Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp.

Transgenic 'Duncan' grapefruit (Citrus paradisi Macf.) and 'Valencia' sweet orange (Citrus sinensis [L.] Osbeck) plants ectopically expressing C. sinensis (cv. Washington navel orange) APETALA1 (CsAP1) or LEAFY (CsLFY) genes under control of the Arabidopsis thaliana stress-inducible promoter AtRD29A flowered under non-inductive (warm temperature, well-watered) greenhouse conditions, whereas their wild-type (WT) counterparts did not. The transgenic plants that flowered exhibited no altered morphological features, except the lack of thorns characteristic of juvenile WT plants. The most precocious T0 line, 'Duncan' grapefruit (Dun134-3) expressing the CsAP1 gene, flowered and fruited when it was 4.5 years old and the T1 siblings from this line flowered and fruited when they were just over 18 months old. In contrast, T1 seedlings from three lines of 'Duncan' grapefruit expressing the CsLFY gene flowered within 3 months after germination, but were unable to support fruit development. Transcript levels of corresponding transgenes in leaves were not correlated with earliness of flowering. To further study the activity of AtRD29A, leaves from three 'Carrizo' citrange (C. sinensis × Poncirus trifoliata) rootstock seedlings transformed with the green fluorescent protein (GFP) gene under regulation of the AtRD29A promoter were subjected to drought stress or well-watered conditions. Expression of GFP was not stress-dependent, consistent with the observation of flowering of CsAP1 and CsLFY transgenic plants under non-inductive conditions. Taken together, the results suggest that AtRD29A is constitutively expressed in a citrus background. Despite the loss of control over flowering time, transgenic citrus lines ectopically expressing C. sinensis AP1 or LFY genes under control of the A. thaliana RD29A promoter exhibit precocious flowering, fruit development and viable transgenic seed formation. These transformed lines can be useful tools to reduce the time between generations to accelerate breeding.

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Cry1Ba1-mediated toxicity of transgenic Bergera koenigii and Citrus sinensis to the Asian citrus psyllid Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan and Valencia expressing Cry1Ba1 exhibited a "delayed senescence" phenotype, similar to observations made for transgenic B. koenigii. In bioassays, significant reductions in the survival of adult psyllids were noted on transgenic B. koenigii and Valencia sweet orange plants expressing Cry1Ba1, but not on transgenic Duncan grapefruit or Carrizo citrange. In contrast to psyllids fed on wild type plants, the gut epithelium of psyllids fed on transgenic plants was damaged, consistent with the mode of action of Cry1Ba1. These results indicate that the transgenic expression of a bacterial pesticidal protein in B. koenigii and Valencia sweet orange offers a viable option for management of D. citri, that may contribute to solutions that counter citrus greening disease.

Genetic Modification of Bergera koenigii for Expression of the Bacterial Pesticidal Protein Cry1Ba1.

The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphorina citri, which vectors the bacterial causative agent of citrus greening or huanglongbing disease. This disease has decimated citrus production in Florida and in other citrus-producing countries. As D. citri exhibits high affinity for feeding on young leaves of B. koenigii, transgenic B. koenigii expressing bacteria-derived pesticidal proteins such as Cry1Ba1 have potential for D. citri management when planted in or adjacent to citrus groves. Importantly, the plant pathogenic bacterium that causes citrus greening does not replicate in B. koenigii. Transgenic plants of B. koenigii were produced by insertion of the gene encoding the active core of the pesticidal protein Cry1Ba1 derived from Bacillus thuringiensis. The transformation success rate was low relative to that of other citrus, at 0.89%. T-DNA integration into the genome and cry1ba1 transcription in transgenic plants were confirmed. Transgenic plants expressing Cry1Ba1 differed from wild-type plants, differed in photosynthesis parameters and hormone levels in some instances, and a marked delay in wilting of detached leaves. The gut epithelium of D. citri fed on transgenic plants was severely damaged, consistent with Cry1Ba1-mediated pore formation, confirming expression of the pesticidal protein by transgenic B. koenigii. These results demonstrate that transgenic B. koenigii expressing bacteria-derived pesticidal proteins can be produced for potential use as trap plants for suppression of D. citri populations toward protection of citrus groves from citrus greening.

Enhancement of phenolic and flavonoids compounds, antioxidant and cytotoxic effects in regenerated red cabbage by application of Zeatin.

This study evaluated the roles of zeatin (2 mg/L) on direct organogenesis, phytochemical compounds, antioxidant activity, and cytotoxic activity in regenerated shoots of red cabbage. The results revealed that the extract of explant treated by 2 mg/L zeatin gives the highest content of total phenolics (5.18 mg gallic acid equivalent/g dry weight) and flavonoids (1.52 mg rutin equivalent/g dry weight). Moreover, HPLC and GC-MS analyses indicated that various bioactive compounds in red cabbage are significantly enhanced with increasing zeatin concentration. Besides that, antioxidant activity test showed that in vitro shooting culture using 2 mg/L zeatin displayed higher antioxidant activity in all assays (DPPH, FRAP and ABTS) compared to control with respective values of 68.12%, 73.28%, and 54.1%, respectively. Finally, the cytotoxic properties illustrated that the extracts of red cabbage explant treated by 2 mg/L zeatin exhibited the strongest cytotoxic effect towards cancer cells compared to control.

Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica.

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34ºC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel.