Immature Embryo-derived Calli Research Articles

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

Particle bombardment-mediated co-transformation of the Cht-2 gene in wheat and the associated changes in defense mechanisms in transgenic plants infected with Fusarium graminearum

Abstract Fusarium graminearum is a major global pathogen of cereals and is considered the main causal agent of Fusarium head blight disease in wheat. Infection with F. graminearum causes a significant reduction in crop yield and quality; therefore, it is very important to improve wheat pathogen resistance. In the present study, the plasmid pAHCht-2 harboring the rice chitinase (Cht-2) gene for pathogen resistance and the plasmid pAB6 containing the gus reporter and bar selectable marker genes were used for genetic transformation of immature embryo-derived calli of the Egyptian wheat cultivar Giza 164 using particle bombardment. The presence and integration of transgenes were assessed by PCR analysis using specific primers for the Cht-2, bar and gus genes. The incorporation of the Cht-2 gene into the genome of the transformants was confirmed by dot-blot analyses. The transformation efficiency (number of transgenic plants/number of embryos) was 6.01%. Additionally, associated changes in defense mechanisms in the transgenic plants were investigated. The results showed variations in biochemical characteristics between transgenic and non-transgenic wheat lines (cv. Giza 164). The transgenic plants had significantly decreased total protein content, phenolic compounds and antioxidant enzyme activities (peroxidase and catalase), and significantly increased phenylalanine ammonia lyase and chitinase activities compared with non-transgenic plants under biotic stress conditions caused by F. graminearum infection. Our results show that activating a specific program of gene expression related to particular environmental stress conditions can reduce the cost of the stress on plant metabolism.

Effect of seed size on in vitro seed germination, seedling growth, embryogenic callus induction and plantlet regeneration from embryo of maize (Zea mays L.) seed

Immature embryo-derived callus is more efficient for plant regeneration in maize but appears difficult to obtain in all seasons of the year compared to mature embryos from dry seeds which are readily available throughout the year. This study investigated the effect of seed size on in vitro seed germination, seedling growth, callus induction and plantlet regeneration, as well as the relationships between these parameters in five maize varieties. Seeds were designated either as large or small for each variety based on its 100-seed weights, while seed germination were obtained in petri-dishes placed between two sheets of pre-wetted filter paper. Seeds were disinfected, and mature embryos were excised from the maize endosperm and inoculated on the Murashige and Skoog salt (MS medium) supplemented with 30 g/l sucrose, 8 g/l agar, 0.1 g/l myo-inositol and 3 mg/l 2,4-D for callus induction, while embryogenic calli were transferred to medium containing 0.5 mg/l Benzylaminopurine (BAP) and 0.5 mg/l Kinetin for plant regeneration. The study showed that large seed size had significant effect on almost all the traits studied, while positive and significant correlations were observed between in vitro germination, seedling growth, callus induction and plantlet regeneration. It can be concluded that callus fresh weight may be used as a marker for improving regeneration efficiency in maize. The results from this study suggest that genetic control of in vitro regeneration from maize mature embryo can be utilized to determine inherent genotypic potentials of maize varieties with tissue culture traits for maize improvement.

GENETIC TRANSFORMATION OF EGYPTIAN WHEAT WITH 1DX5 HIGH-MOLECULAR-WEIGHT GLUTENIN SUBUNIT GENE

Variation in the composition of the high-molecular-weight glutenin subunits (HMW-GS) of wheat is associated with large differences in the bread-making properties. The primary aim of this work was to produce transgenic Egyptian wheat cultivar (Giza 164) with HMW-GS gene (1DX5) to improve bread-making quality. Immature embryo-derived calli were cotransformed with pK-DX5 and pACH25 plasmids containing gus and bar genes using biolistic bombardment. Stable integration and expression of marker genes were detected in seven plants. However, the expression of 1DX5 gene was confirmed in five plants using SDS-PAGE analysis.

Transformation of Wheat Thaumatin-Like Protein Gene and Analysis of Reactions to Powdery Mildew and Fusarium Head Blight in Transgenic Plants

A thaumatin-like protein gene, Ta-Tlp, was cloned from wheat in previous studies, which was expressed in a high level in wheat ( Triticum aestivum L.) 6VS/6AL translocation line with high resistance to powdery mildew ( Erysiphe graminis f.sp. tritici Em. Marchal.), implying its close relation to the resistance of the disease. To further understand the gene's function, Ta-Tlp was constructed into an expression vector driven by the strong ubi promoter. The vector pAHC-TlP constructed was transformed into immature embryo-derived calli of common wheat cultivar Yangmai 158 through particle bombardment. After two rounds of herbicide bialaphos selection and regeneration, herbicide-resistance plants were obtained. The Ta-Tlp proved to be integrated into the wheat genome and was expressed in T 1 and T 2 generations by PCR, Southern blot, and RT-PCR analysis. The transgenic plants of T 0, T 1, and T 2 generations were inoculated by E. graminis and Fusarium graminearum for resistance identification. All plants of T 0, T 1, and T 2 generations were resistant to wheat powdery mildew by delaying disease development, but no distinct resistance to Fusarium head blight.

Plant regeneration from immature embryo-derived calli of wheat isogenic lines

The isogenic lines of wheat (Tritkum aestivum L. var. Novosibirskaja 67) carrying reduced height and leaf pubescence character, were evaluated for their ability to regenerate plants from immature embryo-derived calli using protocols based on six different media. The percentage of calli capable of plant regeneration was recorded. The effect of media on the formation of regenerative calli in the various isogenic lines is demonstrated. Significant differences were observed between the initial variety and the isogenic lines with the Rht genes on several media. The distinctive difference of Novosibirskaja 67 and sib of ANK-7A not carrying the marker character with ANK-7A line isogenic for leaf pubescence was revealed on two media. It is suggested that the leaf pubescence gene of ANK-7A or a gene closely linked to it are likely to play an important role in the control of plant regeneration in vitro. The fact that there is the medium where ANK.-7A produced calli regenerating plants while Novosibirskaja 67 and K-26011 (donor of leaf pubescence for ANK-7A) are completely incapable of plant regeneration confirms that the expression of these genes is influenced by the genomic background.

Plant regeneration from immature embryo-derived calli of wheat isogenic lines

Plant regeneration from immature embryo-derived calli of wheat isogenic lines

Effect of sorbitol concentration on regeneration of embryogenic calli in upland rice varieties (Oryza sativa L.)

This study describes the impact of sorbitol on plantlets regeneration frequency (PRF) of four rice cultivars (japonica, Oryza sativa L.) both of which mature and immature embryo-derived calli were investigated. The variance analysis results showed that PRF of the three elite upland rice cultivars, Handao297 (HD297), Handao502 (HD502), Handao65 (HD65) and one lowland rice cultivar Zhongzuo93 (ZZ93) were significantly increased with addition of appropriate amount of sorbitol in culture media. Supplementing appropriate sorbitol in the media of a continous culture from induction and maintenance to regeneration for mature embryo-derived calli could improve PRF dramatically, originally from 27.6% up to a maximum of 71.8%. Especially to low regenerative capacity (LRC) cultivar HD65, the PRF was increased over 7-fold (from 9.7% to 74.0%). The optimum concentrations of sorbitol for calli induction, subculture and differentiation were 5, 20 and 40 g/l, respectively. Adding sorbitol, only in maintenance media at concentration of 20 g/l, also enhanced the PRF greatly in all the cultivars from 27.6% to 43.3%. Similar results were observed when incorporating with maltose in regenerating media both in immature and mature embryo-derived calli. The optimal concentration was 25 g/l sorbitol + 20 g/l maltose and 20 g/l sorbitol + 25 g/l maltose, respectively. HD297 appeared to be the most responsive genotype compared to other cultivars in PRF, 99.2% in immature embryo-derived calli and 76.8% in mature embryo-derived calli. The results and relevant conclusions might be valuable to establish an efficient plant regeneration system from somatic embryogenesis culture in upland rice.

Transformation of powdery mildew resistance-related genes of wheat

Anthocyanin synthesis regulation gene C1-Lc was used as the reporter gene to optimize the parameters of gene-gun transformation protocol through counting of red spots on wheat calli after transient expression. Wheat Beclin1 like gene TaTBL and thiosulfate sulfutransferase gene TaTST proved to have an increased expression level after induction of wheat powdery mildew fungus (Erysiphe graminis f.sp. tritici Em. Marchal.). These two resistance-related genes were constructed into expression vectors driven by the strong ubi promoter and used to perform genetic transformation on wheat cv Yangmai158 immature embryo-derived calli through particle bombardment. After two rounds of herbicide bialaphos selection and regeneration, herbicide-resistance plants were obtained, which were subsequently subjected to PCR analysis. Five TaTBL transgenic plants and six TaTST transgenic plants were identified. Pathogen inoculation of detached leaves showed that the introduction of exogenous gene increased wheat resistance level by delaying the development of powdery mildew symptoms.

Production of fertile transgenic wheat plants by laser micropuncture

A modified, non-damaging, protocol for the production of fertile transgenic wheat (Triticum aestivum L. cultivar Giza 164) plants by laser micropuncture was developed. The new homemade setup secures the transformation of as many as 60 immature embryo-derived calli (10000 cells each) in less than one hour using a UV excimer laser with two dimensional translation stages, a suitable computer program and a proper optical system. Five-day-old calli were irradiated by a focused laser microbeam to puncture momentarily made self-healing holes ( approximately 0.5 microm) in the cell wall and membrane to allow uptake of the exogenous DNA. The plant expression vector pAB6 containing bar gene as a selectable marker for the herbicide bialaphos resistance and GUS (uidA) gene as a reporter gene was used for transformation. No selection pressure was conducted during the four-week callus induction period. Induced calli were transferred to a modified MS medium with 1 mg l(-1) bialaphos for regeneration, followed by selection on 2 mg l(-1) bialaphos for rooting. Three regenerated putative transgenic events were evaluated for the integration and stable expression of both genes and results indicated that this modified procedure of laser-mediated transformation can be successfully used in transforming wheat.

Micromorphology of Somatic Embryo Development of Castanopsis cuspidata (Thunb) var. sieboldii (Makino) Nakai

In vitro somatic embryogenesis of Castanopsis cuspidata var. sieboldii was achieved from immature embryo-derived callus and the morphological course of events during embryo development was evaluated by scanning electron microscopy technique (SEM). Embryogenic callus could be initiated from immature embryos and optimum induction of callus was observed on Murashige and Skoog (MS) medium supplemented with 10 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 1 μM benzyl‐aminopurine (BAP). Somatic embryo development was occurred on half-strength basal MS medium supplemented with 2 μM BAP, 0.25 μM naphthaleneacetic acid (NAA). Somatic embryo maturation was accomplished using 1 μM zeatin, 0.25 μM indolebutyric acid (IBA). SEM observations indicated development of an extracellular material at an early stage of differentiation and a course of differentiation leading to development of somatic embryos.

Phosphomannose-isomerase ( pmi) gene as a selectable marker for rice transformation via Agrobacterium

A new selectable marker system was adapted for use in Agrobacterium-mediated transformation of rice ( Oryza sativa L.). This selection system utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells were capable of utilizing mannose as a carbon source. Transgenic rice plants regenerated from selected transformed immature embryo-derived calli on media containing various concentrations of mannose. The highest transformation frequency of 6.0% was obtained when a combination of 25 g/l mannose and 5 g/l sucrose was used. Molecular and genetic analyses showed that the plants contained the pmi gene and the gene was transmitted to the progeny in Mendelian fashion. The results indicated that the mannose selection system, which was devoid of the disadvantages of antibiotic or herbicide selection, could be used for rice Agrobacterium-mediated immature embryo transformation.

Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium.

Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.

Chlorate resistance as a tool to study the effect of nitrate reductase antisense gene in wheat

The herbicide chlorate has been used extensively for isolation of mutants or for selection of transgenic plants defective in nitrate reduction. Chlorate, acting as an analogue of nitrate, is substrate of the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Plants being lack of NR function are resistant to chlorate. In this work chlorate resistance was used to control the efficiency of gene transfer cnxl antisense in wheat. Cnxl was transferred with cotransformation of pAHC25 containing bar and gus into immature embryo-derived calli of a spring wheat genotype (CY-45) by microprojectile bombardment The nitrate - chlorate interaction was investigated via nitrate reductase activity (NRA) assay and necrotic symptoms of chlorate toxicity. The optimal conditions of chlorate resistance test signalling the decrease of NRA were determined, and found to be 0.4 mM nitrate for NR induction, and 7 mM chlorate content for evoking greatest differences in toxic symptoms. We succeeded to regenerate of 10 plantlets resistant to phosphinothricine (PPT) indicating the expression of bar, and 6 of them were fertile producing 6 PPT resistant transgenic lines. Among the T 1 progenies of PPT transgenics, two (designated as 117 and 124) showed a significant reduction of NRA. Though T 2 progenies had a less decreased NRA in the mean of the population, but a higher chlorate resistance as compared to that of the control was still experienced.

An Improved Media System for High Regeneration Rates from Barley Immature Embryo-Derived Callus Cultures of Commercial Cultivars

Improved plant regeneration from tissues of elite barley cultivars (Hordeum vulgare L.) will facilitate their genetic transformation. Significant improvements in green plant regeneration from tissues of ‘Morex’ and ‘Harrington’ were achieved by modifying iron and boric acid concentrations, and by adding 6-benzyl aminopurine (BAP) to the culture medium. The plant regeneration in response to various concentrations of eight micronutrients indicated that MS concentrations were appropriate for KH2PO4, NaMoO4, MnSO4, KI, CoCl2, and ZnSO4 Increased H3BO3 (0.75 mM) and decreased FeSO4 (0.05 mM) improved green plant regeneration. The addition of 0.1 mg/L BAP to maintenance media resulted in an increase in regeneration. The effect of these changes, in combination with previously reported improvements, was assessed by comparing regeneration responses of eight cultivars and elite breeding lines on the improved media versus the original MS-based media. Green plant regeneration was improved for all tested lines and cultivars, from an average of two green plants to 29 green plants per Petri dish. Regeneration rates from all elite lines and cultivars on the improved media exceeded that of the highly regenerable ‘Golden Promise’ on the original media, indicating that poor plant regeneration should no longer limit the transformation of modern North American cultivars.

Transgene silencing and reactivation in sorghum

Amongst the important cereals, sorghum has been the most recalcitrant to genetic transformation. There are a few reports on sorghum transformation and the majority of these have reported silencing of one of the transgenes. We regenerated plants from two independent transgenic sorghum callus lines that were cotransformed with Ubi promoter :bar and Act1-D promoter :gusA gene constructs using the particle bombardment method. Southern analyses indicated integration of multiple copies of both the transgenes. T0 plants were found to express the bar gene. The gusA gene, however, was silenced. It was possible to activate gusA gene expression in T1 seedlings and in calli derived from immature T1 and T2 embryos by 5-azacytidine (azaC) treatment. In certain cases, spontaneous expression of the gusA gene was observed in T1 and T2 immature embryo-derived calli. Expression of the bar gene, as analyzed by Basta™ tolerance and Phosphinothricin acetyltransferase (PAT) assays, was detected in T0, T1 and T2 plantlets; however, the expression was reduced in the T2 progeny obtained from a homozygous T1 parent. PAT activity was also lower in the immature embryo-derived T2 calli from the same homozygous T1 parent. Again, culture on azaC increased the level of PAT activity in these calli. Moreover, in a separate set of stable transformation experiments, it was possible to recover a much higher than usual number of gusA gene expressing transgenic calli by growing the bombarded tissues in the presence of azaC. Taken together, these results suggest that methylation-based silencing is frequent in sorghum and probably responsible for several cases of transgene inactivation reported earlier for this crop.

Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars

Perennial ryegrass (Lolium perenne L.) is the most important grass species in areas with a temperate climate. Biolistic transfer of a ubiquitin promoter driven nptII expression cassette into mature or immature tissue derived calli of perennial ryegrass followed by paromomycin selection, resulted in the rapid and efficient production of fertile transgenic ryegrass plants. Transformation efficiencies after paromomycin selection in combination with the nptII selectable marker compared favourably with hygromycin selection in combination with the hph selectable marker. In total 83 independent nptII expressing plants were produced. Transformation frequency was highly affected by genotype, explant, selection regime and the duration of the callus induction period. The optimised transformation protocol for mature embryo derived calli of turf-type or forage-type cultivars resulted in an average transformation efficiency of 5.2% or 6.6% respectively. This converts into 1.7 or 2.2 independent transgenic plants per bombardment. Immature inflorescence- and immature embryo-derived calli were also successfully used as target for the gene transfer, resulting in transformation efficiencies of up to 3.7% or 11.42% respectively. Transgenic plants were transferred to soil 12 or 9 weeks after excision of mature and immature embryos or inflorescences respectively. Transgene integration and expression were confirmed by PCR and ELISA or western blot analysis. Southern blot analysis confirmed the independent nature of the transgenic lines. The majority of lines showed the integration of two to six transgene copies, while 21% of the analysed lines had a single copy insert. A short tissue culture period in comparison to recently published reports seems to be beneficial for the production of normal and fertile transgenic ryegrass plants. Consequently we report for the first time molecular evidence for sexual transgene transmission in fertile transgenic perennial ryegrass.

Morphogenesis in Long-term Maintained Immature Embryo-derived Callus of Wheat (Triticum aestivum L) - Histological Evidence for Both Somatic Embryogenesis and Organogenesis

Callus induced from immature embryos of wheat cv Kharchia 65 on Murashige and Skoog’s medium containing 2.5 mg l1 2,4-D was maintained In the regenerable state by subculturing every 5-6 weeks on medium supplemented with 2,4-D (2.5 mg l-1) and proline (10 mg l-1). Addition of proline helped maintain morphogenic competence for over four years. The regenerating callus was analysed histologically about one year after first induction. Both somatic embryogenesis and shoot organogenesis were seen in the same callus tissue that contained typical stages of somatic embryoid development and evidence for the de novo shoot bud formation.

The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.).

Transfer of T-DNA from Agrobacterium tumefaciens and A. rhizogenes to cells of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is demonstrated following the inoculation of immature embryos and immature embryo-derived callus. Agrobacterium T-DNA vectors containing the C1/Lc anthocyanin-biosynthesis regulatory genes, the gusA gene or a synthetic green fluorescent protein gene (sgfp-S65T) were constructed from original binary vectors. The visual T-DNA markers were used as cell-autonomous reporters of early Agrobacterium-mediated transformation events in the wheat and barley cells. This localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece.

Improved Callus Quality and Prolonged Regenerability in Model and Recalcitrant Barley (Hordeum vulgare L.) Cultivars.

In vitro performance of immature embryo-derived callus of two barley cultivars, the amenable cultivar, Golden Promise (GP) and the recalcitrant commercial cultivar, Galena (GL), was tested on fourteen media containing combinations of two auxins, dicamba and 2, 4-dichlorophenoxyacetic acid (2, 4-D) and two cytokinins, 6-benzylaminopurine (BAP) and zeatin. Generalizations for both cultivars include the fact that callus-induction frequencies on dicamba and 2, 4-D alone and dicamba plus zeatin were nearly 100%. Qualitative appearance for both cultivars was nearly identical on comparable media but markedly better on BAP-containing media. The addition of zeatin with either auxin supported vigorous growth due to the proliferation of nonregenerable callus; the presence of BAP produced higher quality callus although it inhibited the relative growth rate. GP calli grown on 2, 4-D plus 0.01mg/L BAP regenerated the largest number of green shoots at most time points relative to other media, while GL tissue on 2, 4-D plus 0.1mg/L BAP yielded the most green shoots at all but one time point. Over time regenerability was lost more rapidly in GL than GP except on 2, 4-D plus BAP media; GL had a higher propensity toward albinism than GP. In both cultivars, the use of 2, 4-D and BAP yielded tissue that proliferated for prolonged periods in a regenerable state.