We give an overview of the advances of an Agrobacterium-mediated transformation system, clarifying its problems and their solutions, and then show the latest version of our transformation system and examples of the introduction of agronomically important traits into chrysanthemums. Typical problems with the Agrobacterium-mediated transformation in chrysanthemum include low transformation efficiency, high chimerism and cultivar specificity. Using a co-cultivation medium containing acetosyringone and casamino acids for high transformation efficiency and an antibiotic-selection step for transgenic calli before plant regeneration to eliminate the chimerism, we established an efficient and stable transformation system for chrysanthemum. In addition, this system was used to successfully introduce useful agronomical traits, such as insect resistance and new flower color, into chrysanthemums. These traits have been stably and highly expressed to confer the expected characteristics upon the transgenic chrysanthemums. Before applying a field trial of the genetically modified (GM) chrysanthemums, male and female sterility were introduced into the transformants to exclude the transgene flow from the GM plants to their wild relatives. So far, using RNAi technology, some of the transgenic chrysanthemums have displayed complete male sterility with very weak female fertility.
Three selected transgenic papaya lines 124-3, 132-2 and 142-3 across the T1 and T2 generations were preliminarily evaluated under Biological Containment Level 2 (BL2) greenhouse approved by the National Committee on Biosafety of the Philippines (NCBP) to determine the partial stability of expression of resistance to papaya ringspot virus (PRSV) and selected phenotypic traits. The PRSV reaction of the three transgenic lines using mechanical inoculation test and enzyme-linked immunosorbent assay was assessed. The qualitative and quantitative horticultural traits at flowering and ripe fruit stages of the transgenic lines were also evaluated. Based from preliminary results, the percentages of resistant progenies to PRSV in the T1 generation were 83% for 124-3, 74% for 132-2, and 70% for 142-3. However, in the same generation, no difference between the transgenic and non-transgenic were observed for stem, leaf and petiole color, fruit shape, peel, flesh and seed color, stem diameter, number of days to first flower, and fruit edible portion, indicating that the transgenic is seemingly stable. The percentage of resistant progenies to PRSV in the T1 generation decreased in the T2 generation, indicating that the inherited resistance of the three transgenic lines was unstable, and a continuous evaluation of the transgene could be done in later generations. In contrast, the light green (RHS 141B) stem color, dark green (RHS 141A) leaves and petioles and creamy white flowers (RHS 155A) were stably expressed in both the T1 and T2 generations. The plant height for lines 124-3 and 142-3, internode length for line 142-3 and number of nodes to first flower for lines 132-2 and 142-3 were also stable in both T1 and T2 generations. In addition, the pyriform fruit shape for hermaphrodite sex form and rounded fruit shape for the female sex form, light yellow orange (RHS 17A) peel color, bright yellow orange (RHS 23A) flesh color and black (RHS 202A) seeds of the three transgenic lines were stably expressed in both generations. The fruit weight, total soluble solids and fruit edible portion for lines 124-3 and 142-3 was stably expressed in both generations.
Portulaca (Portulaca oleracea cv.), a garden plant, efficiently removes endocrine-disrupting chemicals (EDCs) including bisphenol A (BPA) from hydroponic solution. We hypothesized that polyphenol oxidase (PPO) was involved in the initial steps of detoxifying EDCs in portulaca roots. In order to elucidate the molecular basis of portulaca’s ability to metabolize EDCs, we first isolated five PPO genes (PoPPO1–5) that were expressed mainly in portulaca roots. Among these genes, PoPPO2, PoPPO4 and PoPPO5 were introduced into cultured tobacco cells and expressed as active forms. We found that crude extracts from the cells expressing PoPPO2, PoPPO5, and to a lesser extent PoPPO4, could metabolize BPA. In addition, we found that the BPA metabolites from crude extracts of cells expressing PoPPO2, PoPPO4 and PoPPO5 were identical to those of portulaca. Moreover, PoPPO2 and PoPPO5 also caused hydroxylation of octylphenol, nonylphenol and 17β-estradiol. Therefore, these results strongly suggest that PoPPOs significantly contribute to the superior ability of portulaca to metabolize EDCs.
A rapid near-infrared (NIR) spectroscopy method was established to predict the lignin and starch contents and enzymatic saccharification efficiency of transgenic rice (Oryza sativa cv. Nipponbare) straw wherein expression of genes encoding lignin synthetic enzymes are regulated. Strong correlations were obtained between laboratory wet chemistry values and the NIR-predicted values. This method is useful to develop transgenic rice where cell-wall formation is engineered.
ENHANCER OF SHOOT REGENERATION 2 (ESR2), like ESR1, plays several critical roles during in vitro shoot regeneration. We investigated the genetic interaction between ESR2 and PINOID (PID) during shoot regeneration in this study. Both esr2 and pid mutations markedly decreased the efficiency of shoot regeneration from root segments at comparable levels (27% and 35% of shoot numbers on wild type, respectively); while esr2-2 pid double mutants dramatically decreased the efficiency of shoot regeneration (4.7% of shoot numbers on wild type). Our results demonstrated an additive or synergistic effect of these two mutations on shoot regeneration. Expression of ESR2 in pid explants during shoot regeneration was abnormal after day 5, although pid mutation did not affect ESR2 expression until day 5. In conclusion, PID appears to be required for late development of the shoot apical meristem during shoot regeneration.
NpLRX1 (Nicotiana plumbaginifolia Leucine-Rich Repeat-Extensin 1) encodes a chimeric leucine-rich repeat (LRR)/extensin protein and is thought to regulate cell morphogenesis that is essential for plant tissue formation. We have reported that abnormal cell morphogenesis was observed in NpLRX1-RNAi-introduced tobacco adventitious buds. A similar phenotype was observed when truncated NpLRX1 (N/LRR), a short N-terminal domain with LRR-domain, was expressed. Large gaps were observed between the cells with abnormal cell shapes in the leaves of the transgenic adventitious buds. The epidermal cells and the cuticle layer were defective in these leaves. The cells frequently swelled and the direction of cell division was sometimes out of order with an abnormal arrangement of microtubules in N/LRR-introduced tobacco BY-2 cultured cells. NpLRX1-GFP fusion proteins were detected on the cell membrane and in cell membrane-derived endocytic vesicles with partial degradation. These results suggest that ectopic expression of N/LRR causes defects in cell morphogenesis of tobacco tissues by the association of the LRR-domain with cell membrane.
Lactoferricin B, the antimicrobially active N-terminal peptide derived from acid pepsin hydrolysis of bovine lactoferrin, has broad spectrum antimicrobial activities. We expressed the lactoferricin gene in tobacco plants to evaluate its antimicrobial activity. The coding region for the chimeric peptide gene of the signal peptide from tobacco pathogen-related protein (PR-1) and bovine lactoferricin was synthesized using the recombinant polymerase chain reaction (PCR) method. Transgenic tobacco plants expressing the lactoferricin gene were developed using the Agrobacterium-mediated transformation method. The lactoferricin B gene was integrated into the tobacco genome and its transcription was detected by PCR, Southern blot analysis, and reverse transcription PCR (RT-PCR), respectively. The transgenic tobacco plants were challenged with the pathogenic bacteria Pseudomonas syringae pv. tabaci and Botrytis cinerea. At 30 or 28 days post-inoculation, the transgenic plants were still green and continued to grow, whereas the control plants were infected with bacterial or fungal pathogens from the roots to the tips, resulting in death of the plants. In conclusion, transgenic tobacco plants that overexpressed the lactoferricin gene, linked to the signal peptide of tobacco PR-1 protein under the control of a high expression constitutive promoter, showed enhanced resistance to bacterial (P. syringae pv. tabaci) and fungal (B. cinerea) diseases.
Although both lignin and silica accumulate on cell walls and confer rigidity, mechanical strength, and resistance to pathogen invasion in rice, it remains unclear whether silicon deficiency affects lignin accumulation. We demonstrate that low silicon rice mutated in a silicon influx transporter, Lsi1, or a silicon efflux transporter, Lsi2, contained larger amounts of lignin in the rice straws. Furthermore, wild-type rice cultivated on low silicon media also accumulated larger amounts of lignin in shoots. Significant accumulation of guaiacyl lignin upon silicon deficiency was determined by nitrobenzene oxidation analysis. These data indicate a negative correlation between silicon accumulation and lignin deposition.
The tobacco bZIP transcription factor, TBZF, functions in leaf senescence and floral development. In order to identify TBZF-interaction partner(s), we screened a cDNA library constructed from leaves of tobacco (Nicotiana tabacum cv. Xanthi) by the yeast two hybrid method. Among 16 clones initially identified, one clone was further characterized because of its highly specific binding to the bait. The full length cDNA of 776 bp encoded a polypeptide of 12.5 kDa, which was designated NtTIP1 (Nicotiana tabacumTBZF-Interaction Protein 1). A high similarity was scored to potato CI21A, a member of the ASR/CI21 family of proteins, most of which are induced by biotic and abiotic stresses. NtTIP1 was revealed to be localized in the nucleus by fluorescence assay of NtTIP1-GFP fusion protein in onion epidermal cells. Both NtTIP1 and TBZF transcripts accumulated at high levels in flowers and senescing leaves. The expressional correlation was also observed particularly after 6 h cold stress, and after ABA, JA and ethylene treatments. The results suggest that NtTIP1 and TBZF form a complex in vivo, and they possibly function in flower development and stress response.
Somatic embryogenesis in Ryukyumatsu (Pinus luchuensis Mayr.), an endemic species in Ryukyu Island, Japan, was initiated from megagametophytes containing zygotic embryos on a medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid and 5 µM 6-benzylaminopurine. Embryogenic cultures were maintained and proliferated by subcultures at 2- to 3-week-intervals on the same fresh medium. The maturation of somatic embryos occurred on media containing maltose, activated charcoal, abscisic acid and polyethylene glycol. High frequencies of germination were obtained after the post-maturation treatment of somatic embryos under conditions of high relative humidity, and around 90% of them were converted into plants. Growth of the somatic embryo-derived regenerated plants has been monitored in the field.
The CAPRICE-LIKE MYB gene family, including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 and 2 (ETC1 and ETC2), CPC-LIKE MYB3/ENHANCER OF TRY AND CPC3 (CPL3/ETC3), encodes a small protein with an R3-type MYB motif that regulates leaf trichome differentiation in Arabidopsis thaliana. To understand the role of CPC-LIKE MYB genes in trichome development, we focused on the effect of these genes on trichome branching and clustering. Trichomes of the try etc1 double mutant consistently had more branches than wild-type and the try single mutant, suggesting that the TRY and ETC1 genes cooperatively regulate trichome branch development. The ETC2 gene has little to no involvement in trichome branching. TRY and CPC are known to have strong effects on trichome cluster formation. Double and triple mutant analyses revealed that the ETC1, ETC2 and CPL3 genes have some degree of functional redundancy with TRY in trichome cluster formation.
A precise mechanism for the biosynthesis of natural rubber has not yet been elucidated. The cis-prenyltransferase (cPT), HRT2, identified from latex of Hevea brasiliensis, is thought to be a key enzyme in the biosynthesis of natural rubber. This is due to the observation that recombinant HRT2, expressed in Escherichia coli, is significantly activated in the presence of a centrifuged latex fraction, resulting in the formation of polyisoprenes corresponding to natural rubber. The precise enzymatic characterization of cPT function for HRT2, however, has not been investigated because HRT2 expressed in E. coli does not exhibit significant activity independently. Herein, the enzymatic characterization of HRTs expressed in eukaryotic cell systems is reported. Both HRT2 and HRT1, another cPT from Hevea latex, expressed in Saccharomyces cerevisiae and Arabidopsis T87 cultured cells showed distinct cPT activity, producing polyisoprenoids with chain-lengths of C80–100, although failing to catalyze the formation of natural rubber. The chain lengths of the HRT1/HRT2 products were not altered by the addition of centrifuged latex fractions, and the HRT1/HRT2 expressed in yeast competed with the rubber transferase activity of the latex fraction. These results indicate that HRT1/HRT2 requires additional co-factors from the eukaryotic cells to produce distinct cPT activity, and that latex specific co-factor(s) may be required to enable HRT1/HRT2 rubber transferase activity.
Thioacidolysis is a method to detect the β-O-4 substructures of lignin, and has been employed as a diagnostic test for the presence of lignin. However, the conventional thioacidolysis protocol is low-throughput and is a bottleneck in the characterization of lignins in a large number of samples such as transgenic lines. Recently, a rapid analysis protocol for thioacidolysis was reported. In this study, we modified mainly the work-up process. Our microscale protocol showed higher yields of thioacidolysis products than the conventional protocol.