Plant Cell Reports

Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T2 generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously.

The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. ‘Solo’ and cv. ‘Sunrise’) were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.

Programmed cell death (PCD) is a process that occurs both in animals and in plants and is an essential element in developmental processes. Pollination is a key factor in fruit production and self-incompatibility is one of the main limiting factors of this process. PCD has recently been put forward as a possible cause of pollen-growth arrest. As far as the olive is concerned, no data have been published concerning the mechanisms involved in hindering the growth of pollen tubes in incompatible pollen. Thus, we have studied olive pistils excised from freely pollinated flowers at different stages before and during the progamic phase using different cytochemical techniques, including trypan blue staining. To discover whether the elimination of incompatible pollen might be associated to PCD, we applied different tests to the excised pistils: (1) TUNEL assay; (2) DNA degradation analysis; (3) detection of caspase-3-like activity. Once we had determined that PCD was involved in pollen selection after free pollination, we conducted experiments after controlled pollination in pistils excised from flowers: (a) developing in the absence of pollen; (b) pollinated with sterile pollen that does not germinate; (c) self-pollinated; (d) pollinated with compatible pollen. Our results demonstrate that the growth of tubes in incompatible pollen is halted in the stylar area in a way that suggests the intervention of PCD. Furthermore, any pollen, even if sterile, seemed to accelerate PCD in papillar cells in the olive.

Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings. Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

Complete plastomes of Rheum species facilitated to clarify the phylogeny of Polygonaceae, and comparative chloroplast genomics contributed to develop genetic markers for the authentication of Rheum species. Rheum (Polygonaceae) is widely distributed throughout the temperate and subtropical areas of Asian interior. Rheum species are usually perennial herbs, and half of them are endemic to China with important medicinal properties. On account of similar morphological characteristics, species delimitation of Rheum still remains unclear. Chloroplast genomes of eight Rheum species, Rumex crispus and Oxyria digyna were characterized. Based on the comparison of genome structure of these species and the two published Rheum species, it is shown that plastome sequences of these species are relatively conserved with the same gene order, and three Sect. Palmata species remarkably showed high sequence similarities. Some hotspots could be used to discriminate the Rheum species, and 17 plastid genes were subject to positive selection. The phylogenetic analyses indicated that all the Polygonaceae species were clustered in the same group and showed that Rheum species, except for Rheum wittrockii, formed a monophyletic group with high maximum parsimony/maximum likelihood bootstrap support values and Bayesian posterior probabilities. The molecular dating based on plastomes indicated that the divergences within Polygonaceae species were dated to the Upper Cretaceous period [73.86–77.99 million years ago (Ma)]. The divergence of Sect. Palmata species was estimated to have occurred around 1.60 Ma, indicating that its diversification was affected by the repeated climatic fluctuation in the Quaternary.

Morphine and codeine accumulation in Papaver somniferum suspension cultures increased markedly after removal of hormones from the medium. Cultures developed hormone self-sufficiency without organogenesis or development of meristemoids; enhanced synthesis of morphinan alkaloids was not dependent on formation of shoots, roots or embryos. Without exogenous hormones, maximum codeine and morphine concentrations were 3.0 mg g−1 dry weight and 2.5 mg g−1 dry weight respectively, up to three times higher than in cultures supplied with hormones. Hormone-deprived cells produced a higher ratio of codeine:morphine than cultures supplied with auxin and cytokinin. Improved alkaloid production was correlated with slower overall growth rate.

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l–1 N6-benzylaminopurine (BAP) and 0.5 mg l–1 indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l–1 BAP and 0.5 mg l–1 kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l–1 naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l–1) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.

Using Illumina sequencing technology, we have generated the large-scale transcriptome sequencing data and indentified many putative genes involved in isoflavones biosynthesis in Pueraria lobata. Pueraria lobata, a member of the Leguminosae family, is a traditional Chinese herb which has been used since ancient times. P. lobata root has extensive clinical usages, because it contains a rich source of isoflavones, including daidzin and puerarin. However, the knowledge of isoflavone metabolism and the characterization of corresponding genes in such a pathway remain largely unknown. In this study, de novo transcriptome of P. lobata root and leaf was sequenced using the Solexa sequencing platform. Over 140 million high-quality reads were assembled into 163,625 unigenes, of which about 43.1 % were aligned to the Nr protein database. Using the RPKM (reads per kilo bases per million reads) method, 3,148 unigenes were found to be upregulated, and 2,011 genes were downregulated in the leaf as compared to those in the root. Towards a further understanding of these differentially expressed genes, Gene ontology enrichment and metabolic pathway enrichment analyses were performed. Based on these results, 47 novel structural genes were identified in the biosynthesis of isoflavones. Also, 22 putative UDP glycosyltransferases and 45 O-methyltransferases unigenes were identified as the candidates most likely to be involved in the tailoring processes of isoflavonoid downstream pathway. Moreover, MYB transcription factors were analyzed, and 133 of them were found to have higher expression levels in the roots than in the leaves. In conclusion, the de novo transcriptome investigation of these unique transcripts provided an invaluable resource for the global discovery of functional genes related to isoflavones biosynthesis in P. lobata.