Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes

Insertion of foreign DNA into plant genomes frequently results in the recovery of transgenic plants with silenced transgenes. To investigate to what extent regeneration under selective conditions limits the recovery of transgenic plants showing gene silencing in woody species, Mexican lime [ Citrus aurantifolia (Christm.) Swing.] plants were transformed with the p25 coat protein gene of Citrus tristeza virus (CTV) with or without selection for nptII and uidA. Strikingly, more than 30% of the transgenic limes regenerated under non-selective conditions had silenced transgenes, and in all cases silencing affected all the three transgenes incorporated. These results indicate that the frequency of transgene silencing may be greatly underestimated when the rate of silencing is estimated from the number of regenerants obtained under selective conditions. To our knowledge, this is the first report in which the frequency of gene silencing after transformation has been quantified. When the integration pattern of T-DNA was analyzed in silenced and non-silenced lines, it was observed that inverted repeats as well as direct repeats and even single integrations were able to trigger gene silencing. Gene silencing has often been associated with the insertion of DNA sequences as inverted repeats. Interestingly, here, direct repeats and single-copy insertions were found in both silenced and non-silenced lines, suggesting that the presence of inverted-repeat T-DNAs and the subsequent formation of dsRNAs triggering gene silencing cannot account for all silencing events.     

Frequencies,Timing, and Spatial Patterns of Co-Suppression of Nitrate Reductase and Nitrite Reductase in Transgenic Tobacco Plants

Frequencies, timing, and spatial patterns of co-suppression of the nitrate (Nia) and nitrite (Nii) genes were analyzed in transgenic tobacco (Nicotiana tabacum) plants carrying either Nia or Nii cDNAs under the control of the 35S promoter, or a Nii gene with its own regulatory signals (promoter, introns, and terminator) cloned downstream of two copies of the enhancer of the 35S promoter. We show that (a) the frequencies of transgenic lines affected by co- suppression are similar for the three constructs, ranging from 19 to 25%; (b) Nia and Nii co-suppression are triggered stochastically during a phenocritical period of 2 weeks between germination and flowering; (c) the timing of co-suppression (i.e. the percentage of isogenic plants affected by co-suppression reported as a function of the number of days of culture) differs from one transgenic line to another; (d) the percentage of isogenic plants affected by co-suppression is increased by growing the plants in vitro prior to their transfer to the greenhouse and to the field; and (e) at the end of the culture period, plants are either unaffected, completely co-suppressed, or variegated. Suppressed and nonsuppressed parts of these variegated plants are separated by a vertical plane through the stem in Nia co-suppression, and separated by a horizontal plane in Nii co-suppression.     

Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana

CONSTANS (CO) is an important floral regulator in the photoperiod pathway, integrating the circadian clock and light signal into a control for flowering time. It is known that CO promotes flowering in Arabidopsis under long-day conditions. CONSTANS-LIKE 9 (COL9) is a member of the CONSTANS-LIKE gene family, encoding a nuclear protein. The expression of COL9 is regulated by the circadian clock in the photoperiod pathway and is detected in various organs. Unexpectedly, overexpression of COL9 in transgenic Arabidopsis resulted in delayed flowering, while co-suppression lines and a transferred DNA (T-DNA) knockout line showed earlier flowering under long-day conditions. Overexpression of COL9 did not enhance the late-flowering phenotype in a co mutant background. Double overexpressors produced by overexpression of CO in COL9 transgenic lines showed an early flowering phenotype similar to single CO overexpressors. The pattern of oscillation of a number of circadian-associated genes remained unchanged in the COL9 transgenic lines. Compared with wild-type plants, the abundance of CO and FLOWERING LOCUS T (FT) mRNA was reduced in the COL9 overexpression lines. Our results indicate that COL9 is involved in regulation of flowering time by repressing the expression of CO, concomitantly reducing the expression of FT and delaying floral transition.     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking CrylAb expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the crylAb gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of crylAb gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%-30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low con     

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.     

Production of transgenic pineapple (Ananas cosmos (L.) Merr.) plants via adventitious bud regeneration

A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement.     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking Cry1Ab expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the cry1Ab gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of cry1Ab gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%–30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low concentration of 5-azacytidine (45 mg/L for 1 d and 2 d) could lead to the highest reactivation ratio and the highest expression level of the cry1Ab gene.     

病毒诱导的PVX cp转基因沉默及其DNA甲基化

利用PCR方法获得了马铃薯X病毒(PVX)外壳蛋白(CP)基因(cp),并将其构建到植物表达载体中,利用农杆菌介导的叶盘法转化烟草(Nicotiana tabacum L.)。Northern杂交及Run on实验表明有3株转基因烟草发生了转录后基因沉默。发生沉默的cp基因的甲基化分析结果表明,发生转录后基因沉默的cp基因发生了不同程度的甲基化,说明DNA甲基化并没有完全抑制cp基因的转录。利用PVX病毒对外壳蛋白正常表达的转基因烟草进行接毒,Northern杂交检测结果表明,病毒诱导cp发生了基因沉默。进一步的Run on结果表明,转基因烟草中cp基因在沉默前后转录速率并没有发生变化,说明病毒诱导的沉默是一种转录后沉默。对cp基因沉默前后的甲基化分析表明,病毒的侵染导致了cp基因甲基化程度的增加。     

The role of epigenetic processes in controlling flowering time in plants exposed to stress

Plants interact with their environment by modifying gene expression patterns. One mechanism for this interaction involves epigenetic modifications that affect a number of aspects of plant growth and development. Thus, the epigenome is highly dynamic in response to environmental cues and developmental changes. Flowering is controlled by a set of genes that are affected by environmental conditions through an alteration in their expression pattern. This ensures the production of flowers even when plants are growing under adverse conditions, and thereby enhances transgenerational seed production. In this review recent findings on the epigenetic changes associated with flowering in Arabidopsis thaliana grown under abiotic stress conditions such as cold, drought, and high salinity are discussed. These epigenetic modifications include DNA methylation, histone modifications, and the production of micro RNAs (miRNAs) that mediate epigenetic modifications. The roles played by the phytohormones abscisic acid (ABA) and auxin in chromatin remodelling are also discussed. It is shown that there is a crucial relationship between the epigenetic modifications associated with floral initiation and development and modifications associated with stress tolerance. This relationship is demonstrated by the common epigenetic pathways through which plants control both flowering and stress tolerance, and can be used to identify new epigenomic players.