Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

Background

Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.

Methodology/Principal Findings

We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.

Conclusions/Significance

We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.     

葡萄基因工程研究进展

植物基因工程技术为培育优良葡萄品种开辟了一条全新而有效的途径。葡萄基因转化受体系统的建立主要包括器官发生途径和胚状体发生途径,建立良好的受体系统是葡萄基因转化成功的关键,遗传转化途径主要有根癌农杆菌介导的遗传转化和基因枪法。概述了迄今国内外葡萄基因工程的研究进展,着重对葡萄基因转化受体系统的建立、转化的方法、转化植株的筛选和检测、影响葡萄基因转化的主要因素等进行了综述,并展望了葡萄基因工程的发展前景。     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Genetic engineering approaches to enhance oil content in oilseed crops

Oilseed crops play an important role in the agricultural economy. Apart from being an integral component of human diet and industrial applications, they are also gaining importance as replacement to fossil fuels for meeting the energy needs. The last two decades have been marked by several important events in genetic engineering and identification of gene targets for enhancing seed oil content in oilseed crops, and will aid the successful development of new generation high yielding oil crops. Specifically, genetic engineering has shown real breakthrough in enhancing oil content in oilseed rape, camelina, soybean and maize. Moreover, ongoing research efforts to decipher the possibilities of genetic modifications of key regulators of oil accumulation along with physiological and biochemical studies to understand lipid biosynthesis will set a platform to produce transgenic oilseed crops with enhanced oil content. In this review, we briefly describe different genetic engineering approaches explored by different researchers for enhancing oil content. Further, we discuss a few promising and potential approaches and challenges for engineering oil content in oilseed crops.     

人参的遗传改良*

遗传改良是人参育种的重要手段之一,而遗传转化和再生体系的建立是开展人参遗传改良工作的前提和基础。人参植株再生可以通过器官发生和体细胞胚发生,间接体细胞胚发生是人参植株再生的主要途径,从不同外植体,不同碳源,体细胞胚优化和无激素再生等方面进行了综述。在人参遗传转化方面,发根农杆菌和根癌农杆菌对人参的遗传转化均已成功,人参皂苷合成途径中的关键酶基因和抗除草剂基因也已陆续导入人参,得到了遗传改良的转化人参。发根培养系统可用于大量生产人参皂苷,讨论了rolC基因对人参发根诱导的作用,发根植株再生能力及生物反应器培养,最后指出了人参基因工程研究中存在的问题。     

Genetic transformation of Vitis vinifera via organogenesis

Background  

Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.     

Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance

Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO₂. The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.     

Multiple locus genome-wide association studies for important economic traits of oil palm

Palm oil has a balanced fatty acid composition and has no trans fat. As a result, its use in food has increased as food-labeling laws have changed to specify trans fat content. Increasing oil production is the main goal in oil palm breeding. Genetic mapping and genomic studies in palm trees are necessary to understand the genetic architecture of economic traits of importance for palm oil production. To help achieve this, we sampled 422 oil palms from MPOB (Malaysian Palm Oil Board)­Angola germplasm collection and measured 13 economic traits from these palms. Multi-locus genome-wide association studies (GWAS) were conducted using least absolute shrinkage and selection operator (LASSO) and genome-wide efficient mixed model analysis. We identified 19 quantitative trait loci (QTLs) for 8 traits. Of these, four Angola-specific QTLs associated with bunch components were detected on chromosomes 4, 8, and 11. These QTLs are potentially useful for introgression of desirable genes from the Angola palms to advanced breeding populations for improvement of bunch and oil yield traits. The majority of the QTLs were detected by LASSO-A, in which the p values of individual markers were calculated based on bootstrapped standard errors. Many of the detected QTLs are nearby known QTLs detected from linkage studies reported by other research groups. We also conducted genomic selection (GS) for the 13 traits and concluded that GS can be an effective tool for oil palm breeding. This is the first GWAS and GS study conducted on oil palm germplasm from Angola, and the results can be very useful in oil palm genetic studies and breeding.     

Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.     

Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

ABSTRACT: BACKGROUND: As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus the establishment of an efficient genetic transformation method is required. RESULTS: To develop a genetic transformation method for C. roseus, A. tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report beta-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 muM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance mediums, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. CONCLUSIONS: In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11.11 % of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.     

Millets genetic engineering: the progress made and prospects for the future

Millets comprise a highly variable small-seeded group of Poaceae members that can grow in extreme environmental conditions of drought, high temperature and low soil fertility hence, recognized as climate-resilient. Among millets, the phylogenetic closeness of Setaria with other agronomically important grasses like maize, sugarcane, and sorghum helped in its adoption as a translational model plant. Established efficient gene transfer methodology is a prerequisite for embracing plant species as models. However, genetic engineering of some of the economically important millets has been started in the 1990s, but inadequate progress made this group lag behind other members of Poaceae as rice, maize and wheat. Genetic transformation in millets has generally been achieved by a physical method of microprojectile bombardment, recently Agrobacterium-mediated gene transfer technique has also established in some of the millets but with very few reports. The central hindrance in millet transformation is its recalcitrant nature to regeneration through tissue culture techniques. Optimization of highly efficient regeneration procedure for each millet species is thus, necessary to establish advanced transformation system for them. The possibility of alternative transformation approaches is also discussed. The establishment of robust gene transfer methods whether it’s conventional in-vitro tissue culture dependent or in-planta are important for functional validation studies and would enable development of crop improvement strategies. This review presents the progress made on millet genetic transformation, discussing the major challenges that need to be overcome and future opportunities of transgenic techniques in various millets.

     

Recent advances in wheat transformation

Summary Since the first report of wheat transformation in the early 1990s, genetic engineering of wheat has evolved rapidly. Several laboratories worldwide have reported the production of fertile transgenic wheat plants using a variety of methods. While there are several innovative and promising approaches for wheat transformation using different explants as targets for transformation, different methods of transformation, and different selection schemes, the most common approach to wheat transformation is the bombardment of tissue derived from immature embryos followed by selection based on resistance to the bar gene. Even with all these successful reports, hurdles still exist for this recalcitrant crop. Of these hurdles, low transformation rates, tools for transgene expression, and transgene silencing in subsequent generations are probably the most critical. This review will provide an overview of wheat transformation in the past decade, addressing both positive and negative factors that effect transformation while highlighting the successes of the past and prospects for the future.     

Detection of coconut cadang-cadang viroid-like sequences in oil and coconut palm and other monocotyledons in the south-west Pacific

Fractionation, electroblotting and molecular hybridisation of nucleic acids extracted from tissue of African oil palm and coconut palm and some other monocotyledonous species, collected in several areas of the south-west Pacific region, demonstrated the presence of small nucleic acids with nucleotide sequences and secondary structure similar to coconut cadang-cadang viroid (CCCVd). The oil palms which contained CCCVd-related molecules showed orange leaf spots resembling those described for oil palm naturally infected with CCCVd in the Philippines, and also characteristic of a condition known as "genetic orange spotting" (GOS). We provide preliminary evidence that GOS is an infectious disorder caused by a viroid. The coconut palms did not show symptoms typical of cadang-cadang disease, but sometimes were chlorotic, stunted, or had a reduced yield. The possibility that the isolates represent variants of CCCVd is discussed. The data suggest that viroids with nucleotide sequences similar to CCCVd occur widely in palms and other monocotyledons outside the Philippines.     

Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

The association between DNA methylation status and embryogenic competency in oil palm tissue culture was examined through Representational Difference Analysis (RDA) approach, using methylation-sensitive restriction endonucleases. “Difference Products” (DPs) of RDA derived from palms of similar genetic backgrounds but exhibiting different embryogenesis rates during the regeneration process were isolated. The DPs were sequenced using a pyrosequencing platform. To our knowledge, this is the first study profiling partial HpaII methylation sites in oil palm young leaf tissues which are potentially associated with embryogenic amenability through a genomic subtractive approach. Quantitative real-time PCR analysis demonstrated that the methylation status of a novel fragment, EgNB3, was higher in highly embryogenic leaf explants compared to low embryogenesis rate materials. These differences are likely to be contributed by the 5′-^mCCGG-3′ and/or 5′-^mC^mCGG-3′ methylation patterns. Our data suggest that the differentially methylated site in EgNB3 has potential as a molecular biomarker for the screening of oil palm leaf explants for their embryogenic potentials.     

Switching from monoculture to polyculture farming benefits birds in oil palm production landscapes: Evidence from mist netting data

Monoculture farming is pervasive in industrial oil palm agriculture, including those RSPO plantations certified as sustainably managed. This farming practice does not promote the maintenance of farmland biodiversity. However, little scientific attention has been given to polyculture farming in oil palm production landscapes. Polyculture farming is likely to increase the floristic diversity and stand structural complexity that underpins biodiversity. Mist nets were used to sample birds at 120 smallholdings in Peninsular Malaysia. At each site, 12 vegetation structure characteristics were measured. We compared bird species richness, abundance, and composition between monoculture and polyculture smallholdings and used predictive models to examine the effects of habitat quality on avian biodiversity. Bird species richness was significantly greater in polyculture than that of monoculture smallholdings. The number of fallen and standing, dead oil palms were also important positive predictors of species richness. Bird abundance was also strongly increased by standing and dead oil palms and decreased with oil palm stand height. Our results indicate that polyculture farming can improve bird species richness in oil palm production landscapes. In addition, key habitat variables that are closely associated with farming practices, such as the removal of dead trees, should and can be managed by oil palm growers in order to promote biodiversity. To increase the sustainability of oil palm agriculture, it is imperative that stakeholders modify the way oil palms are currently planted and managed. Our findings can guide policy makers and certification bodies to promote oil palm production landscapes that will function more sustainably and increase existing biodiversity of oil palm landscapes.     

First production of wild hemmer (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. dicoccoides) transgenic plants

Plant genetic transformation and regeneration has become a valuable research tool for functional genomics. A successful transformation event involves the transfer of the target gene into a suitable explant, the integration and expression of the transgene into the host genome and the regeneration of the fertile transgenic plants from the transformed tissues. Wheat is considered as a recalcitrant species even if many efforts have been done in recent years to improve transformation efficiency. The transformation of its progenitors has never been attempted, even though the possibility to transform wild hemmer represents a valuable tool to evaluate structural and functional variability occurring in wild hemmer and explaining its higher adaptation to abiotic stresses. In this paper we report, as far as we know, for the first time, the microparticle transformation of immature embryos of the wild hemmer Triticum dicoccoides with the Tapgip1 gene. The transformation method was successfully transferred from durum wheat and several transgenic lines were obtained. Its application for the exploitation of wheat progenitors for molecular breeding is of great relevance for genomic and functional genomics studies. This result, indeed, opens new perspectives in complementation studies for the comprehension of durum and bread wheat adaptation mechanisms to stresses.     

Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (β-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (β-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.     

Efficient regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated stable transformation of perennial ryegrass

We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine. Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin, a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques, we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596. The text was submitted by the authors in English.