Transgenic Plantlets Research Articles

Establishment of Glyoxalase I gene transformation and regeneration of cotton (Gossypium hirsutum L.)

Aim: The current study was carried out to develop transgenic cotton plantlets with glyoxalase I (gly I) gene using Agrobacterium-mediated transformation. Methodology: Seeds of cotton were inoculated on MS medium and the explants such as shoot tip (3-5 mm), hypocotyl and leaf were aseptically removed from in vitro plantlets. The pre-cultured and infected explants with Agrobacterium harboring gly I gene and the shoot tip were inoculated on MS media for shoot initiation and subcultured on elongation medium with growth hormones, and antibiotics. Healthy and well-grown shoots were subcultured on medium with indole butyric acid (IBA) (0.3 mgl-1) for root formation and the plantlets were hardened in plastic cups with sterile soil. The putative transgenic plantlets were analyzed histochemically for gus gene and the PCR analysis was performed for gly 1 gene. Results: The transformation efficiency of cotton ranged 48.57 to 64.53 %. The regenerated plantlets showed the presence of gus gene in terms of blue coloration in shoots, whole leaf and leaf segments. The PCR was performed in putative transgenic plant lets with both gus gene as well as gly I gene primers. The PCR results showed the presence of 1031 bp DNA band with gus gene primers and 800 bp DNA band with the gly I gene primers. Interpretation: The current study has proven the reproducible procedure for the Agrobacterium-mediated gene transfer and regeneration of Indian cotton varieties. The PCR results revealed the presence of glyoxalase I gene in the transformants. Key words: Cotton varieties, Glyoxalase I gene, PCR analysis, Regeneration, Transformation

Functional Identification of EjGIF1 in Arabidopsis and Preliminary Analysis of Its Regulatory Mechanisms in the Formation of Triploid Loquat Leaf Heterosis.

Although several results have been obtained in triploid loquat heterosis (i.e., leaf size of triploid loquat) studies in the past years, the underlying mechanisms of the heterosis are still largely unknown, especially the regulation effects of one specific gene on the corresponding morphology heterosis. In this study, we sought to further illustrate the regulatory mechanisms of one specific gene on the leaf size heterosis of triploid loquats. A leaf size development-related gene (EjGIF1) and its promoter were successfully cloned. Ectopic expression of EjGIF1 in Arabidopsis showed that the leaf size of transgenic plantlets was larger than that of WTs, and the transgenic plantlets had more leaves than WTs. Quantitative Reverse Transcription PCR (qRT-PCR) showed that the expression level of EjGIF1 showed an AHP expression pattern in most of the hybrids, and this was consistent with our previous phenotype observations. Structure analysis of EjGIF1 promoter showed that there were significantly more light-responsive elements than other elements. To further ascertain the regulatory mechanisms of EjGIF1 on triploid loquat heterosis, the methylation levels of EjGIF1 promoter in different ploidy loquats were analyzed by using bisulfite sequencing. Surprisingly, the total methylation levels of EjGIF1 promoter in triploid showed a decreasing trend compared with the mid-parent value (MPV), and this was also consistent with the qRT-PCR results of EjGIF1. Taken together, our results suggested that EjGIF1 played an important role in promoting leaf size development of loquat, and demethylation of EjGIF1 promoter in triploid loquats caused EjGIF1 to exhibit over-dominance expression pattern and then further to promote leaf heterosis formation. In conclusion, EjGIF1 played an important role in the formation of triploid loquat leaf size heterosis.

Development of an efficient in-planta Agrobacterium-mediated transformation method for Iranian purslane (Portulaca oleracea L.) using sonication and vacuum infiltration

Purslane (Portulaca oleracea L.), a valuable medicinal herb, is used as a source of pharmaceutical components such as flavonoids, alkaloids, fatty acids, terpenoids, and sterols. Regeneration of transgenic purslane plantlets from transformed cells is a time-consuming procedure and needs hard work. In this study, in-planta transformation of purslane, using sonication and vacuum infiltration, is reported. The purslane seeds were infected through Agrobacterium tumefaciens strain LBA4404 having the uidA gene on pBI121 vector. Effective selection of transformants was performed by supplementing MS media with 250 mg l−1 kanamycin. Several factors affecting the in-planta procedure including pre-culture duration, acetosyringone dose, sonication, and vacuum infiltration duration were investigated. The results demonstrated that the highest number of GUS-positive purslane plantlets was obtained when the pre-cultured seeds were sonicated and vacuum-infiltered for 2 min in agro-bacterial cell suspension, and then co-cultivated in MS media having 100 µM acetosyringone. The integration and expression of uidA gene in transgenic purslane was successfully corroborated by southern blot and GUS histochemical analyses.

Agrobacterium-Mediated Genetic Transformation of Bread Wheat (Triticum aestivum L.) Using Immature Embryos

Wheat is considered the most important food crop globally. Genetic transformation provides a method for genetic manipulation of wheat to enhance its agronomic performance. Wheat is a difficult crop for genetic transformation. Agrobacterium-mediated transformation facilitates precise integration of genes into the plant genome compared to biolistic approach. In the present research, we studied the effect of acetosyringone concentrations, genotypes and embryo explant types (intact or longitudinally halved-embryos or fragmented latitudinal pieces) on transformation frequency for immature embryos derived from four different genotypes of bread wheat (Achtar, Amal, Mehdia, and Rajae). Embryo explant types were inoculated with A. tumefaciens strain EHA101 harboring the plasmid vector pTF101.1 carrying the barley HVA1 drought tolerance gene and bar-selection marker for resistance to the herbicide phosphinothricin. The effects of the genotype, embryo explant type, and acetosyringone concentrations on the frequency of phosphinothricin resistant bread wheat plantlets regeneration after transformation with Agrobacterium were significant. Transgenic plants were generated from longitudinally-halved immature embryo and intact-immature embryos, whereas, no transgenic plantlets were obtained from fragmented immature embryos. Based on the PCR analysis, the transformation efficiency of T0 plants for the two genotypes varied from 0.66% (‘Rajae’ from the longitudinally halved-immature embryo cultured with no acetosyringone) to 0.33% (‘Achtar’ genotype from intact-immature cultured with 200 μM of acetosyringone).

Gene Transformation of Alfalfa Plants (Medicago varia L.) DomesticCultivar andlsquo;Burgaltaiandrsquo; for Increasing Leaf Size

‘Burgaltai’ cultivar of alfalfa is the most important forage in Mongolia and its yield had decreasing last decades due to cross pollination. This study was conducted for increasing leaf size of ‘Burgaltai’ cultivar through Agrobacterium mediated genetic transformation transferring growth regulating factor AtGRF2 gene of Arabidopsis thaliana. In vitro regeneration was derived from seven day old hypocotyl and cotyledon explants for callus induction. Induced calli were used for shoot induction and proliferation. Proliferated shoots carried to root induction medium. The best calli induction medium was MSB5 (Murashige Skoog’s medium with Gamborg’s vitamin) medium supplemented with 2 mg/L 2,4-D, 0.2 mg/L BAP and 1 mg/L NAA, shoot induction and proliferation media from calli were MSB5 medium supplemented with 1mg/L BAP, 0.1 mg/L NAA, proliferated shoots were rooted on half strength MSB5 medium supplemented with 0.5 mg/L NAA. For the genetic transformation, hypocotyl explants used as an initial source. Totally 235 explants were tested to transformation and 30 explants (12.7%) were resistant to hygromycin antibiotics and regenerate plantlets. Plantlets were transferred to the soil and fresh young leaf used for GUS histochemical assay and RT-PCR for confirmation. Among them fully regenerated seven independent transgenic plantlets (T6, 8, 11, 12, 13, 15, 22) were confirmed. Transgenic ‘Burgaltai’ cv. of alfalfa leaf size was increased by 0.7 cm2 comparing to the wild type. T6 and 11 lines showed 3:1 segregation ratio in T1 progenies (X2=0.04-0.42, p ≤ 0.52-0.84).

Physiological responses and P5CS gene expression of transgenic oil palm plantlet induced by drought stress

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.

Agrobacterium-mediated transformation of Persian walnut using BADH gene for salt and drought tolerance

ABSTRACT Transgenic walnuts (Juglans regia L.) were produced in the presence of the Agrobacterium strain LBA4404 and the plasmid pBI121. This plasmid contains a BADH gene driven by a CaMV 35S promoter. It further includes an NPTII gene as a selectable marker which, in turn, is driven by a NOS promoter. Somatic embryos of the walnut cultivar ‘Chandler’ were inoculated using an Agrobacterium suspension. Secondary embryos that developed on a selection medium were driven towards germination so as to develop micro-shoot cultures. The regeneration rate was 5.5%. PCRand southern blot analysis were used for confirming that the BADH transgene is successfully integrated into the plant genome. Transgenic and wild-type plants raised from somatic embryos were exposed to four osmotic stress levels (i.e. 0%, 2%, 4%, and 8% PEG) and four salinity levels (i.e. 0, 50, 100, and 200 mM NaCl). After 21 days, the transgenic plants grew almost vigorously in saline and drought-stress conditions, whereas the wild-type ones showed retarded levels of growth and did not survive after the cradle stage. These results show that the BADH gene can be expressed effectively in transgenic plantlets of walnut and can be used for breeding walnuts against drought and salinity.

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)

Grapevine (Vitis vinifera), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by Erysiphe necator. In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of MLO (mildew resistance Locus O) family genes that are thought to serve as S factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes, VvMLO3 and VvMLO4, in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 VvMLO3/4-edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four VvMLO3-edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and H2O2 accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.

Transient expression of human serum albumin (HSA) in tobacco leaves.

Today, recombinant human proteins make up a considerable part of FDA-approved biotechnological drugs. The selection of proper expression platform for manufacturing recombinant protein is a vital factor in achieving the optimal yield and quality of a biopharmaceutical in a timely fashion. This experiment was aimed to compare the transient expression level of human serum albumin gene in different tobacco genotype. For this, the Agrobacterium tumefaciens strains LB4404 and GV3101 harboring pBI121-HSA binary vector were infiltered in leaves of three tobacco genotypes, including Nicotiana benthamiana and N. tabacum cv Xanthi and Samsun. The qRT-PCR, SDS-PAGE, western blotting and ELISA analysis were performed to evaluate the expression of HSA gene in transgenic plantlets. Our results illustrated that the expression level of rHSA in tobacco leaves was highly dependent on Agrobacterium strains, plant genotypes and harvesting time. The highest production of recombinant HSA protein was obtained in Samsun leaves infected with A. tumefaciens strain GV3101 after 3days of infiltration.

Plant regeneration and Agrobacterium-mediated genetic transformation systems in liliaceous ornamental plants.

The family Liliaceae (Cronquist system) contains various important ornamental plants. We have been examining for about 20 years the establishment of plant regeneration and genetic transformation systems in liliaceous ornamental plants for their biotechnological breeding and elucidation of the molecular mechanisms determining ornamental traits. In this review, studies on in vitro plant regeneration in 7 genera and on Agrobacterium-mediated production of transgenic plants in 4 genera are described. Plant regeneration was achieved via callus cultures in Agapanthus, Hemerocallis, Hosta, Lilium, Muscari and Tricyrtis. Auxins (2,4-dichrolophenoxyacetic acid, α-naphthaleneacetic acid and/or picloram) were effective for inducing regenerable calli. Tulipa species and cultivars were very recalcitrant to callus induction and plant regeneration. Agrobacterium-mediated transformation was examined in Agapanthus, Lilium, Muscari and Tricyrtis, and transgenic plants were obtained in all genera by using regenerable calli as a target material for Agrobacterium inoculation, inoculation and co-cultivation with Agrobacterium in the presence of acetosyringone, and selection of transgenic tissues and plantlets on hygromycin-containing media. Among 4 genera, Tricyrtis has several advantages for transformation studies: higher transformation efficiency, relatively small plant size, ease of cultivation, and taking only 1 year from in vitro regeneration to flowering. We are now investigating the molecular mechanisms for determining plant form, flower color and flower form by using Tricyrtis spp. as liliaceous model plants.

Effective reduction in chimeric mutants of poplar trees produced by CRISPR/Cas9 through a second round of shoot regeneration

The clustered regulatory interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system has been applied to edit the genomes of quite a few plant species, including perennial woody poplar trees. However, chimeras often exist in primary transgenic plants. For perennial woody trees such as poplar trees, it is difficult to obtain homozygous mutants by self-pollination for due to their long vegetative life and low-seed setting rates. In this study, we report an effective approach to reduce the frequency of chimeric mutants of poplar trees by CRISPR/Cas9 with a second round of shoot regeneration using leaves as the explants. PdbPDS1 was used as the target gene, and only one homozygous PdbPDS1 mutant was obtained from 15 primary transgenic plantlets, which was verified by both the phenotype and the DNA sequence of the PCR product. This indicates that the majority of primary transgenic plantlets in the T0 generation were chimeras. After the second round of shoot regeneration of the chimeric mutants generated by CRISPR/Cas9, approximately 27.0% or 19.1% of the regenerated shoots were homozygous mutants with or without kanamycin selection, respectively. The results showed that a second regeneration could produce homozygous mutant shoots at a high frequency and that kanamycin selection could increase the frequency of homozygous mutant shoots.

Development and identification of glyphosate-tolerant transgenic soybean via direct selection with glyphosate

Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD600 of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T0 transgenic plants, consisting of spotting plantlets with 1 µL Roundup®. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.

Biolistic Transformation of Japonica Rice Varieties.

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of interest into rice mature embryos using Biolistic® PDS-1000/He particle delivery system. We also provide information required for recovery of transformed plants and production of transgenic seed for next generation analysis. Using this protocol, typical 50-70 putative independent transgenic callus lines can be generated from 100 bombarded embryos. Transgenic rice plantlets can be produced within 2months after the initiation of seed germination for transformation.

Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

BackgroundRecent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. ResultsAbiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. ConclusionsThe present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula

BackgroundTissue culture transformation of plants has an element of art to it, with protocols passed on between labs but often not directly compared. As Medicago truncatula has become popular as a model system for legumes, rapid transformation is critical, and many protocols exist, with varying results.ResultsThe M. truncatula ecotypes, R108 and A17, were utilized to compare the effect of a modification to a previously used protocol based on shoot explants on the percentage of transformed plants produced from calli. This percentage was then compared to that of two additional transformation protocols based on root explants in the R108 ecotype. Variations in embryonic tissue sources, media components, time for transformation, and vectors were analyzed.ConclusionsWhile no A17 transgenic plants were obtained, transgenic plantlets from the R108 ecotype were produced in as little as 4 months with a comparison of the two widely studied ecotypes under a single set of conditions. While the protocols tested gave similar results in percentage of transformed plants produced, considerations of labor and time to transgenics that vary between the root explant protocols tested were discovered. These considerations may influence which protocol to choose for introducing a single transgene versus creating lines with multiple mutations utilizing a CRISPR/Cas9 construct.

Infection of Embryonic Callus with Agrobacterium Enables High-Speed Transformation of Maize.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Agrobacterium-MEDIATED GENETIC TRANSFORMATION OF SEAWEED Kappaphycus alvarezii USING Gα GENE AND CALLUS CULTURES

Cottonii seaweed (Kappaphycus alvarezii Doty) is one of the most important commercial sources of carrageenans which are widely used in the pharmaceuticals and food industries. A problem in the cultivation of this seaweed is the ice-ice disease, which is caused by extreme changes in environmental conditions such as temperature and seawater salinity. Gene transformation to produce Cottonii seaweed transgenics that are tolerant to environmental stress is a potential solution to this problem. Gα gene encodes for the heterotrimeric G protein α subunit is a gene that plays a role in tolerance to biotic and abiotic environmental stresses. This study aimed to: (a) introduce the Gα gene into the callus cells of K. alvarezii and regenerate transformed callus cells to transgenic plantlets; (b) determine the appropriate concentration of acetosyringone and Agrobacterium tumefaciens strain for gene transfer into the callus of K. alvarezii. The callus cells of K. alvarezii were transformed using Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying the expression vector pGWB502-Gα with a CaMV-35S promoter. The calli and A. tumefaciens were co-cultivated in several concentrations of acetosyringone (20, 40, 60 mg/L). The regeneration of transformed callus cells into transgenic plantlets was successfully performed using the somatic embryogenesis technique. The results showed that the highest percentage of putative transgenic micropropagule formation occurred at the 20-40 mg/L concentration of acetosyringone. Polymerase chain reaction (PCR) analysis on the twenty transgenic plantlets indicated that the Gα gene was successfully introduced into the genomic DNA of all of them. The highest transformation efficiency was in the co-cultivation treatment of 20-40 mg/L acetosyringone (22-28%). The transformation efficiency produced by Agrobacterium tumefaciens EHA105 (23%) was not significantly different from that produced by the LBA4404 (15%).

The regulatory module MdPUB29‐MdbHLH3 connects ethylene biosynthesis with fruit quality in apple

The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality. Overexpression of apple MdbHLH3 gene, an anthocyanin-related basic helix-loop-helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene-related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis. High glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein. Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose-inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

Genetic transformation of the astaxanthin biosynthetic genes bkt and crtR-B into apple tree to increase photooxidation resistance

Apples may suffer sunburn during growing season because of photooxidation damage. Astaxanthin have a stronger antioxidant activity against photooxidation compared to its precursors zeaxanthin and β-carotene. Here, we transferred β-carotene ketolase (bkt) and β-carotene hydroxylase (crtR-B), two genes encoding key enzymes for astaxanthin biosynthesis, into “Brookfield Gala” apple plants using pCAMBIA1302-bkt-crtR-B via transformation mediated by Agrobacterium tumefaciens EHA105. We aimed to confer a stronger antioxidant ability against sunburn to apple plants by regulating metabolic pathways of carotenoids. RT-PCR and qPCR analysis confirmed that bkt and crtR-B genes were expressed simultaneously in transgenic plants. Southern blotting analysis confirmed that the transgene was stably integrated and determined transgene copy number. Transgenic plants synthesized novel ketocarotenoids, resulting in an accumulation of astaxanthin up to 12.06 μg/g and of canthaxanthin up to 6.38 μg/g per leaf fresh weight. The light antioxidant ability of transgenic plantlets was stronger than that of non-transgenic ones in high light conditions. We speculate that the transgenic apples accumulating astaxanthin are resistant to photooxidation and can therefore prevent sunburn.