappearance of particular polypeptides. Some variations were fixed in homozygous conditions but in a majority of somaclonal lines the variations remained in heterozygous conditions.
Hairy roots of Lithospermum erythrorhizon cultured in “Culture Bag” containing NH4NO3-depleted MS medium without agitation were found to produce shikonin. Although the amount in the Culture Bag was less than that in shaken flasks, the ratio of shikonin secreted into the medium in the former was higher than that in the latter, suggesting that the “Culture Bag” is useful for studies of the physiological effect of shaking or agitation on plant tissue and cell cultures in a liquid medium.