Efficient Transformation System Research Articles

Targeted creating new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants.

Fruits and vegetables in the Cucurbitaceae family, such as cucumber, melon, watermelon, and squash, contribute greatly to the human diet. The widespread use of genome editing technologies has greatly accelerated gene functional characterization and crop improvement. However, most economically important cucurbit plants, including melon and squash, remain recalcitrant to standard Agrobacterium tumefaciens-mediated transformation, limiting the effective use of genome editing technology. In this study, we used an “optimal infiltration intensity” strategy to establish an efficient genetic transformation system for melon and squash. We harnessed the power of this method to target homologs of the ERECTA family of receptor kinase genes and created alleles that resulted in a compact plant architecture with shorter internodes in melon, squash, and cucumber. The optimized transformation method presented here enables stable CRISPR/Cas9-mediated mutagenesis and provides a solid foundation for functional gene manipulation in cucurbit crops.

An efficient root transformation system for CRISPR/Cas9-based analyses of shoot-root communication in cucurbit crops.

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot–root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K+ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock and by overexpressing the pumpkin tonoplast Na+/H+ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function. However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed. Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium (MS basal salts, 30 g L−1 sucrose, 8 g L−1 agar, 5 mg L−1 6-benzylaminopurine (6-BA), 2 mg L−1 thidiazuron (TDZ), and 1 mg L−1 1-naphthlcetic acid (NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime (150 mg L−1) and kanamycin (15 mg L−1). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.

Regeneration and Agrobacterium-mediated genetic transformation of twelve <i>Eucalyptus</i> species

<italic>Eucalyptus</italic> is a genus of over 900 species and hybrids, and many of them are valuable fast-growing hardwoods in the world. Due to its economic importance, <italic>Eucalyptus</italic> is one of the early tree species whose genomes were deciphered. However, the lack of efficient genetic transformation system severely restricts the functional genomic research on the plant. The success of <italic>Eucalyptus</italic> regeneration and transformation depends greatly on the genotypes and explants. In this study, we systematically screened 26 genotypes from 12 <italic>Eucalyptus</italic> species with an attempt to obtain <italic>Eucalyptus</italic> genotypes with high regeneration potential. We developed two common regeneration media that can be applied to most tested <italic>Eucalyptus</italic> genotypes for both seeding hypocotyls and cloned internodes as explants. Then, we implemented DsRed2 as a visual marker for genetic transformation efficiency test. Our results suggest that <italic>E. camaldulen</italic> and <italic>E. robusta</italic> are amenable for genetic transformation. Finally, we successfully set up a stable <italic>Agrobacterium</italic>-mediated genetic transformation procedure for both <italic>E. camaldulen</italic> and <italic>E. robusta</italic> using seeding hypocotyls and cloned internodes respectively. Taken together, our study provides valuable means for vegetative propagation, gene transformation, CRISPR based gene mutagenesis, activation and suppression, as well as functional characterization of genes in <italic>Eucalyptus</italic>.

Genotype Screening of Recipient Resources with High Regeneration and Transformation Efficiency in Chrysanthemum

Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum, which relies on an optimal regeneration and transformation system. However, the regeneration system of different chrysanthemum cultivars varies, and the regeneration time of most cultivars is long. To screen cultivars with highly efficient regeneration, leaves and shoot tip thin cell layers (tTCL) from eight chrysanthemum cultivars with different flower colors and flower types were cultured on Murashige and Skoog media (MS) supplemented with 1.0–5.0 mg L−1 6-benzylaminopurine (6-BA) and 0.1–1.0 mg L−1 α-naphthaleneacetic (NAA). The results showed that the most efficient regeneration media were MS + 6-BA 1.0 mg L−1 + NAA 0.5 mg L−1 for leaf explants and MS + 6-BA 5.0 mg L−1 + NAA 0.1 mg L−1 for tTCL explants. Subsequently, another 13 chrysanthemum cultivars were screened by using the media, and finally, three cultivars with high regeneration efficiency were obtained from 21 cultivars. Among these, C1 had the highest regeneration efficiency: the regeneration rate of leaf explants reached 80.0% after 42 days of culture, and the regeneration rate of tTCL explants reached 100% after 31 days of culture. Furthermore, we also established the transformation system for C1 as follows: preculturing for one day, infecting with Agrobacterium suspension (OD600 = 0.6) for 10 min, and cultivating in the regeneration medium with 350 mg L−1 carbenicillin and 10 mg L−1 kanamycin, thus ultimately achieving a transformation rate of 4.0%. In this study, a new chrysanthemum cultivar with an efficient regeneration and transformation system was screened, which is beneficial to enrich the flower color of chrysanthemum transgenic plant recipients and to the functional research of flower color or type-related genes.

Molecular Farming in Seed Crops: Gene Transfer into Barley (Hordeum vulgare ) and Wheat (Triticum aestivum ).

The production of recombinant proteins in seed crops has a long history and cereal grains are now one of the platforms in commercial use. Specific advantages include excellent storage properties, a well-developed endomembrane system with a high biosynthetic capacity and well-established cultivation procedures worldwide. However, the production of transgenic cereals is a time-consuming procedure and the lack of efficient transformation systems is still a significant bottleneck. Barley can be transformed at high efficiency but the protocols are genotype-dependent. Wheat is generally more challenging to transform, but considerable progress has been made in enhancing transformation efficiencies and in controlling transgene expression. In this chapter, we describe and discuss standard procedures for generating transgenic barley and wheat for the production of recombinant proteins.

Transcriptome analysis of melatonin regulating the transformation of glucoraphanin to sulforaphane in broccoli hairy roots.

Sulforaphane (SF) is one of the most effective natural products in preventing and fighting cancer, foundin cruciferous plants. In this study, broccoli hairy roots grown for 20 d were used as the experimental material, and it was treated with 500μmol/L melatonin (MT) for 0, 12 and 32h to explore the effect of MT on the conversion of glucoraphanin (GRA) to SF. Results showed that the yields of GRA and SF were the largest under MT treatment for 12h, which were 1.53 and 1.93-fold,respectively, compared to 0h. However, Myrosinases activity was the highest under MT treatment for 32h, which was 1.42-fold compared tothat of the 0h. The differential expression of key genes involved in GRA conversion to SF in broccoli hairy roots was identified transcriptome sequencing, and the path of the transformation from GRA to SF was simulated, which provided a theoretical basis for establishing an efficient transformation system from GRA to SF.

High-efficiency &lt;i&gt;Agrobacterium&lt;/i&gt;-mediated transformation of chrysanthemum via vacuum infiltration of internode

Leaf disc transformation is one of the traditional methods that are now widely used in chrysanthemum with highly economical and ornamental value in world flower production, but it depends on plant genotypes and is time consuming and complicated. In addition, the transformation success rate of this method is low, generally ranging from 0.1% to 6.25%. Therefore, a highly efficient transformation system is needed. In this study, we are the first to establish a high-efficient chrysanthemum <i>Agrobacterium</i>-mediated transformation system via vacuum infiltration. Chrysanthemum stem internode explants were used as research material and <i>CmLEC1</i> was used as a reporter gene. After approximately 3 months of culture and selection, the positive transgenic plants were obtained. Additionally, the positive probability was about 42%. The transformation efficiency was up to 37.7%, and if the escapes were removed, it was 16%. Furthermore, stable expression of <i>CmLEC1</i> in transgenic 'Yuhualuoying' was confirmed by qRT-PCR analysis. These results suggest that this genetic transformation system via vacuum infiltration of chrysanthemum stem internode is highly efficient and convenient, and much better than traditional leaf disc transformation, and it will play an important role in chrysanthemum transformation and functional genetics research.

Genome-editing in millets: current knowledge and future perspectives.

Millets are small seeded cereal crops predominantly cultivated and consumed by resource-poor farmers in the semi-arid tropics of Asia and Africa. Millets possess rich nutrients and a climate resilience property when compared to the other cereals such as rice and wheat. Millet improvement using modern genetic and genomic tools is falling behind other cereal crops due to their cultivation being restricted to less developed countries. Genome editing tools have been successfully applied to major cereal crops and, as a result, many key traits have been introduced into rice, wheat and maize. However, genome editing tools have not yet been used for most millets although they possess rich nutrients. The foxtail millet is the only millet utilised up to now for genome editing works. Limited genomic resources and lack of efficient transformation systems may slow down genome editing in millets. As millets possess many important traits of agricultural importance, high resolution studies with genome editing tools will help to understand the specific mechanism and transfer such traits to major cereals in the future. This review covers the current status of genome editing studies in millets and discusses the future prospects of genome editing in millets to understand key traits of nutrient fortification and develop climate resilient crops in the future.

Establishment of a Novel and Efficient Agrobacterium-Mediated in Planta Transformation System for Passion Fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is an important fruit crop with high economic value. Genetic engineering plays an important role in crop improvement with desired traits and gene functional studies. The lack of a simple, efficient, and stable transformation system for passion fruit has greatly limited gene functional studies. In this study, a simple and efficient Agrobacterium-mediated in planta transformation system for passion fruit was established, using Agrobacterium virulent strain EHA105 harboring the binary vectors pCAMBIA1301 and pCAMBIA1302 with GUS and GFP reporter genes. The system requires less time and labor costs than conventional transformation systems, and no additional phytohormones and sterile conditions are required. Regeneration efficiency of 86% and transformation efficiency of 29% were achieved, when the wounds were wrapped with Parafilm and the plants were kept in darkness for 15 days. Approximately 75% of the regenerated plants had a single shoot and 26% multiple shoots. The transformation was confirmed at the DNA and RNA levels as well as by GUS staining and GFP fluorescent measurements. The developed protocol will contribute to the genetic improvement of passion fruit breeding.

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

BackgroundLitchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi.ResultsIn this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots.ConclusionWe first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

Establishment of Agrobacterium tumefaciens – mediated genetic transformation of apple pathogen Marssonina coronaria using marker genes under the control of CaMV 35S promoter

Premature leaf fall of apple caused by Marssonina coronaria is economically very important apple disease and all the commercially available apple cultivars are susceptible to this disease. The non-availability of an efficient transformation system for this fungus hinders the functional genomics research. Herein, we report for the first time, the successful Agrobacterium-mediated transformation in apple leaf blotch fungus M. coronaria by transferring T-DNA harbouring the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA) and green fluorescent protein (gfp) under the control of CaMV 35S promoter. The key factors that affect the transformation efficiency including type of recipient fungal material, acetosyringone concentration, the conditions for co-cultivation, Agrobacterium concentration, Agrobacterium strains and membrane types as support were investigated. The present results have recommended that 250 μM concentration of acetosyringone, 24 °C temperature and 48 h time, 0.5 OD600 of A. tumefaciens, EHA105 Agrobacterium strain and Whatman filter paper were the optimal co-cultivation conditions for the transformation of M. coronaria by using fragmented mycelia suspension and mycelial plugs. We observed that conidia were tedious to transform as compared to the fragmented mycelia and mycelial plugs of this slow growing fungus. These optimized parameters yielded 54 and 70 average transformants per 60 mycelial plugs and 104 fragmented mycelia, respectively. Fungal transformants were analysed for T-DNA integration, gus gene expression and gfp gene expression. Strong gus histochemical staining and green fluorescence expression indicated that the CaMV 35S promoter can drive gene expression in M. croronaria. Some mutants showed difference in the morphology of the colony as compared to the wild type control. This report will be very useful to inspect molecular basis of apple-M. coronaria interactions by deciphering the functional roles of various genes in this pathogenic fungus.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

Efficient plant regeneration and genetic transformation system of the precious fast-growing tree Toona ciliata

Toona ciliata has considerable economic value, providing high-quality materials and medicines. However, the growth and sustainable development of T. ciliata is limited due to environmental changes, poor natural regeneration ability, and excessive logging. Therefore, this study used sterile seedling leaves from a good provenance of T. ciliata as explants to establish a highly efficient regeneration and genetic transformation system. The effects of different hormones and concentrations on leaf callus induction were investigated. A genetic transformation system using kanamycin (Kan) as the selective agent was established based on the regeneration system. The results showed that Murashige and Skoog (MS) medium supplemented with 3 mg/L N-(Phenylmethyl)-9H-purin-6-amine (6-BA), 1 mg/L kinetin (KT) and 0.05 mg/L 1-naphthlcetic acid (NAA) (T1 medium) were suitable for callus formation and adventitious bud production from whole leaves at 45 days. In addition, pCAMBIA2301-EYFP was introduced into competent cells of Agrobacterium tumefaciens strain GV3101 and used for genetic transformation. Healthy leaves pre-cultured on T1 medium for three days were directly infected with A. tumefaciens infection solution (OD600 = 0.6) for 20 min through the addition of 50 mmol/L of Acetosyringone (AS). Thereafter, the infected leaves were co-cultivated for one day on T1 medium supplemented with 150 mmol/L AS, and the infected leaves were transferred to fresh T1 medium containing 10 mg/L Kan and 100 mg/L Cefotaxime (Cef). Twenty-four positive transformed plants were selected through histological staining and PCR, laser confocal microscopy of EYFP from 259 putative transformants was conducted, and the transformed plants were obtained after approximately three months.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in halophyte Puccinellia tenuiflora.

The online version contains supplementary material available at 10.1007/s11032-021-01247-8.

An efficient sorghum transformation system using embryogenic calli derived from mature seeds.

Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The nptII and gus gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T1 seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.

Establishment of efficient callus genetic transformation system for Pyrus armeniacaefolia

The purpose of the present study was to establish an efficient and stable callus genetic transformation system for Pyrus armeniacaefolia. Fruits 15 days after flowering were used for callus inducing. Major factors that influence growth state of callus (concentrations of plant growth regulators, subculture cycle) and transformation (Agrobacterium tumefaciens concentration, infection time, co-culture time, AS and kanamycin concentrations) were examined. The results showed that the optimum mediums for callus induction was MS supplemented with 3.0 mgL−1 2,4-Dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgL−1 6-Benzylaminopurine (6-BA). The appropriate proliferation medium was MS containing 2.5 mgL−1 2,4-D and 0.5 mgL−1 6-BA with 21 d subculture cycle. Transformation was successfully achieved using the following protocol: callus were soaked in A. tumefaciens strain EHA105 (OD600 = 0.8) harboring pBI121 plasmid for 15 min. After 3 d co-culture on proliferation medium supplemented with 20 mgL−1 AS, the callus were then screened on the selection medium (proliferation medium containing 25 mgL−1 kanamycin and 200 mgL−1 cefotaxime) to select the resistant callus. The GUS-positive rate was 96%. And the GUS-positive callus were further identification at DNA and transcription levels. We believe that the present study provides technical support for functional researches on fruit quality related genes.

Development of an efficient Agrobacterium rhizogenes-mediated gene transformation system for millets

Development of an efficient Agrobacterium rhizogenes-mediated gene transformation system for millets

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

Genetic transformation of legumes: an update.

This review summarizes the recent advances in legume genetic transformation and provides an insight into the critical factors that play a major role in the process. It also sheds light on some of the potential areas which may ameliorate the transformation of legumes. Legumes are an important group of dicotyledonous plants, highly enriched in proteins and minerals. Majority of the legume plants are cultivated in the arid and semi-arid parts of the world, and hence said to be climate resilient. They have the capability of atmospheric nitrogen fixation and thus play a vital role in the ecological sphere. However, the worldwide production of legumes is somehow not up to the mark and the yields are greatly affected by various biotic and abiotic stress factors. Genetic engineering strategies have emerged as a core of plant biology and remarkably facilitate the crop improvement programmes. A significant progress has been made towards the optimization of efficient transformation system for legume plants over the years but this group is still underutilized in comparison to other crops. Among the variety of available DNA delivery systems, Agrobacterium-mediated and particle bombardment have been primarily deployed for optimization and trait improvement. However, recalcitrance and genotype-dependence are some of the major bottlenecks for successful transformation. In this context, the present review summarizes the advances taken place in the area of legume transformation and provides an insight into the present scenario. The challenges and future possibilities for yield improvement have also been discussed.